University of Southern California Dissertations and Theses

The polymeric immunoglobulin receptor in ocular tissues

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The polymeric immunoglobulin receptor in ocular tissues

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THE POLYMERIC IMMUNOGLOBULIN RECEPTOR IN OCULAR TISSUES

by

Wenzheng Zhang

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

December 2006

Copyright 2006 Wenzheng Zhang

ii
DEDICATION
To my dearest parents, Hongkui Zhang and Su Liu, and my beloved
husband, Shaojun Zhu, who give me endless love.

iii
ACKNOWLEDGEMENTS
A “Thank You” could not convey my deepest appreciation to my mentors,
Dr. Vincent H. L. Lee and Dr. Curtis T. Okamoto, for their insightful guidance
and warm encouragement in my Ph.D. study. I started my training with little
knowledge in pharmaceutical sciences. With great patience and understanding,
Dr. Lee and Dr. Okamoto led me through the transition from an engineering
student to an independent pharmaceutical researcher.
Gratitude is owed to my other dissertation committee members, Dr.
Kwang-Jin Kim, Dr. Austin K. Mircheff, Dr. Wei-Chiang Shen, and Dr. Melvin D.
Trousdale, for their invaluable direction on my Ph.D. work. Special thanks to Dr.
Sarah Hamm-Alvarez’s team who has been supportive during the course of my
Ph.D. research.
All the members from Dr. Lee’s and Dr. Okamoto’s labs are thanked for
their assistance and the share of all happy memories.

iv
TABLE OF CONTENTS
Page
Dedication
Acknowledgements
List of Figures
Abstract
Chapter I
Chapter II

Chapter III

Chapter IV

Chapter V
Bibliography

Introduction

Identification of the polymeric
immunoglobulin receptor in rabbit
conjunctival epithelial cells

Direct interaction between rab3D and the
polymeric immunoglobulin receptor may
regulate its trafficking through regulated
secretory vesicles in lacrimal gland acinar
cells

Cross-talk between rab11 and rab3D in
regulation of the polymeric
immunoglobulin receptor trafficking may
reflects communication between regulated
and constitutive secretory pathways in
lacrimal gland acinar cells

Conclusions and future directions

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25

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72

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101

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LIST OF FIGURES
Page
Chapter I - Introduction

Figure 1.1 Schematic diagram of the anterior segment of the eye
(sectional view)

Figure 1.2 Electron micrograph of adjacent acinar cells from the
rat lacrimal gland

Figure 1.3 Model of cellular pathways for stimulating lacrimal
gland protein secretion

Figure 1.4 Domain structure of pIgR

Figure 1.5 Pathway of the polymeric immunoglobulin receptor
(pIgR) through a polarized epithelial cell

Figure 1.6 A model for how IgA binding to the pIgR activates
intracellular signaling pathways and stimulates transcytosis

Figure 1.7 Functions of dIgA/sIgA and pIgR

Chapter II - Identification of the polymeric immunoglobulin
receptor in rabbit conjunctival epithelial cells

Figure 2.1 pIgR transcytosis

Figure 2.2 Western blot detection of pIgR in RCEC

Figure 2.3 Immunoprecipitation of SC from apical (A) and
basolateral (B) medium of cultured RCEC on Transwell filters

Figure 2.4 Immunolocalization of pIgR in cultured RCEC on
Transwell filters by confocal laser-scanning microscopy

Figure 2.5 Layer distribution of pIgR in cultured RCEC on
Transwell filters by confocal laser-scanning microscopy

3

5

9

11

14

16

19

29

36

38

39

41

vi
Chapter III – Direct interaction between rab3D and the
polymeric immunoglobulin receptor may regulate its
trafficking through regulated secretory vesicles in lacrimal
gland acinar cells

Figure 3.1 Colocalization of pIgR and rab3D

Figure 3.2 Recombinant rab3DWT pull-down of pIgR

Figure 3.3 Recombinant wild-type and mutant rab3D pull-down of
pIgR

Figure 3.4 Recombinant rab3DWT pull-down of pIgR from pIgR-
transfected MDCK cells

Figure 3.5 Direct interaction between rab3D and pIgR

Figure 3.6 Effect of CCH on rab3D-pIgR interaction

Figure 3.7 Regulated merocrine and constitutive transcytotic
pathways involved in the regulation of pIgR trafficking and SC
secretion in LGAC

Chapter IV – Cross-talk between rab11 and rab3D in
regulation of the polymeric immunoglobulin receptor
trafficking may reflect communication between regulated and
constitutive secretory pathways in lacrimal gland acinar cells

Figure 4.1 Colocalization of rab11 and pIgR

Figure 4.2 Co-immunoprecipitation of pIgR with anti-rab11 mAb

Figure 4.3 CCH induces a movement of rab11 and rab3D from
membrane to cytosol

Figure 4.4 Recombinant rab3DWT pull-down of rab11 and pIgR

Figure 4.5 Recombinant wild-type and mutant rab3D pull-down of
rab11 and pIgR

Figure 4.6 Two major pathways involved in the regulation of pIgR
trafficking in LGAC are the constitutive transcytotic pathway (red
arrow) and regulated merocrine pathway (blue arrows)

56

57

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60

62

63

71

82

83

85

86

88

94
vii
ABSTRACT
Conjunctiva and lacrimal gland are two major ocular epithelial tissues
included in the mucosal immunity system. The polymeric immunoglobulin
receptor (pIgR) is expressed in a wide variety of secretory epithelial cells. The
pIgR is the precursor to secretory component (SC) and secretory IgA (sIgA). It
regulates secretion of SC and sIgA through its transcytosis across epithelial cells.
The pIgR therefore plays an important role in mucosal immunity. To explore its
role in ocular surface immunity, we sought to identify and characterize pIgR in
ocular tissues.
In rabbit conjunctival epithelial cells, the pIgR and SC were molecularly
identified and localized, which provide the potential of utilizing endogenously
expressed pIgR for prevention of ocular surface-related diseases. Thus, a pIgR-
based model of ocular mucosal immunity can be developed and characterized. In
lacrimal gland acinar cells, we found that two members of small ras-like GTPases,
rab3D (a regulated secretory vesicle marker) and rab11 (an apical endosome
marker), are involved in the regulation of pIgR trafficking and therefore SC
secretion in a stimulation-dependent manner. Furthermore, rab3D and rab11 may
cross-talk in modulating pIgR trafficking through a regulated merocrine pathway
and a constitutive transcytotic pathway, respectively, initiating a novel model of
pIgR trafficking.
1
CHAPTER I - INTRODUCTION
A. Conjunctiva
Conjunctiva is a thin, mucus-secreting, vascularized, and multilayered
tissue that covers most of the inner surface of the eyelids and is part of the
anterior sclera where the cornea begins [Srinivasan et al., 1982]. The conjunctiva
occupies a 9-fold larger surface area than the cornea in rabbits and a 17 times
larger surface area in humans [Watsky et al., 1988]. The portion of the
conjunctiva covering the posterior surface of the eyelids and the anterior surface
of sclera are the palpebral conjunctiva and bulbar conjunctiva, respectively. The
area where palpebral and bulbar conjunctivae meet is the fornix conjunctiva
(figure 1.1). The conjunctiva can be divided into three layers [Bron et al., 1985]:
(1) an outer epithelium, forming a permeability barrier; (2) the substantia propria,
containing structural and cellular elements, nerves, lymphatics, and blood vessels;
and (3) the submucosa, providing a loose attachment to the underlying sclera.
Superficial, wing, and basal cells are the three principal epithelial cell types in the
conjunctiva. Interspersed among the superficial cells are round or oval mucus-
secreting goblet cells, while the wing and basal cells may contain pigment
[Kompella et al., 2000]. The surface cells of the conjunctiva are connected by
tight junctions which render the epithelium a relatively impermeable barrier
[Hogan et al., 1971]. In contrast to cornea, the conjunctiva is highly vascularized
and capable of mucus secretion. However, similar to the cornea, the conjunctival
2
epithelium is a nonkeratinized, stratified epithelium with microvilli covered with
glycocalyx and mucus [Bron et al., 1985; Nichols et al., 1983].
Conjunctiva contributes to the regulation of electrolyte and fluid balance
in the microenvironment of its mucosal surface [Shiue et al., 2000], as well as
serves as a passive physical protective barrier and participates in the maintenance
of tear film stability due to the mucus secreted by the resident goblet cells
[Srinivasan et al., 1982]. The conjunctiva plays a vital role in both vision and
immunity. It physically protects the delicate structures of the eye by serving as a
mechanical barrier to foreign substances, and it provides epithelium to cover
corneal wounds [Kinoshita et al. 1983]. Its goblet cells secrete mucus that forms
the inner layer of the tear film and is essential for normal vision. The abundant
blood supply of the conjunctiva delivers protective substances such as antibodies,
complement, and white blood cells to the eye to combat infections and to remove
dead or damaged tissue [Dawson 1976, 1984]. Lastly, the conjunctival, intestinal,
bronchiolar, and other mucous membranes constitute the mucosal immune system,
found in the regions of the body most commonly invaded by microorganisms
[Chandler et al., 1980; Wolf et al., 1984].

B. Lacrimal gland
The main lacrimal gland is an almond-shaped gland and, in most species,
is located on the anterior and lateral parts of the roof of the orbit of the eye (figure

Figure 1.1 Schematic diagram of the anterior segment of the eye (sectional view).

1.1). Its function is to synthesize, store, and secrete proteins, water, and
electrolytes that are released into the tears and onto the surface of the eye. Similar
to other exocrine glands, such as the salivary glands and the pancreas, the lacrimal
gland is a multilobed, tubuloacinar structure with ducts that secrete onto external
body surface. The lacrimal gland contains various types of cells, such as acinar,
ductal, and myoepithelial cells. In cross section, the acinar cells appear as a ring,
in a structure termed an acinus. These cells comprise about 80% of the mass of
the lacrimal gland. The acinar cells of the lacrimal gland have tight junctions that
3
4
surround the cells at the lumen dividing the plasma membrane into apical (lumen)
and basolateral (blood) membranes. These tight junctions are responsible for
generating a polarized secretory cell that allows vectorial secretion of proteins and
fluid. The nucleus, Golgi apparatus, and endoplasmic reticulum reside mainly in
the basal portion of the acinar cell while the apical portion of the cell has
numerous secretory granules containing proteins (figure 1.2). Although the ductal
cells are known to secrete a limited amount of protein, they do not contain many
secretory vesicles. The main function of these cells is to secrete water and
electrolytes. Human lacrimal glands have multiple ducts leading to the ocular
surface while rat and rabbit lacrimal glands contain a single excretory duct. The
myoepithelial cells are involved in the contraction of the acinar and ductal cells to
facilitate the secretory process.
In addition to acinar, ductal, and myoepthelial cells, the lacrimal gland
also contains various types of lymphocytes (expressing immunoglobulin (Ig)A,
IgD, IgE, IgG, and IgM), plasma cells, mast cells, and macrophages. In human
lacrimal gland, more than 50% of the mononuclear cells are plasma cells, the
majority of which are IgA positive [Wieczorek et al., 1988]. These cells are very
important as they synthesize and secrete IgA, which is a critical component of the
immune system of the eye.

Figure 1.2 Electron micrograph of adjacent acinar cells from the rat lacrimal gland. The
nucleus (N) lies in the basal portion of the cell and is surrounded by endoplasmic reticulum
(ER). Secretory granules (SG) lie in the apical portion of the cell. Tight junctions (TJ) are
present adjacent to the lumen (L) and separate the apical membrane (AM) from the
basolateral membrane (BLM) [Dartt, 1994].

C. Lacrimal gland protein secretion
Many of the proteins secreted by the lacrimal gland are antibacterial and
5
group II secretory phospholipase A2, which serve to protect the ocular surface
from infection [Nevalainen et al., 1994]. As the lacrimal gland is the primary
player for the secretory immune system of the ocular surface, many secreted
proteins are also involved in immunological protection of the eye, such as
monomeric and dimeric IgA, secretory IgA (sIgA), IgE, IgM and IgG. It is
interesting that secretion of lacrimal gland fluid differs not only between species
but also between sexes. Lacrimal glands from rabbits do not secrete peroxidase
whereas lacrimal glands from rats, humans and guinea pigs do. Rat lacrimal
6
glands synthesize and secrete amylase whereas lacrimal glands from rabbits and
guinea pigs do not [Thorig et al., 1984, 1985]. It has been shown that male rats
secrete higher levels of proteins and more tear than female rats. Males secrete
more secretory component (SC), IgA, and cystatin-related protein and peroxidase
than females [Sullivan et al., 1998].
There are two types of protein secretion from the lacrimal gland, regulated
and constitutive. Constitutively secreted proteins are synthesized in the same
organelles as regulated secretory proteins, but in the trans-Golgi apparatus
constitutive and regulated proteins are segregated into different vesicles. The
constitutive vesicles are larger and more electronlucent than regulated granules
and have a shorter half-life [Dartt, 1989]. Constitutive secretory proteins are
synthesized in the gland and sorted into granules that are not stored but fuse with
the plasma membrane releasing their contents to the exterior of the cell.
Constitutive secretory proteins are secreted slowly in response to steroid
hormones. Thus regulation of their secretion occurs at the level of gene
transcription and translation. Stimuli control the rate of synthesis of these
proteins. Previously, the best-studied examples of the constitutively secreted
lacrimal gland proteins are sIgA and SC [Sullivan et al., 1984]. Regulated
secretory proteins are synthesized in the lacrimal gland and stored in secretory
granules. In response to stimulation by classic neurotransmitters, biologically
active peptides, or peptide hormones, regulated secretory proteins are secreted
rapidly. Stimuli control the rate of release of these proteins.
7
D. Cellular pathways for stimulating lacrimal gland protein secretion
Lacrimal gland protein secretion is under neural and hormonal control.
Proteins in lacrimal gland fluid are secreted primarily by the acinar cells [Dartt,
1989]. The lacrimal gland is densely innervated with parasympathetic
(cholinergic) nerves and less densely innervated with sympathetic (adrenergic)
nerves [Ding et al. 2001]. Afferent sensory nerves in the cornea and conjunctiva
stimulate efferent parasympathetic and sympathetic nerves in the lacrimal gland.
Cholinergic agonists, released from parasympathetic nerves, and norepinephrine,
released from sympathetic nerves, are major stimuli of lacrimal gland secretion
[Dartt, 2004].
In the lacrimal gland, cholinergic agonists stimulate protein secretion.
Cholinergic agonists cause a rapid increase in protein secretion from the lacrimal
gland that gradually decreases with time [Dartt et al., 1984]. Of the five
muscarinic receptor subtypes, only the M
3
subtype is present in the lacrimal gland
[Mauduit et al., 1993]. Upon cholinergic agonists binding, the M
3
receptor,
through G
q/11
α subtype of G-proteins, activates phospholipase C (PLC) to break
down phosphatidylinositol bisphosphate (PIP
2
) into inositol trisphosphate (1,4,5-
IP
3
) and diacylglycerol (DAG). 1,4,5-IP
3
causes release of Ca
2+
from intracellular
stores. This Ca
2+
, perhaps in conjunction with calmodulin (CaM), activates
specific protein kinases that may be involved in secretion. DAG activates protein
kinase C that stimulates protein secretion (figure 1.3).
8
Vasoactive intestinal peptide (VIP), β-adrenergic agonists, α-melanocyte
stimulating hormone, and adrenocorticotropic hormone are also lacrimal gland
seretagogues. They activate adenylate cyclase (AC) to produce cAMP (figure
1.3). cAMP stimulates protein kinase A, which perhaps regulates protein
secretion. Cholinergic agonists and VIP also stimulate lacrimal gland fluid
secretion, and the same signal transduction pathways utilized by these agonists to
stimulate protein secretion are most likely used for electrolyte and water secretion.
α1-Adrenergic agonists also stimulate lacrimal gland protein secretion.
These agonists use a pathway that is separate from that utilized by cholinergic
agonists and VIP. The specific pathway has not been identified but may be DAG
and protein kinase C (figure 1.3). Thus, three separate cellular pathways
stimulate lacrimal gland protein secretion.

E. Immunoglobulin A (IgA) and secretory IgA (sIgA)
Among Ig isotypes, IgA, including secretory IgA (sIgA), which
predominates in humans and represents more than 60% of all of the antibody
isotypes [Conley et al., 1987; Mestecky et al. 1987], demonstrates a unique
developmental adaptation of immunoglobulins for effective function and optimal
survival in the relatively harsh environment of mucosal secretions. Other
polymeric immunoglobulins, notably IgM, which is frequently associated with the
secretory component (SC), may function similarly, especially in patients with IgA
deficiency [Ogra, 1979]. The role of the monomeric immunoglobulins (IgD, IgE,

Figure 1.3 Model of cellular pathways for stimulating lacrimal gland protein secretion. PIP
2
,
phosphatidylinositol bisphosphate; IP
3
, inositol trisphosphate; CaM, calmodulin; DAG,
diacylglycerol; AC, adenylate cyclase. See text for details [Dartt, 1989].

and IgG) that exist in external secretions in variable amounts, appear to be
secondary in mucosal immunity [Tomasi, 1976]. Compared to IgG, IgM, and IgE,
antibodies of the IgA class are much less proinflammatory and can even be
considered anti-inflammatory [Kilian et al., 1994].
Serum IgA is monomeric and is produced by monomeric IgA-secreting
plasma cells in bone marrow [Benner et al., 1981], but secretory IgA is the major
immunoglobulin of tears [Little et al., 1969] and is a dimer derived from local
synthesis by plasma cells [Allansmith et al., 1980]. Only polymeric IgA (usually
dimeric) is transported across epithelial cells by the pIgR [Mestecky et al., 1991].
It was found that antibodies in tears and saliva were entirely of the IgA isotype
without production of serum antibodies [Franklin, 1989].
9
10
The major class of immunoglobulin found in a wide variety of mucosal
secretions, such as gastrointestinal and respiratory secretions, milk, saliva, tears
and bile is dIgA (MW~320 kDa) which is composed of two monomeric IgA
subunits, joined by a small polypeptide J chain [Ahnen et al., 1985; Bienenstock,
1984; Brandtzaeg, 1981]. dIgA is produced by activated B lymphocytes (plasma
cells) that reside in the mucosal lamina propria [Bienenstock, 1984]. After
secretion, dIgA is taken up by an overlying epithelial cell, transported across the
cell, and released into external secretions, where the dIgA forms the first specific
immunologic defense against infection [Brandtzaeg, 1981].

F. Polymeric immunoglobulin receptor (pIgR) and secretory component
(SC)
Macromolecules can be transported across epithelial cells with tight
junctions in a process termed transcytosis [Mostov et al., 1985]. Transcytosis can
occur in either direction, from the apical to basolateral cell surface, or from the
basolateral to apical cell surface. One of the best-studied examples of transcytosis
is the basal-to-apical transport of dIgA across various mucosa [Childers et al.,
1989].
The polymeric immunoglobulin (Ig) receptor (pIgR) is synthesized by
epithelial cells, including those lining the salivary, lacrimal, respiratory,
gastrointestinal, hepatic, mammary and urogenital tracts [Mestecky et al., 1987;
Underdown et al., 1986]. The pIgR is an integral membrane protein that has an
extracellular ligand-binding domain, a single membrane-spanning segment and a
cytoplasmic COOH-terminal domain of 103 amino acids (figure 1.4). The
extracellular ligand-binding portion, known as secretory component (SC),
contains five homologous repeating domains of 100-110 residues each [Mostov et
al., 1984]. The pIgR is responsible for the basal-to-apical transcytosis of dIgA
and pentameric IgM. Therefore, SC, a soluble secretory protein, is a proteolytic
fragment of the pIgR [Mostov et al., 1980]. Brandtzaeg and Pryda provided
direct evidence for an integrated function of J chain and pIgR in epithelial
transport of immunoglobulins, by demonstrating that only polymeric IgA and IgM
containing J chain could bind to the surface of human intestinal epithelial cells
expressing pIgR [Brandtzaeg et al., 1984].

Figure 1.4 Domain structure of pIgR. The pIgR is a membrane protein that has a large
extracellular region arranged in five domains (that are homologous to the variable-like
domains of the Ig superfamily), a transmembrane region and a 103-amino-acid cytoplasmic
region [Rojas et al., 2002].

11
12
The pathway of pIgR-mediated transport of dIgA across polarized
epithelial cells has now been characterized thoroughly (figure 1.5). After
synthesis in the endoplasmic reticulum and exit from the Golgi, pIgR is delivered
directly from the trans-Golgi network to the basolateral surface of epithelial cells.
At that surface, pIgR can bind its ligands (mainly dIgA but also pentameric IgM if
present, although transport of the latter is less efficient) [Mostov et al., 1999].
With or without bound dIgA, the pIgR-dIgA complex or pIgR alone is
endocytosed and delivered to basolateral early endosomes (BEE), then transported
through a common endosomal (CE) compartment and sorted into apical recycling
endosomes (ARE) for delivery to the apical membrane. This transport is
unidirectional, occurring only in the basolateral to apical direction and with a
half-time of ~30 min for pIgR-dIgA (should be longer for pIgR alone) [Mostov et
al., 1986]. At the apical surface, pIgR is proteolytically cleaved and the
extracellular binding domain of the receptor in free form or bound to dIgA is
released into the mucosal secretions. This cleaved extracellular domain of the
receptor is called secretory component (SC). Secreted dIgA in association with
the SC is known as secretory IgA (sIgA). It has the additional function of
stabilizing the dIgA against denaturation or proteolysis in the harsh external
environment [Brandtzaeg, 1977; Underdown et al., 1974]. Comparative serologic
studies have suggested that sIgA possesses a significantly greater agglutinating
capacity than monomeric IgA and IgG [Tomasi, 1976]. A fraction of the pIgR at
the apical surface may be re-internalized into apical early endosome (AEE), then
13
delivered back to the apical surface through the ARE [Kaetzel, 2005].
Interestingly, unoccupied pIgR is also efficiently transcytosed, leading to the
release of free SC in the lumenal secretions [Goldman, 1993; Luton et al., 1999].
Given its intracellular pathway (synthesis on rough endoplasmic reticulum,
processing of oligosaccharide chains in the Golgi apparatus, delivery to the
basolateral membrane from the trans-Golgi network, endocytosis from the
basolateral membrane, and transcytosis to the apical membrane), the pIgR could
serve as a model protein for identification and characterization of signals involved
in any of these steps [Okamoto et al., 1994].

G. Regulation and signaling pathway in pIgR transcytosis
Transcytosis of the pIgR is regulated at multiple levels and provides an
excellent model to study the regulation of membrane traffic. At least three
mechanisms stimulate transcytosis in pIgR-transfected MDCK cells, a cell culture
model to sudy basal-to-apical transcytosis.
First, Ser-664, which is located in the 17-residue basolateral sorting signal,
is phosphorylated [Casanova et al., 1990]. Mutation of Ser-664 to a
nonphosphorylatable alanine decreases transcytosis. Mutation to an aspartic acid,
whose negative charge may mimic a phosphate, increases transcytosis [Casanova
et al., 1990].

Figure 1.5 Pathway of the polymeric immunoglobulin receptor (pIgR) through a polarized
epithelial cell. A simplified epithelial cell is illustrated, with the apical surface at the top and
the basolateral surface at the bottom. Newly synthesized pIgR is targeted to the basolateral
surface, where ligand binding may occur. Following receptor-mediated endocytosis, ligand-
bound or unoccupied pIgR is transported through a series of intracellular vesicles, in which
neutralization of pathogens and antigens can take place. At the apical surface, pIgR is
proteolytically cleaved to secretory component (SC). At the mucosal surface and in external
secretions, free SC and secretory IgA (sIgA) contribute to immune defense. pIgA, polymeric
IgA; BEE, basolateral early endosome; CE, common endosome; ARE, apical recycling
endosome; AEE, apical early endosome [Kaetzel, 2005].

The second major control mechanism for transcytosis is binding of the
ligand dIgA [Song, Bomsel et al., 1994]. Although a significant rate of
transcytosis occurs when the pIgR is not bound to its ligand, binding of dIgA
augments this rate of transcytosis. This stimulation does not depend on
14
15
phosphorylation of Ser-664. However, maximal transcytosis of pIgR is achieved
only when dIgA is bound and phosphorylation of Ser-664 occurs. The finding
that dIgA binding to the pIgR stimulates transcytosis suggests that the pIgR is
capable of transducing a signal across the plasma membrane to the cytoplasmic
sorting machinery (figure 1.6). Binding of dIgA to the pIgR activates the non-
receptor tyrosine kinase p62yes [Luton, Verges et al., 1999] and very rapidly
causes the tyrosine-phosphorylation of several proteins, including a
phosphatidylinositol-specific phospholipase C γ
1
(PLC γ
1
). This enzyme causes
hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP
2
) to diacylglycerol
(DAG) and inositol 1,4,5-trisphosphate (IP
3
). The DAG in turn leads to activation
of protein kinase C (PKC) which can also be activated by phorbol esters (PMA),
to stimulate transcytosis. Thus, activation of PKC by dIgA binding to pIgR can
stimulate transcytosis [Cardone et al., 1994]. The production of IP
3
probably
causes the release of Ca
2+
from intracellular stores and an increase in intracellular
Ca
2+
([Ca
2+
]
i
). Artificially increasing [Ca
2+
]
i
with the drug thapsigargin rapidly
stimulates transcytosis, so it is likely that an increase in [Ca
2+
]
i
caused by dIgA
binding to pIgR also stimulates transcytosis. The increased [Ca
2+
]
i
has many
possible targets, including calmodulin (CaM). The CaM binds to and sequesters
the basolateral targeting signal of the pIgR [Apodaca, Enrich et al., 1994]. This
may promote transcytosis and/or act as a negative feedback inhibition of signaling.
The stimulation of transcytosis by dIgA binding, activation of PKC, or increase in
[Ca
2+
]
i
all seem to activate transcytosis [Song, Apodaca et al., 1994].
A third mechanism for regulating transcytosis comprises extracellular
signals other than dIgA. For example, binding of bradykinin or cholinergic drugs
to their receptors stimulates transcytosis, possibly by activation of PKC and
increasing [Ca
2+
]
i
[Mostov et al., 1995]. Activation of the heterotrimeric G
s

protein also stimulates transcytosis [Bomsel et al., 1993]. Part of this stimulation
is apparently due to activation of adenylate cyclase (AC) by G
s
.

Figure 1.6 A model for how IgA binding to the pIgR activates intracellular signaling
pathways and stimulates transcytosis. When IgA binds to the pIgR, the pIgR interacts with
a tyrosine kinase (TK). The TK phosphorylates PLC, which then hydrolyzes PIP
2
, yielding
DAG and IP
3
. The DAG activates PKC, which stimulates transcytosis. The IP
3
causes an
increase in [Ca
2+
]
i
, which activates CaM. The CaM binds to and sequesters the basolateral
targeting signal of the pIgR. This may promote transcytosis and/or act as a negative
feedback inhibition of signaling [Mostov et al., 1995].

H. Ocular mucosal immunity and immune functions of dIgA, sIgA and
free SC
It has been estimated that the mucosal immune system spans a surface area
of greater than 400 m
2
, or more than 250 times larger than the skin surface in
humans [McGhee et al., 1994]. A striking feature of the mucosal immune system
16
17
is the presence of large quantities of secretory IgA, a unique characteristic of the
mucosal surfaces [Ogra et al., 1980]. Ocular tissues are involved in sophisticated
immune responses in pathophysiological conditions. The conjunctiva and the
lacrimal gland participate in the common mucosal immune system [McClellan,
1997].
dIgA, the predominant mucosal antibody and the major mediator of virus
neutralization, functions in host defense at three levels in relation to the mucosal
epithelium. The first is immune exclusion. dIgA antibodies secreted into the
lumen can prevent antigens and microbes from adhering to and penetrating the
epithelium [Enriquez et al., 1998; Fubara et al., 1973; Outlaw et al., 1990] (figure
1.7 a). The second is intracellular neutralization, most likely occurring within the
epithelial cells [Burns et al., 1996], where antiviral dIgA antibodies in transit
through epithelial cells can act intracellularly to interfere with virus replication,
assembly, or release, presumably by binding to newly synthesized viral proteins
[Alfsen et al., 2001; Fujioka et al., 1998; Hocini et al., 1999; Mazanec et al., 1992;
Vaerman et al., 1985; Williams et al., 1972] (figure 1.7 b). Such intra-epithelial
cell neutralization has been strongly correlated with the presence of specific dIgA
antibody in mucosal secretions, and has been demonstrated by IgA monoclonal
antibodies against Sendai virus, influenza virus, rotavirus, and human
immunodeficiency virus (HIV) [Bomsel et al., 1998; Burns et al., 1996; Fujioka
et al., 1998; Mazanec et al., 1995]. The third is virus excretion. dIgA antibodies
in the lamina propria can bind antigens and excrete them through the epithelium
18
into the lumen [Kaetzel et al., 1991] (figure 1.7 c). Thus, dIgA antibodies appear
to be capable of providing an excretory immune system that can rid the body of
foreign substances and minimize exposure to a potentially harmful burden of local
or systemic immune complexes.
Surface plasmon resonance-based binding experiments indicated that
purified sIgA and dIgA had identical binding affinities for immobilized antigen
[Lullau et al., 1996]. Similarly, sIgA was found to be no more effective than
dIgA in its ability to neutralize influenza virus in vitro [Renegar et al., 1998].
However, the presence of SC has been shown to enhance both the stability and
effector functions of dIgA. The presence of bound SC has been shown to inhibit
degradation of dIgA by neutrophil elastase, thus enhancing the effectiveness of
humoral immunity in the respiratrory tract [Pilette et al., 2003]. In addition to the
functions of sIgA in antigen-specific immunity, free SC and sIgA have been
shown to contribute in novel ways to the regulations of innate, ‘non-specific’
responses to pathogens [Phalipon et al., 2003].

I. Rab proteins
Rab proteins form the largest branch of the ras-like family of small GTPases. The
human genome is predicted to contain more than 60 rab genes [Zerial M, et al.,
2001], and rab proteins seem to be involved in nearly every aspect of membrane
traffic: vesicle formation, motility, tethering, docking, and fusion events. Rab
proteins act by virtue of their guanine nucleotide-specific interaction with effector

Figure 1.7 Functions of dIgA/sIgA and pIgR. (a) Formation of sIgA and luminal pathogen
attachment prevention. dIgA is secreted by the plasma cells, where it binds to the pIgR at
the basolateral membrane of secretory epithelial cells. dIgA is transported through a series
of endosomal compartments (step 1) en route to the apical plasma membrane domain (step
2), where a proteinase cleaves the large extracellular domain of the receptor, thereby
releasing it, bound to dIgA, into secretions (step 3). dIgA, in association with the cleaved
receptor fragment (also known as secretory component, SC) form sIgA. In secretions, sIgA
interacts with antigens/pathogens, preventing them from adhering to and penetrating the
mucosal epithelium. (b) Intracellular virus neutralization. pIgR-dIgA complexes are
delivered to endocytic compartments (step 1) where they encounter infecting viruses (step 2)
or newly synthesized viral membrane proteins. The interaction of pIgR-dIgA with the virus
prevents virus assembly/disassembly and the exit from the cell, possibly by targeting pIgR-
dIgA-virus complexes to lysosomes (step 3) where they are degraded. (c) Antigen secretion.
Pathogenic antigens that penetrate the epithelium and enter the laminal propria are bound
to pIgR-dIgA, internalized (step 1), transported across the cell (step 2) and released at the
apical pole of the cell (step 3) where they are cleared [Rojas et al., 2002].

proteins which include a variety of proteins that appear unrelated to each other,
ranging from large protein complexes (e.g., TRAPP, exocyst) and cytoskeletal
elements (e.g., kinesin, myosin V) to lipid kinases (e.g., phosphatidylinositol 3-
kinase). The nucleotide state, i.e., the balance between GTP binding and
hydrolysis, and thus the activity of rab proteins, is regulated by GTPase-activating
19
20
factors (GAPs) and guanine nucleotide exchange factors (GEFs). Most rab
proteins studied so far appear to control a specific step in membrane traffic, which
is reflected by their specific subcellular localizations. While many rab proteins
appear to be ubiquitously expressed, the expression of others appears restricted to
specific (epithelial) cell types and/or is developmentally regulated.
Several rab proteins have been reported to be exclusively expressed in or
to perform specific functions in epithelial cells. These include rab3B, rab3D, rab4,
rab8, rab11a, rab13, rab17, rab18, and rab25. The rab8 and rab13 are likely to
play a role in polarized sorting in the biosynthetic pathway. The others all
localize to the transcytotic pathway, in particular to the common and the apical
recycling endosomes, with variable degrees of overlap. Several effector proteins
have been identified, some of which may bind to more than one of these rab
proteins or may interact with each other. This underscores the complexity of the
endocytic system in epithelial cells and the importance of rab proteins in
organizing and regulating membrane traffic events.

J. Rab3
The rab3 family consists of four members: rab3A, rab3B, rab3C, and
rab3D. Rab3 family members are typically involved in the process of regulated
secretion and, accordingly, are expressed predominantly in cells with specialized
secretory functions, such as neurons and endocrine cells [Geppert et al., 1998;
Lledo et al., 1994].
21
Interestingly, rab3B and 3D have been reported to also perform epithelial-
specific functions [Larkin et al., 2000; Smythe, 2002; Weber et al., 1994; van
IJzendoorn et al., 2002]. Rab3D appears to be associated with the transcytotic
pathway, as followed by the polymeric immunoglobulin receptor [Larkin et al.,
2000]. Thus, rab3D was found in a hepatocyte membrane fraction enriched in
transcytotic vesicles, and immunoisolation of rab3D-containing vesicles was
found to be enriched in transcytosed pIgR.
Another rab3 family member, rab3B, is preferentially expressed in
cultured epithelial cells and native epithelial tissue, including the liver, intestine,
and kidney [Weber et al., 1994]. Data from studies in which rab3B was
overexpressed in PC12 cells suggested that this GTPase may influence cell
signaling pathways that, in turn, modulate cytoskeleton arrangement and
junctional protein targeting [Sunshine et al., 2000]. Detailed information about a
role of rab3B in apical trafficking and transcytosis was described in MDCK cells
[van IJzendoorn et al., 2002]. In these cells, rab3B was found associated with
vesicles that were concentrated in the apical cytoplasm and near the centrosomes.
These vesicles contain transcytosing pIgR. GTP-bound rab3B interacts directly
with cytoplasmic domain of pIgR, suggesting that rab3B may modulate the
polarized transport of the receptor.

22
K. Rab11
Rab11a was initially characterized as a 24-kDa GTP-binding protein from
bovine brain membranes [Kikuchi et al., 1988], and was subsequently cloned
from a number of sources, including MDCK [Chavrier et al., 1990], mouse
kidney [Chavrier et al., 1992], and rabbit gastric parietal cells [Goldenring et al.,
1994]. There are currently three identified members of the mammalian rab11
family, rab11a, rab11b, and rab25 [Bhartur et al., 2000], which mainly differ
within a 20-amino acid stretch near the carboxyl terminus. While rab11a and
rab11b are ubiquitously expressed, rab25 is expressed only in certain epithelial
cells [Goldenring et al., 1993], indicating that individual members of the family
may be involved in different pathways. Rab11a was the first of the family cloned
and so most of the work done on this family was focused on rab11a. Studies in
nonpolarized cells have established that rab11a is a marker of the plasma
membrane recycling system involved in trafficking of the transferrin receptor
[Green et al., 1997; Ren et al., 1998; Ullrich et al., 1996]. A rab11 has also been
associated with the trans-Golgi network and vesicle movement out of the Golgi
[Chen et al., 2001; Urbe et al., 1993]. In polarized MDCK cells both rab11a and
rab25 are associated with the apical recycling endosome and the trafficking of
polymeric IgA, but not transferrin [Casanove et al., 1999; Leung et al., 2000;
Wang et al., 2000]. Rab11b does not colocalize with either transferrin receptor or
the polymeric IgA receptor [Lapierre et al., 2003].
23
A specific role for rab11a in epithelial cells is indicated by the observation
that this small GTPase specifically localized to apical vesicle populations in
discrete epithelial cell populations [Goldenring et al., 1996]. In gastric parietal
cells, rab11a is present in subapical tubulovesicles that are involved in transport to
the apical domain [Calhoun et al., 1996]. In other polarized epithelia, the rab11a-
specific compartment was identified as the apical recycling endosome and is
accessible to basolaterally endocytosed dIgA-pIgR [Casanova et al., 1999; Wang
et al., 2000] and apically internalized membrane-associated markers [Rahner et
al., 2000], but not for the basolaterally internalized transferrin receptor [Brown et
al., 2000; Wang et al., 2000]. The latter is in contrast to the localization and
function of rab11a in nonpolarized fibroblasts, where rab11a is involved in the
recycling of transferrin receptors from recycling endosomes. These data suggest a
functional role for rab11a in regulating transport between the apical recycling
endosome and the apical surface and underscore the segregation of rab11a from
the transferrin receptor recycling pathway in polarized epithelial cells.
Although the association of rab11a with the apical recycling system in
polarized epithelial cells is well established, still little is known about its function.
However, several rab11a effector proteins have been identified: rab11-binding
protein (rab11BP) or rabphilin 11 [Mammoto et al., 1999; Zeng et al., 1999],
myosin Vb [Lapierre et al., 2001], and a family of four rab11a-binding proteins
which consists of rab11-family-interacting protein 1 (rab11-FIP1), rab11-FIP2,
rab11-FIP3, and rab11-interacting protein (Rip11) [Hales et al., 2001]. Some of
24
the rab11a effectors can either self-interact or interact with each other [Wallace et
al., 2002]. Rab11-FIP1, rab11-FIP2, rab11-FIP3 and Rip11 all interact with GTP-
rab11a, as well as with rab11b and rab25. Moreover, like myosin Vb, rab11-FIP1,
rab11-FIP2, and Rip11 colocalize with rab11a in the ARE in kidney epithelial
cells and in the subapical tubulovesicular compartments in parietal cells [Hales et
al., 2001; Prekeris et al., 2000]. Although the role of these rab effector proteins in
epithelial cells awaits further investigation, it is suggested that different rab11a
effector proteins may control different aspects of endosome functioning.

25
CHAPTER II - IDENTIFICATION OF THE POLYMERIC
IMMUNOGLOBULIN RECEPTOR IN RABBIT CONJUNCTIVAL
EPITHELIAL CELLS
A. Abstract
Purpose. The polymeric immunoglobulin receptor (pIgR) is expressed in
a wide variety of secretory epithelial cells, where it regulates secretory component
(SC) and secretory IgA (sIgA) secretion through the transcytosis of polymeric
immunoglobulins. The pIgR therefore plays a major role in mucosal immunity.
To begin to characterize its role in ocular surface immunity, we sought to identify
and localize pIgR in conjunctiva. Methods. Western blot, immunoprecipitation
and immunocytochemistry were performed on freshly isolated and primary
cultured pigmented rabbit conjunctival epithelial cells (RCEC). Results.
Western blot showed two distinct bands at ~120 kDa and ~95 kDa for pIgR in
both freshly isolated and cultured RCEC, but not in corneal epithelium. The sizes
of these bands are consistent with the known molecular forms of pIgR. Two
distinct proteins at ~80 kDa and ~55 kDa, consistent with the sizes of SC, were
immunoprecipitated from the apical medium of RCEC cultured on Transwell
filters, providing evidence for pIgR transcytosis to the apical membrane followed
by proteolytic cleavage of the extracellular domain to SC. Moreover, SC
accumulation in the apical medium increased with time and provided further
evidence of pIgR transcytosis and SC processing. Immunocytochemistry
indicated that pIgR predominantly exists in the superficial layer of the
26
multilayered RCEC but not all cells express pIgR. Conclusions. The pIgR and
SC were molecularly identified in the rabbit conjunctival epithelium. These are
the first data to show SC secretion from a multilayer epithelium. Therefore, the
potential of utilizing endogenously expressed pIgR in conjunctiva for prevention
of ocular surface-related diseases can be explored, and a pIgR-based model of
ocular mucosal immunity can be developed.

B. Introduction
Conjunctiva is a thin, mucus-secreting, vascularized, and multilayered
tissue that covers most of the inner surface of the eyelids and is part of the
anterior sclera where the cornea begins [Srinivasan et al., 1982]. Conjunctiva
contributes to the regulation of electrolyte and fluid balance in the
microenvironment of its mucosal surface [Shiue et al., 2000]. It also serves as a
passive physical protective barrier, and participates in the maintenance of tear
film stability due to the mucus secreted by the resident goblet cells [Srinivasan et
al., 1982]. The conjunctiva can be divided into three layers: an outer epithelium,
the substantia propria, and the submucosa [Bron et al., 1985]. The surface cells of
the conjunctiva are connected by tight junctions which render the epithelium a
relatively impermeable barrier [Hogan et al., 1971]. In substantia propria, the
abundant blood supply of the conjunctiva delivers protective substances such as
antibodies, complement, and white blood cells to protect the eye from infections
and to remove dead or damaged tissue [Dawson 1976, 1984]. The submucosa
27
provides a loose attachment to the underlying sclera. The conjunctival and other
mucous membranes, such as intestinal and bronchiolar, which constitute the
mucosal immune system, are the most commonly invaded by microorganisms
[Chandler et al., 1980; Wolf et al., 1984]. Thereby, the conjunctiva plays a vital
role in both vision and ocular immunity.
The immunoglobulin A (IgA) and secretory IgA (sIgA) are predominant
immunoglobulin isotypes in humans and represent more than 60% of all the
antibody isotypes [Conley et al., 1987; Mestecky et al. 1987]. It was found that
antibodies in tears and saliva were entirely of the IgA (including sIgA) isotype
[Franklin, 1989]. IgA in tears is a dimer, produced by local B lymphocytes
(plasma cells) in mucosal lamina propria [Allansmith et al., 1980]. After
synthesis by plasma cells, dimeric IgA (dIgA) is taken up by an overlying
epithelial cell, transported across the cell, and released into external secretions,
where the dIgA forms the first specific immunologic defense against infection
[Brandtzaeg, 1981]. The dIgA is transported across epithelial cells by the
polymeric immunoglobulin receptor (pIgR) [Mestecky et al., 1991].
The pIgR is expressed in a wide variety of secretory epithelial cells,
including those lining the salivary, lacrimal, respiratory, gastrointestinal, hepatic,
mammary and urogenital tracts [Mestecky et al., 1987; Underdown et al., 1986].
The pIgR is an integral membrane protein that has an extracellular ligand-binding
domain, a single transmembrane domain and a cytoplasmic domain of 103-amino-
acid [Rojas et al., 2002]. The extracellular ligand-binding domain, called
28
secretory component (SC), contains five homologous repeating regions, and is
responsible for the basal-to-apical transcytosis of dIgA and pentameric IgM
[Mostov et al., 1984].
The pIgR can be transported across epithelial cells by vesicular transport
in a process termed transcytosis as shown in figure 2.1. After synthesis in the
endoplasmic reticulum (ER) and exit from the Golgi, pIgR is delivered directly
from the trans-Golgi network (TGN) to the basolateral surface where it can bind
its ligand, dIgA, that forms the first specific immunologic defense against
pathogens infection through mucosal surfaces [Brandtzaeg, 1981]. With or
without bound dIgA, the pIgR-dIgA complex or pIgR alone is then endocytosed
and transported through a series of endosomal compartments (e.g. basolateral
endosome (BE) and apical endosome (AE)) across the cell to the apical surface by
the process of transcytosis. This transport is unidirectional, occurring only in the
basolateral to apical direction. At the apical surface, the extracellular ligand-
binding domain of the pIgR, known as SC, is cleaved and released in free form or
in association with dIgA into the mucosal secretions. Secreted dIgA bound with
the SC is known as sIgA which is the major effector molecule in mucosal defense
[Mestecky et al., 1987]. The sIgA has the additional function of stabilizing the
dIgA against denaturation or proteolysis in the harsh external environment
[Brandtzaeg, 1977; Underdown et al., 1974].
The wide expression of pIgR in various secretory epithelial cells, where it
regulates SC and sIgA secretion through the transcytosis of pIgR, determines its
major role in mucosal immunity. Although SC has been detected in histological
sections of human conjunctiva [Knop et al., 2000], the existence and role of pIgR
in conjunctival epithelium to date has not been explored. In this study, we sought
to identify and localize pIgR in conjunctiva and to show that SC is secreted by
this epithelium, to determine its contribution to the immunological defense of the
conjunctiva and the ocular surface.

pIgR
dIgA
SC
sIgA
TGN
AE
BE
Apical
Basolateral
ER

Figure 2.1 pIgR transcytosis. After synthesis in the endoplasmic reticulum (ER) and exit
from the Golgi, pIgR is delivered directly from the trans-Golgi network (TGN) to the
basolateral surface where it binds its ligand, dimeric IgA (dIgA) that represents the primary
defense against pathogens infection through mucosal surfaces. The pIgR-dIgA complex is
then endocytosed and transported through a series of endosomal compartments (e.g.
basolateral endosome (BE) and apical endosome (AE)) across the cell to the apical surface by
the process of transcytosis. At the apical surface, pIgR is proteolytically cleaved and the
extracellular binding domain of the receptor that is bound to dIgA is released into the
mucosal secretions. This cleaved extracellular domain of the receptor is known as the
secretory component (SC). Secreted dIgA in association with the SC is known as secretory
IgA (sIgA) which is critical for the immune response at mucosal surface.
29
30
C. Materials and methods
1. Animals and reagents
Male Dutch-belted pigmented rabbits, weighing 2.0 - 2.5 kg, were
purchased from Irish Farms (Los Angeles, CA). The rabbits were handled in
accordance with the Guiding Principles in the Care and Use of Animals
(Department of Health, Education and Welfare Publication, National Institutes of
Health 80-23) and the Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Visual Research
(http://www.arvo.org/eweb/dynamicpage.aspx?site=arvo2&webcode=AnimalsRe
search).
Sheep anti-rabbit SC antibody was generated against purified SC from
rabbit bile (Pel-Freeze, Rogers, AK) by preparative gel electrophoresis and used
to produce sheep anti-rabbit SC polyclonal antiserum (Capralogics, Hardwick,
MA). The antiserum was of sufficient titer to use diluted for Western blotting.
Donkey anti-sheep secondary antibody conjugated to HRP, FITC or Cy3 was
from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA).
ProLong antifade mounting kit, rhodamine phalloidin and BODIPY
phallacidin were from Molecular Probes (Eugene, OR). Protein G-Sepharose was
purchased from Pharmacia LKB Biotechnologies Inc. (Alameda, CA).
Protease (type XIV), deoxyribonuclease (DNase I), and hydrocortisone
were obtained from Sigma Chemical Co (St. Louis, MO). ITS
+
(insulin 6.5 µg/ml,
transferrin 6.5 µg/ml, selenium acid 6.5 ng/ml, BSA 1.25 mg/ml, and linoleic acid
31
5.35 mg/ml), bovine pituitary extract (BPE), epidermal growth factor (EGF), rat
tail collagen type I and cell strainer (40 μm pore size) were purchased from BD
Biosciences (Bedford, MA). Dulbecco’s Modified Eagle Medium/Nutrient
Mixture F12 (DMEM-F12) was from Mediatech Inc. (Herndon, VA). Transwell
filters (12 mm diameter, 0.4 μm pore size) were from Costar (Cambridge MA).
Other cell culture reagents and supplies were from Invitrogen Inc. (Carlsbad, CA).

2. Primary cultures of rabbit conjunctiva epithelial cells
Rabbit conjunctival epithelial cells (RCEC) were harvested using a
protocol modified from that developed by Yang et al. [Yang et al., 2000]. Briefly,
Male Dutch-belted pigmented rabbits, weighing 2.0 to 2.5 kg, were euthanized
with an injection of 85 mg/kg sodium pentobarbital solution into a marginal ear
vein. Following excision, the conjunctiva was washed in ice-cold Ca
2+
/Mg
2+
- free
Hanks’ balanced salt solution (HBSS) and treated with 0.2% protease (type XIV)
for 60 min at 37 °C in 95% air and 5% CO
2
to dissociate the cells. Epithelial cells
were then scraped off using a sterile scalpel blade. The isolated cells were treated
with S-Minimum Essential Medium (S-MEM) containing 10% fetal bovine serum
(FBS) to stop protease action and 1 mg/ml deoxyribonuclease (DNase I) to
hydrolyze extracellular DNA. The cells were washed twice with S-MEM
containing 10% FBS, centrifuged at 100 × g for 10 min at room temperature (RT)
and filtered through a 40µm cell strainer. The final cell pellet was resuspended in
DMEM-F12 medium supplemented with 100 U/ml penicillin-streptomycin, 0.5%
32
gentamicin, 1% ITS
+
(insulin 6.5 µg/ml, transferrin 6.5 µg/ml, selenium acid 6.5
ng/ml, BSA 1.25 mg/ml, and linoleic acid 5.35 mg/ml), 30 µg/ml bovine pituitary
extract (BPE), 1 µM hydrocortisone, and 1 ng/ml epidermal growth factor (EGF).
These cells were seeded at a density of 1.2×10
6
cells/cm
2
on Transwell pre-coated
with 2% rat tail collagen type I (day 0, culture inserts), and cultured in 5% CO
2

and 95% air at 37 °C. On day 4 and thereafter, the cells were switched to an air-
interfaced condition, in which medium was only added to basolateral side to
mimic the in vivo situation. The cell layers were used until cells grew to
confluence with transepithelial electrical resistances (TEER) > 1.0 k Ω × cm
2

within 7-10 days of culture on Transwell filters. Bioelectrical parameters in
cultured RCEC on Transwell filters were monitored daily with EVOM (World
Precision Instruments Inc., Sarasota, FL).

3. Western blot
The conjunctiva and cornea were excised from male Dutch-belted
pigmented rabbits and washed in ice-cold HBSS. The conjunctival and corneal
epithelial cells were carefully scraped off. Microsomal pellets from
tubulovesicular membranes of rabbit gastric mucosa, which are enriched in pIgR,
were prepared as described by Wolosin et al. [Wolosin et al., 1981], and used as
positive blotting control. The freshly isolated rabbit corneal epithelial cells (Cr),
freshly isolated RCEC (Cj), RCEC grown to confluence with TEER > 1.0 k Ω ×
cm
2
within 7-10 days of culture on Transwell filters (cCj), and positive control
33
(Control) were lysed by 0.5% SDS lysis buffer (0.5% SDS, 100 mM NaCl, 50
mM Triethanolamine-HCl, pH 8.1, 5 mM EDTA, pH 8.0, 0.2% NaN
3
). The
protein concentration of each was measured by BCA protein assay (BioRad,
Hercules, CA), using bovine serum albumin (BSA) as a standard. 40 μg proteins
were electrophoresed on 10% SDS-polyacrylamide gel (SDS-PAGE), and
subsequently electrotransferred to a nitrocellulose membrane (Amersham,
Downers Grove, IL). The membrane was incubated for 1 hour at RT in
phosphate-buffered saline Tween-20 (PBST) containing 0.5% nonfat dry milk to
prevent nonspecific binding of antibodies. Incubation with primary sheep anti-
rabbit SC antibodies (1:2000 dilution) was at 4 °C overnight in PBST.
Immunoreacivity was visualized with HRP-conjugated donkey anti-sheep
immunoglobulins (1:100,000 dilution). Immunoblot procedure based on the
enhanced chemiluminescence (ECL) method was performed by exposing the
protein side of the nitrocellulose membrane to the ECL detection reagent
(Amersham, Downers Grove, IL) for 1 min, followed by immediate exposure to
X-ray film.

4. Immunoprecipitation
The confluent RCEC grown on the Transwell filters were washed twice
and replenished with fresh supplemented DMEM-F12 medium at 37 °C. At
different time intervals (0 hour (0 h), 2 hours (2 h), 6 hours (6 h), and 20 hours
(20 h)), apical medium was harvested. The collected medium was subjected to
34
immunoprecipitation with sheep anti-rabbit SC antibodies covalently coupled to
Protein G-Sepharose at 4 °C overnight. The beads were collected by
centrifugation, and after four washes, SDS sample buffer was added. Then
samples were processed for SDS-PAGE and transferred onto nitrocellulose
membrane followed by the analysis on Western blots using primary sheep anti-
rabbit SC antibodies and secondary donkey anti-sheep antibodies.

5. Immunocytochemistry
The confluent RCEC grown on the Transwell filters were fixed by 4%
formaldehyde, and permeabilized by 0.5% Triton X-100, followed by incubating
with 0.066% fish skin gelatin (FSG) to block non-specific binding sites at RT.
Then the cell layers were reacted with primary sheep anti-rabbit SC antibodies
(1:300 dilution), and secondary FITC- or Cy3-conjugated donkey anti-sheep
(1:300 dilution) antibodies on both apical and basolateral sides. Actin was stained
with rhodamine phalloidin or BODIPY phallacidin. After washing, the cell layers
on the permeable support were cut out and mounted on the coverslips.
Immunofluorescence was observed by confocal microscopy (Nikon PCM2000,
Japan) to detect pIgR localization in RCEC. Confocal images were obtained with
a Simple PCI imaging system. Panels were compiled in MetaMorph Software
(Molecular Devices Corporation, Downingtown, PA).

35
D. Results
1. The pIgR is detected in conjunctiva by Western blot
To determine the existence of pIgR in rabbit conjunctiva epithelium,
Western blot was performed to provide molecular evidence for pIgR expression.
Freshly isolated rabbit corneal epithelial cells (Cr), freshly isolated RCEC (Cj),
RCEC grown to confluence with TEER > 1.0 k Ω × cm
2
within 7-10 days of
culture on Transwell filters (cCj), and positive control (Control, from
tubulovesicular membranes from rabbit gastric mucosa) were analyzed on
Western blot using a sheep anti-rabbit SC antibody. This antibody was generated
against the epitope in extracellular domain of the pIgR. Therefore, it can
recognize both full-length pIgR and free SC.
In figure 2.2, Western blot shows two distinct bands at ~120 kDa and ~95
kDa for pIgR in both freshly isolated (Cj) and cultured RCEC (cCj), as well as in
the positive control (Control), but not in corneal epithelium (Cr), suggesting
significant expression of pIgR in conjunctival epithelium, and a lack of expression
in corneal epithelium. The sizes of these bands are consistent with the molecular
weight of known molecular forms of rabbit pIgR. The band positions of pIgR
from conjunctiva are slightly different from those from gastric cells as
demonstrated in Control. This may be due to the pIgR from different organs
migrating differently on the SDS-PAGE.

- pIgR
- pIgR
100 kDa –
Cr Cj cCj Control
150 kDa –
75 kDa –

Figure 2.2 Western blot detection of pIgR in RCEC. Freshly isolated rabbit cornea
epithelial cells (Cr), freshly isolated RCEC (Cj), RCEC grown to confluence with
transepithelial electrical resistances (TEER) > 1.0 k Ω × cm
2
within 7-10 days of culture on
Transwell filters (cCj), and positive control (Control, tubulovesicular membranes from
rabbit gastric mucosa) were lysed by 0.5% SDS lysis buffer. Then samples were processed
for SDS-PAGE and transferred onto nitrocellulose membrane followed by the analysis on
Western blots using a sheep anti-rabbit SC antibody. The positions of immunoreactive
bands for rabbit pIgR in conjunctiva are indicated on the right Results shown are
representative of at least three independent experiments.

2. Secretory component is detected in the apical medium of primary
cultured conjunctival cells
To determine whether the cultured conjunctival cells are functional with
respect to SC secretion, SC secretion into the culture medium was assayed.
Apical and basolateral medium of the confluent RCEC grown on the Transwell
filters was collected at different time intervals (0 hour (0 h), 2 hours (2 h), 6 hours
(6 h), and 20 hours (20 h)), respectively. The collected medium was subjected to
immunoprecipitation with a sheep anti-rabbit SC antibody covalently coupled to
Protein G-Sepharose, followed by analysis on Western blots.
36
37
As shown in figure 2.3 A & B, two distinct proteins at ~80 kDa and ~55
kDa, consistent with the sizes of rabbit SC, were immunoprecipitated
predominantly from the apical medium (figure 2.3 A) of RCEC cultured on
Transwell filters, providing evidence for polarized SC secretion that movement of
pIgR from the basolateral surface to the apical surface is followed by proteolytic
cleavage of the extracellular domain to SC. Moreover, compared to 0 h, SC
accumulation in the apical medium increased with time (figure 2.3 A), but there
was no significant change in the basolateral medium (figure 2.3 B), suggesting
that pIgR transcytosis followed by cleavage to release SC is a constitutive process
in conjunctival epithelium. The weak bands in the basolateral medium (figure 2.3
B) are probably due to the nonspecific binding of the polyclonal primary and/or
secondary antibodies to other proteins existing in supplemented DMEM/F12
medium as observed in the 0 h sample.

3. Localization of pIgR in cultured RCEC by immunocytochemistry
Immunocytochemistry was used to explore the localization of pIgR in
RCEC. The confluent RCEC grown on the filters were processed as described in
Methods. In figure 2.4, panel A is the immunofluorescence image observed with
treatment of primary sheep anti-rabbit SC antibody and secondary FITC-
conjugated donkey anti-sheep antibody (green). F-actin was stained with
rhodamine phalloidin to outline individual cells (red). Panel A showed positive
staining of pIgR (green) in most RCEC, but not all cells express pIgR. Panel B is
38
- SC
- SC
75 kDa –
0 h 2 h 6 h 20 h

Figure 2.3 Immunoprecipitation of SC from apical (A) and basolateral (B) medium of
cultured RCEC on Transwell filters. The confluent RCEC grown on the Transwell filters
were washed twice and replenished with fresh supplemented DMEM-F12 medium at 37 °C.
At different time intervals (0 hour (0 h), 2 hours (2 h), 6 hours (6 h), and 20 hours (20 h)),
apical (A) and basolateral (B) medium was harvested, respectively. The collected medium
was subjected to immunoprecipitation overnight at 4 °C with a sheep anti-rabbit SC
antibody covalently coupled to Protein G-Sepharose. The beads were collected by
centrifugation, and after four washes, SDS sample buffer was added. Then samples were
processed for SDS-PAGE and transferred onto nitrocellulose membrane followed by the
analysis on Western blots using a sheep anti-rabbit SC antibody. The positions of
immunoreactive bands for SC in apical medium (A) are indicated on the right. Results
shown are representative of at least three independent experiments.

is the immuofluorescence Z-section image at the blue line cut of panel A. As
shown in Panel B, several layers of RCEC overlap, and the top layer of RCEC has
pIgR positive staining (green), whereas the bottom layers do not, demonstrating
50 kDa –
A
75 kDa –
50 kDa –
B
that RCEC are multilayered, and only the uppermost cell layer of the conjunctival
epithelium expresses pIgR.

A
B

Figure 2.4 Immunolocalization of pIgR in cultured RCEC on Transwell filters by confocal
laser-scanning microscopy. The confluent RCEC grown on filters were fixed by 4%
paraformaldhyde, and permeabilized by 0.5% Triton X-100, followed by blocking with
0.066% fish skin gelatin. Then cells were treated with appropriate primary and secondary
antibody. After washing with PBS, the cells on filters were cut out and mounted on a
coverslip. Immunofluorescence was observed by confocal microscopy (Nikon PCM2000,
Japan) to detect pIgR localization in RCEC. Panel A is the immunofluorescence image
observed with treatment of primary sheep anti-rabbit SC antibody and secondary FITC-
conjugated donkey anti-sheep antibody (green). F-actin was stained with rhodamine
phalloidin (red). Panel B is the immuofluorescence Z-section image at the blue line cut of
panel A. Results shown are representative of at least three independent experiments.

39

40
4. Distribution of pIgR in multilayered cultured RCEC
The following study was to investigate further the pIgR distribution in
multilayered RCEC. Panels 1-42 in figure 2.5 are the immunofluorescence
images scanned from top (apical) to bottom (basolateral) of cultured RCEC on
Transwell filters with increment of 0.5 µm between each image collected. Cells
were treated with primary sheep anti-rabbit SC antibody and secondary Cy3-
conjugated donkey anti-sheep antibody (red). F-actin was stained with BODIPY
phallacidin (green). The positive staining of pIgR (red) shows up from panel 1 to
panel 28, which is the superficial layer of RCEC. The subjacent layers appear in
panels 29-42. Immunofluorescent staining in figure 2.5 provided additional
evidence that pIgR predominantly exists in the superficial layer of the
multilayered RCEC, but not all cells express pIgR, which can also be observed in
figure 2.4.

E. Discussion
This study provides molecular and immunocytochemical evidence for the
existence and localization of pIgR in RCEC. The rabbit pIgR is heterogeneous.
Rabbits have a minimum of two pIgR translation products, one is high molecular
weight (MW) (~120 kDa), the other is lower MW (~95 kDa) [Kuhn et al., 1983].
A cDNA clone of the smaller pIgR isoform lacks two of the five IgG-like
domains of of the extracellular region. These two domains were on a single exon
which can be spliced in or out [Deitcher et al., 1986]. As shown in figure 2.2, two

Figure 2.5 Layer distribution of pIgR in cultured RCEC on Transwell filters by confocal
laser-scanning microscopy. The confluent RCEC grown on the filters were processed the
same way as described in Figure 2.4 legend. Panels 1-42 are the immunofluorescence images
scanned from top (apical) to bottom (basolateral) of cultured RCEC on Transwell filters
with increment of 0.5 µm between each image collected. Cells were treated with primary
sheep anti-rabbit SC antibody and secondary Cy3-conjugated donkey anti-sheep antibody
(red). F-actin was stained with BODIPY phallacidin (green). Results shown are
representative of at least three independent experiments.

distinct bands at ~120 kDa and ~95 kDa for pIgR were evident in both freshly
isolated and cultured RCEC, but not in the cornea epithelium, suggesting the
existence and significant expression of pIgR in RCEC. The data suggest that the
pIgR in rabbit conjunctival epithelium is also heterogeneous, consistent with the
mentioned findings that the gene can be spliced in two ways: the exon coding for
domains II and III of extracellular region of pIgR can be left in to produce the 120
41
42
kDa translation product, or spliced out to produce the 95kDa product [Deitcher et
al., 1986]. Lack of expression of pIgR in rabbit corneal epithelium suggests that
this tissue is unlikely to contribute to the transcytosis of dIgA or SC in ocular
secretions.
Rabbit SC constitutes a highly heterogeneous population of glycoprotein
molecules that are present in secretions as free or bound forms to polymeric
immunoglobulin [Frutiger et al., 1986]. Two SC families are known, one of high
MW (~80 kDa) composed of five domains related to Ig variable domains, and one
of low MW (~55 kDa) [Kuhn et al., 1983]. The low MW SC polypeptide is
derived from the high MW SC by the internal deletion of the entire second and
third domains [Coyne et al., 1994]. In our experiments, immunoprecipitation
(figure 2.3) revealed two distinct major bands at ~80 kDa and ~55 kDa for SC, in
concert with the known size of rabbit SC, predominantly in apical medium of
cultured RCEC, providing evidence for polarized SC secretion, presumably
resulting from the transcytosis of pIgR from the basolateral surface to the apical
surface, and its cleavage at the apical surface to SC. Moreover, the longer the
time interval, the more the SC accumulated in the apical medium of cultured
RCEC, indicating that pIgR transcytosis followed by SC secretion is a constitutive
process in conjunctival epithelium.
The thickness of the conjunctival epithelium varies from region to region,
being 10-15 layers thick toward the cornea and 5-6 layers thick at the lids in
human [Kompella et al., 2000]. In guinea pig, the epithelium is generally three to
43
six cells thick, whereas the epithelium over a lymphoid follicle is thinner
consisting of two or three cell layers and sometimes only a single layer [Nichols,
1996]. Our air-interfaced culture of RCEC showed stratified cell layers (figure
2.4, 2.5), which is consistent with previous findings that air-interfaced culture
appeared to have more differentiated cell layers than liquid-covered culture (Yang,
unpublished data). The immunofluorescence images (figure 2.4, 2.5) clearly
show that RCEC expresses pIgR, but not all cells express pIgR, and that the
conjunctival epithelium is a multilayered epithelium, with the pIgR predominantly
existing in the superficial layer. We used different colored conjugates to stain
pIgR in figure 2.4 (green) and figure 2.5 (red) to ensure that the staining pattern
was not due to non-specific binding of secondary antibodies.
The typical components of a secretory immune tissue are epithelial cells
producing pIgR, and subjacent tissue containing dIgA-producing plasma cells
[Franklin, 1989]. It has always been considered that the pIgR and SC are
produced by lacrimal gland in ocular system. Our study provides the first
biochemical evidence for the functional expression of pIgR in RCEC. It also
confirms earlier histochemical studies showing the presence of SC in conjunctiva,
predominantly localized to the uppermost layer of cells [Knop et al., 2000].
Analysis of conjunctiva obtained from humans revealed that a lymphoid layer
consisting of T cells and IgA
+
B cells is below the epithelial layer of the
conjunctiva, indicating that dIgA is actively secreted [Meek et al., 2003].
Furthermore, morphological and functional studies have demonstrated a
44
conjunctival-associated lymphoid tissue in rabbits [Franklin et al., 1984].
Therefore, RCEC fulfills the criteria for inclusion in the secretory immune system.
Our identification of pIgR and SC in conjunctival epithelium has
significant ocular immunological and pharmacological significance. pIgR
knockout mice have been shown to lack mucosal immunoglobulins and
accumulate dIgA in circulation at levels 100-fold higher than those in normal
mice [Uren et al., 2003]. In addition, pIgR knockout mice have reduced
protection against influenza A and B viruses [Asahi et al., 2002; Asahi-Ozaki et
al., 2004]. Therefore, the existence of pIgR in conjunctival epithelium ensures
continuous delivery of dIgA to the epithelial surface and external secretions, and
may promote extracellular exclusion, intracellular neutralization and transcellular
excretion of antigens and pathogens. In addition to the functions of sIgA in
antigen-specific immunity, free SC and sIgA exhibits novel functions in the
regulation of innate, non-specific responses to pathogens [Phalipon et al., 2003].
Free SC has been shown to limit infection or reduce morbidity via binding to
bacterial components such as Clostridium difficile toxin A, and fimbriae of
enterotoxigenic Escherichia coli [Dallas et al., 1998; de Oliveira et al., 2001].
Thereby, free SC derived from pIgR in conjunctival epithelium may be seen as a
potential non-specific microbial scavenger to protect ocular surface, including
conjunctiva and cornea, from pathogen infection, which may be directly clinically
relevant. It has been reported that tear sIgA can prevent bacteria adhesion to
contact lens [Lan et al., 1999], and extended wear of contact lenses can reduce
45
sIgA levels in ocular surface fluid and increase susceptibility to ocular infection
[Pearce et al., 1999]. Moreover, tear sIgA concentration in dry eye patients is
significantly decreased [Grus et al., 2001]. By furthering our study of the
expression of pIgR and SC in conjunctival epithelium, new kinds of artificial tear
formulations enriched in SC are also feasible if this protein is implicated in
conjunctival and corneal protection from pathogens.
In conclusion, we have identified pIgR in the superficial layer of
multilayered rabbit conjunctival epithelial cells, indicating an important role of
conjunctiva in ocular mucosal immune protection via pIgR. Therefore, the
lacrimal gland is no more the only ocular tissue having pIgR expression.
Furthermore, based on the identification of the pIgR and SC in the rabbit
conjunctival epithelium, the potential of utilizing endogenously expressed pIgR in
conjunctiva for the prevention of ocular surface-related diseases can be explored,
and a pIgR-based model of ocular mucosal immunity can be developed.
46
CHAPTER III - DIRECT INTERACTION BETWEEN RAB3D AND THE
POLYMERIC IMMUNOGLOBULIN RECEPTOR MAY REGULATE ITS
TRAFFICKING THROUGH REGULATED SECRETORY VESICLES IN
LACRIMAL GLAND ACINAR CELLS
A. Abstract
The lacrimal gland is responsible for tear production, and a major protein
found in tears is secretory component (SC), the proteolytically cleaved fragment
of the extracellular domain of the polymeric immunoglobulin receptor (pIgR), the
receptor responsible for the basal-to-apical transcytosis of polymeric
immunoglobulins across epithelial cells. Immunofluorescence studies indicated
that the small ras-like GTPase rab3D, a regulated secretory vesicle marker, and
the pIgR are colocalized in a population of membrane vesicles subjacent to the
apical plasma membrane of lacrimal gland acinar cells (LGAC) from rabbit.
Therefore, we characterized the biochemical and functional interaction between
rab3D and pIgR to determine the role of rab3D in the regulation of pIgR
trafficking in LGAC. Pull-down of pIgR from resting LGAC lysates with
recombinant (His)
6
-tagged wild-type rab3D (rab3DWT) or mutated rab3D
proteins showed that rab3DWT and a dominant-negative GDP-locked mutant
rab3D, rab3DT36N, bind to pIgR; however, the constitutively-active GTP-locked
rab3D mutant, rab3DQ81L, does not bind to pIgR. Furthermore, when GTP γS or
GTP is included in the pull-down assays with rab3DWT, binding of pIgR is
significantly inhibited. These binding characteristics of pIgR to rab3D were
47
reproduced when cell lysates of MDCK cells-stably transfected with pIgR were
used in pull-down assays. In blot overlay assays, recombinant rab3DWT bound
to immunoprecipitated pIgR suggesting that rab3D and pIgR interact directly. In
pull-down assays with rab3DWT using lysates from LGAC stimulated with 100
μM carbachol (CCH) for up to 1 hr prior to lysis, binding of pIgR to rab3DWT
was decreased relative to untreated controls, suggesting that the pIgR-rab3D
interaction is also modulated by secretagogues. These data demonstrate that
rab3D directly binds to pIgR and it may regulate pIgR trafficking and therefore
SC secretion by LGAC in a stimulation-dependent manner.

B. Introduction
The lacrimal gland is the primary source of the tear film’s aqueous portion
which contains water, electrolytes and proteins, necessary for the health and
maintenance of the ocular surface [Dartt, 2004]. Lacrimal gland protein secretion
is under neural and hormonal control. Proteins in lacrimal gland fluid are secreted
predominantly by the acinar cells [Dartt, 1989]. The lacrimal gland is densely
innervated with parasympathetic (cholinergic) nerves and less densely innervated
with sympathetic (adrenergic) nerves [Ding et al., 2001]. Afferent sensory nerves
in the cornea and conjunctiva stimulate efferent parasympathetic and sympathetic
nerves in the lacrimal gland. Cholinergic agonists, released from parasympathetic
nerves, and norepinephrine, released from sympathetic nerves, are major stimuli
of lacrimal gland secretion [Dartt, 2004].
48
The polymeric immunoglobulin receptor (pIgR) is expressed in a wide
variety of secretory epithelial cells, including those lining the salivary, lacrimal,
respiratory, gastrointestinal, hepatic, mammary and urogenital tracts [Mestecky et
al., 1987; Underdown et al., 1986]. After synthesis in the endoplasmic reticulum
and exit from the Golgi, the pIgR is delivered from the trans-Golgi network to the
basolateral surface where it binds its ligands, dimeric IgA (dIgA) or pentameric
IgM, which represent the primary defense against pathogens at mucosal surfaces
[Childers et al., 1989; Corthesy et al., 1999]. The pIgR delivers dIgA from the
basolateral to the apical surface of epithelial cells by the process of transcytosis.
After dIgA binds to pIgR at the basolateral surface, the pIgR-dIgA complex is
then endocytosed and transported through a series of endosomal compartments
across the cell to the apical surface. At the apical surface, the extracellular
domain of the pIgR that is bound to dIgA is proteolytically cleaved, and secretory
IgA (sIgA) is released into the mucosal secretion. If the receptor did not bind to
dIgA while at the basolateral membrane, this cleaved extracellular domain of the
receptor is known as secretory component (SC).
Rab proteins are members of the ras superfamily of small molecular
weight guanosine triphosphatases (GTPases) that function as key regulators of
vesicular traffic [Gonzalez et al., 1999; Novick et al., 1997; Schimmoller et al.,
1998; Zahraoui et al., 1989]. More than 60 rab proteins have been identified and
each is associated with a specific membrane compartment [Zerial et al., 2001;
Pereira-Leal et al., 2001]. Among the many rab proteins identified to date, 4
49
highly homologous rab3 isoforms (rab3A, rab3B, rab3C, rab3D) are expressed in
cells with regulated secretory pathways [Larkin et al., 2000]. Rab3A and rab3C
are both associated with synaptic vesicles and secretory granules in brain and
neuroendocrine cells [Bean et al., 1997]. Rab3B is abundant in epithelial cells
[Weber E, et al., 1994]. Rab3D is localized to secretory vesicles of various
exocrine secretory cells, such as the acinar cells of the pancreas, parotid and
lacrimal glands, and the chief cells of the stomach [Ohnishi et al., 1996;
Raffaniello et al., 1996; Tang et al., 1996; Valentijn et al., 1996].
Rab proteins act as molecular switches that cycle between active GTP-
and inactive GDP-bound states [Boguski et al., 1993; Bourne et al., 1991]. Small
GTPase rab proteins play an important role in various aspects of membrane traffic,
including cargo selection, vesicle budding, vesicle motility, tethering, docking,
and fusion [van Ijzendoorn et al., 2003]. However, the relationship between the
rab3D GTP- and GDP-bound states and its function involved in pIgR trafficking
in lacrimal gland has not been explored to date.
Tear fluid is relatively rich in sIgA and SC, and the SC concentration in
rat tear fluid is 10 times higher than its concentration in saliva [Gudmundsson et
al., 1985]. We and others have reported the presence of the pIgR and SC in
LGAC from rabbit and rat [Sullivan et al., 1984; Qian et al., 2003]. LGAC may
therefore represent a good, physiologically relevant, organ-based model system to
characterize the mechanism of regulation of pIgR trafficking and SC secretion.
Of particular interest is the regulation of membrane trafficking and protein sorting
50
by the rab family of small ras-like GTPases in LGAC. A number of rab proteins,
including rab1, rab2, rab6, rab3B, rab3D, rab11, rab13, rab17, and rab25, in
hepatocytes have been implicated as possible transcytotic regulators [Jin et al.,
1996; Larkin et al., 2000; Tuma et al., 2001]. Rab3 isotypes have been shown to
have both positive and negative regulatory functions in a number of steps in
regulated secretion [Darchen et al., 2000]. Rab3B has been further identified to
interact directly with the pIgR in transfected Madin-Darby Canine Kidney
(MDCK) cells, and the dissociation of rab3B from pIgR appears to require GTP
hydrolysis [van Ijzendoorn et al., 2002].
In this work, we use primary cultured rabbit lacrimal acinar cells as a
model system, as well as MDCK cells as a supplementary system, to investigate
the interaction between rab3D and pIgR, and to explore the role of rab3D in the
regulation of pIgR trafficking and SC secretion. We present biochemical and
functional evidence for an interaction between the pIgR and rab3D in rabbit
LGAC.

C. Materials and methods
1. Reagents
CCH, GTP γS, GTP, Ni
2+
-nitriloacetate (NTA) beads, and other chemical
reagents were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-
Sepharose was from Pharmacia LKB Biotechnologies Inc. (Alameda, CA).
51
Sheep anti-rabbit SC polyclonal antiserum was generated by a commercial
vendor (Capralogics, Hardwick, MA) against SC purified from rabbit bile (Pel-
Freeze, Rogers, AK) by preparative gel electrophoresis. The antiserum was of
sufficient titer to use diluted for Western blotting, immunoprecipitation and
immunofluorescence.
Plasmids encoding (His)
6
epitope-tagged forms of wild-type rab3D
(rab3DWT), the constitutively-active mutant Q81L (rab3DQ81L), and the
dominant-negative mutant T36N (rab3DT36N) were gifts from Dr. John A
Williams (University of Michigan, Ann Arbor, Michigan). They were expressed
in Escherichia coli (E. Coli) and purified on Ni
2+
-nitriloacetate (NTA) beads.
Anti-rab3D polyclonal antibodies were generated in rabbits against recombinant
(His)
6
epitope-tagged wild-type rab3D expressed in E. coli and purified by
chromatography over protein A/G agarose (Antibodies Inc., Davis, CA).
ProLong antifade mounting kit, goat anti-rabbit secondary antibody
conjugated to Alexa Fluor-568 were from Molecular Probes (Eugene, OR).
Donkey anti-sheep secondary antibody conjugated to FITC was from Jackson
ImmunoResearch Laboratories Inc. (West Grove, PA). Goat anti-rabbit
IRDye800- and donkey anti-sheep IRDye700-conjugated secondary antibodies
were purchased from Rockland (Gilbertsville, PA). Cell culture reagents were
from Invitrogen Inc. (Carlsbad, CA).
52
2. Lacrimal gland acinar cell isolation and primary culture
Isolation of lacrimal acini from female New Zealand white rabbits (1.8-2.2
kg) obtained from Irish Farms (Norco, CA) was in accordance with the Guiding
Principles for Use of Animals in Research. Lacrimal acini were isolated as
described [da Costa et al., 1998; Gierow et al., 1995] and cultured for 2-3 days.
Cells prepared in this way aggregate into acinus-like structures while individual
cells within these structures display distinct apical and basolateral domains and
maintain a robust secretory response [da Costa et al., 1998, 2003; Wang et al.,
2003]. Cell treatment with carbachol (CCH) was used at 100 µM dissolved in
phosphate-buffered saline (PBS).

3. Confocal fluorescence microscopy
Reconstituted rabbit lacrimal acini cultured on Matrigel-coated coverslips
were fixed and processed as described [da Costa et al., 1998, 2003; Wang et al.,
2003]. Acini were incubated with appropriate primary and fluorophore-
conjugated secondary antibodies. Most confocal images were obtained with a
Zeiss LSM 510 Meta NLO imaging system (Germany) equipped with Argon, red
and green HeNe lasers mounted on a vibration-free table and attached to an
incubation chamber controlling temperature, humidity and CO2. The ability of
this system to acquire fluorescence emission signals resolved within narrow
ranges in multitrack mode, and the use of singly-labeled control samples ensured
the validity of co-localization studies by assessing lack of signal bleed-through.
53
Panels were compiled in Adobe Photoshop 7.0 (Adobe Systems Inc., Mountain
View, CA).

4. Pull-down of rab3D and pIgR
(His)
6
epitope-tagged forms of wild-type rab3D (rab3DWT), the
constitutively-active mutant Q81L (rab3DQ81L), and the dominant-negative
mutant T36N (rab3DT36N) were expressed in E. coli and purified on Ni-NTA
bead columns. 3.6×10
7
resting or CCH-stimulated LGAC, or 5.0 ×10
7
MDCK
cells were solubilized in pull-down buffer (PDB) containing 1% Triton X-100,
20mM Na-HEPES, pH 7.4, and 50mM KCl, and incubated overnight at 4 °C with
40 μg of recombinant wild-type or mutant rab3D. In some cases, the lysate was
supplemented with 10 μM nonhydrolyzable GTP γS or 0.5 mM GTP. Rab3D was
recovered from the lysates by incubation with Ni-NTA beads for 1 hour at room
temperature and washed 3 times by PDB. Rab3D and any interacting proteins
were eluted from the beads with SDS-PAGE sample buffer, and analyzed by
Western blots.

5. Immunoprecipitation and overlay
Resting LGAC (7.2 ×10
7
cells) were solubilized in a buffer containing
2.5% Triton X-100, 100 mM triethanolamine, pH 8.6, 100 mM NaCl, 5 mM
EDTA, and 0.02% NaN
3
, and pIgR was immunoprecipitated at 4 °C overnight
with 1 μl of anti-SC polyclonal antibody covalently coupled to 40 μl Protein G-
54
Sepharose. After washing, the immunoprecipitate was run on SDS-
polyacrylamide gel (SDS-PAGE), and transferred to nitrocellulose membrane.
The membrane was soaked in a solution containing 50 mM Tris-HCl, pH 7.5,
150mM NaCl, 1mM DTT, 0.3% Tween-20, 3% bovine serum albumin (BSA),
0.05% NaN
3
for 6 - 7 hours at room temperature, then incubated with 10 μg
recombinant rab3DWT in 10 ml of a solution containing 10 mM Tris-HCl, pH 7.5,
20mM NaCl, 1mM DTT, and 0.5 mM EGTA at 4 °C overnight. After washing,
the membrane was analyzed by Western blot.

6. Nitrocellulose membrane stripping
Nitrocellulose membrane was soaked in a buffer containing 100 mM 2-
mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8, and 2% SDS at 50 °C for 30 min to
remove previously bound primary and secondary antibodies. After washing and
blocking, the membrane was reprobed with appropriate primary and secondary
antibodies and analyzed by Western blot.

7. Western blots
Western blots were processed utilizing appropriate primary antibodies and
secondary antibodies conjugated to either IRDye-800 or IRDye-700. Blots were
quantified using Li-Cor Odyssey Scanning Infrared Fluorescence Imaging System
(Lincoln, NE). For display, fluorescent signals were converted digitally to black
and white images.
55
D. Results
1. Localization of pIgR and Rab3D
To investigate whether rab3D was involved in the trafficking of pIgR, we
compared intracellular localization of rab3D with that of pIgR in LGAC. From
confocal fluorescence microscopic analysis of the immunofluorescence associated
with pIgR (figure 3.1, green), pIgR immunoreactivity was distributed on the
basolateral membrane (BLM), and additional pIgR immunoreactivity was
detected in large apparent vesicles or organelles localized beneath the apical
plasma membrane (APM) (figure 3.1, arrows) surrounding the lumenal regions
(figure 3.1, *). The secretory vesicle (SV) marker, rab3D (figure 3.1, red) also
was localized to this region in similar structures. In the merged image, there
appeared to be significant colocalization of the pIgR and rab3D signals near the
apical membrane (figure 3.1, yellow). In fact, the quantification of the
colocalization between the signals from two proteins revealed that about 40% of
the rab3D was co-localized with pIgR/SC, and vice versa (data not shown).

2. Recombinant rab3D binds to pIgR in pull-down assays
The significant co-localiztion of pIgR and rab3D in LGAC suggests that
they may interact. Attempts at co-immunoprecipitation of pIgR and rab3D were
not successful. Thus, pull-down assays with recombinant (His)
6
-tagged wild-type
rab3D (rab3DWT) were performed. Different amounts (40 μg, 80 μg) of

pIgR rab3D Merge
Figure 3.1 Colocalization of pIgR and rab3D. The LGAC grown on Matrigel-coated
coverslips were fixed and permeabilized by 100% cold ethanol, followed by blocking with
1% BSA in phosphate-buffered saline (PBS). Then cells were treated with primary anti-SC
and anti-rab3D antibodies, and secondary FITC-conjugated donkey anti-sheep and Alexa
Fluor-568-conjugated goat anti-rabbit antibodies, respectively. Immunofluorescence was
observed by confocal microscopy (Zeiss, Germany) to detect pIgR and rab3D localization in
LGAC. Green, pIgR; red, rab3D; arrows, regions of co-localization; *, lumena; bars, 5 μm.
Results shown are representative of at least three independent experiments.

recombinant rab3D protein were used in pull-down assays with lysates from
resting (untreated) LGAC. Rab3DWT could successfully pull down pIgR from
these lysates, indicating that rab3D can associate with pIgR (figure 3.2). In
addition, the amount of pIgR pulled down with rab3DWT was proportional to the
amount of rab3DWT protein added (figure 3.2), consistent with an interaction
between the two proteins.

3. GTP-bound form of rab3D disrupts rab3D-pIgR interaction
To examine how the guanine nucleotide binding state of rab3D influences
rab3D-pIgR interaction, the binding of pIgR to rab3DWT or mutated rab3D
proteins was tested in pull-down assays. Pull-down of pIgR from resting LGAC
56

57

- pIgR
- rab3D
WT 40 μg

WT 80 μg

Beads alone
120 kDa -
28 kDa -

Figure 3.2 Recombinant rab3DWT pull-down of pIgR. WT rab3D expressed as (His)
6
-
tagged proteins in E. coli were purified on Ni
2+
-nitriloacetate (NTA) bead columns, and 40 μg
or 80 μg of each used in pull-down assays with lysates from resting LGAC. After rab3D was
recovered from lysates by incubation with Ni-NTA beads, rab3D and any interacting
proteins were eluted from the beads with SDS-PAGE sample buffer and analyzed by
Western blot using primary anti-SC and anti-rab3D antibodies, and secondary donkey anti-
sheep IRDye700- and goat anti-rabbit IRDye800-conjugated antibodies, respectively.
Results shown are representative of at least three independent experiments.

lysates with either recombinant WT or mutant rab3D proteins showed that
rab3DWT and a putative dominant-negative, GDP-locked rab3D mutant T36N,
bound maximally to pIgR (figure 3.3A). In fact, rab3DT36N can bind 36% ± 7%
more pIgR compared to pIgR binding to rab3DWT (figure 3.3B). Interestingly,
however, the constitutively-active rab3DQ81L, a GTP-locked rab3D mutant, does
not bind to pIgR (figure 3.3A & 3.3B), only 4% ± 5% of the amount of pIgR
bound to rab3DWT. Furthermore, as shown in figure 3.3A and 3.3B, when 10
μM GTP γS or 0.5 mM GTP is included in the pull-down assays with rab3DWT,
to affect the GTP-bound state of rab3D, binding of pIgR is significantly inhibited,
58
only 1% ± 0% and 9% ± 9%, respectively, of the amount of pIgR bound to
rab3DWT. Figure 3.3B is a summary of the experiments plotted as the
percentage of pIgR binding in the various experiments relative to rab3DWT.
These data suggest that pIgR can interact with rab3D when rab3D is in the GDP-
bound form. The GTP-bound form of rab3D, whether induced by a mutation or
by addition of GTP or GTP γS, appears to be incapable of interacting with pIgR.
Similar experiments were performed in pIgR-transfected MDCK cells
with the same concentration of GTP γS or GTP included in the pull-down assays.
Figure 3.4 demonstrated that binding of rab3D and pIgR can be replicated in
pIgR-transfected MDCK cells but not in non-transfected cells. In addition, there
was no interaction of rab3D and pIgR observed when GTP γS or GTP was added
in pull-down assays with ra3DWT. Thus, the binding characteristics of rab3D
and pIgR in LGAC can be reproduced in pIgR-transfected MDCK cells,
indicating that the interaction of rab3D with pIgR may be independent of cell type.
These data indicate that GDP- or GTP-bound form of rab3D determines
whether or not rab3D is able to bind pIgR, and, together with the
immunofluorescence data, also suggest that rab3D may be involved in the
regulation of pIgR trafficking.

WT+GTP γS
WT+GTP
Q81L
0
20
40
60
80
100
120
140
160
WT T36N Q81L WT+GTPrS WT+GTP
pIgR / Rab3D (%)
B
- pIgR
- rab3D
A
Beads alone
WT
T36N
120 kDa -
28 kDa -
Figure 3.3 Recombinant wild-type and mutant rab3D pull-down of pIgR. (A) WT, mutant
dominant-negative T36N and constitutive-active Q81L rab3D expressed as (His)
6
-tagged
proteins in E. coli were purified and 40 μg of each used in pull-down assays with lysates from
resting LGAC. In some cases, the lysate was supplemented with 10 μM GTP γS or 0.5 mM
GTP. Western blot utilized primary anti-SC and anti-rab3D antibodies, and secondary
donkey anti-sheep IRDye700- and goat anti-rabbit IRDye800-conjugated antibodies,
respectively. (B) Data are plotted as a percentage of pIgR binding to WT rab3D. Results
shown are representative of at least three independent experiments.

59

- rab3D
non-transfected
MDCK
- pIgR
pIgR-transfected
MDCK
WT+GTP γS
WT+GTP
WT
Beads alone
WT
120 kDa -
28 kDa -

Figure 3.4 Recombinant rab3DWT pull-down of pIgR from pIgR-transfected MDCK cells.
Lysates from untransfected MDCK or pIgR-transfected MDCK cells were incubated with
recombinant rab3DWT in pull-down asssays. In some cases, the lysate from pIgR-
transfected MDCK cells was supplemented with 10 μM GTP γS or 0.5 mM GTP. Western
blot applied primary anti-SC and anti-rab3D antibodies, and secondary donkey anti-sheep
IRDye700- and goat anti-rabbit IRDye800-conjugated antibodies, respectively. Results
shown are representative of at least three independent experiments.

4. Direct interaction between rab3D and pIgR
The pull-down assays do not distinguish whether the interaction between
rab3D and pIgR is a direct or indirect one. Thus, overlay assays were performed
to test whether rab3D and pIgR interact directly. The pIgR was
immunoprecipitated from LGAC, run on SDS-PAGE, and transferred to
nitrocellulose. Then the membrane was incubated with recombinant rab3D and
probed with anti-rab3D antibody to detect where rab3D bound on the blot. As
shown in figure 3.5 (left lane), rab3D bound to a protein migrating at ~120 kDa
which corresponds to the position of pIgR on gels. To confirm whether this band
60
61
was indeed pIgR, the nitrocellulose was stripped of rab3DWT and was reprobed
with anti-SC antibody to detect pIgR. As shown in the right lane in figure 3.5, the
anti-SC antibodies reacted with the protein migrating at ~120 kDa; upon
comparison of both blots, the 120 kDa protein binding to rab3D and anti-SC
immunoreactivity coincided precisely. A good internal control is provided by the
protein at ~80 kDa that reacts with anti-SC, suggesting that this protein is
immunoprecipitated SC, but it does not bind to rab3D. This further supported the
existence of direct interaction between rab3D and pIgR, and that this interaction is
dependent upon the presence of the cytoplasmic and/or membrane spanning
domain of pIgR.
Thus, the data suggest that the interaction between rab3D and pIgR is
direct, specific, and does not require any additional proteins.

5. Acute treatment of cholinergic agonist abolishes rab3D-pIgR
interaction
It has been reported that 100 μM carbachol (CCH), a cholinergic agonist,
stimulated the release of SC from lacrimal acini for up to 60 min [Jerdeva et al.,
2005; Schechter et al., 2002]. Carbachol may then regulate the interaction of
pIgR with rab3D, ultimately to regulate SC release from lacrimal acinar cells,
which led us to explore further how rab3D controls the trafficking of pIgR, that is,
what is the relationship between CCH-stimulated SC secretion and rab3D-pIgR
interaction.

WB: anti-rab3D anti-SC
rab3D
– pIgR
– SC
150 kDa -
100 kDa -
75 kDa -

Figure 3.5 Direct interaction between rab3D and pIgR. The pIgR was immunoprecipitated
from LGAC with anti-SC antibody, run on SDS-PAGE, and transferred to nitrocellulose
membrane. The membrane was incubated with recombinant rab3DWT and then probed
with primary anti-rab3D antibodies and secondary goat anti-rabbit IRDye800-conjugated
antibodies. Reactivity on the left lane shows where rab3D bound. The membrane was then
stripped and was re-probed with primary anti-SC antibodies and secondary donkey anti-
sheep IRDye700-conjugated antibodies. The positions of immunoreactive bands for pIgR
and SC are indicated on the right. The pIgR reactive signal co-migrated exactly with the
lower of the rab3D binding proteins. Rab3D did not bind to SC. Results shown are
representative of at least three independent experiments.

Stimulation of LGAC secretion with 100 μM CCH for 30 min or 60 min
prior to lysis, followed by incubation of lysates with recombinant rab3DWT in
pull-down assays, resulted in a loss of pIgR binding to rab3DWT from 100% (0
min) to 7% ± 10% (30 min) and 1% ± 0% (60 min) (figure 3.6A and 3.6B). Thus,
the cabachol-dependent physiological signaling pathway in lacrimal acinar cells
modulates the interaction between rab3D and pIgR. The carbachol-dependent
loss of interaction between rab3D and pIgR could provide a mechanism for the
62
observed stimulation of SC secretion in lacrimal acinar cells, particularly if the
rab3D-pIgR interaction negatively regulates the terminal steps in SC release.

0
20
40
60
80
100
120
0 30 min 60 min
pIgR/Rab3D (% of 0 min)
B
A
Beads alone
- rab3D
- pIgR
0 min
30 min
60 min
CCH (100 μM)
120 kDa -
28 kDa -
Figure 3.6 Effect of CCH on rab3D-pIgR interaction. (A) Lysates from unstimulated (0 min)
or 100 μM CCH-stimulated (30 min or 60 min) LGAC were incubated with recombinant
rab3DWT in pull-down assays. Western blot used primary anti-SC and anti-rab3D
antibodies, and secondary donkey anti-sheep IRDye700- and goat anti-rabbit IRDye800-
conjugated antibodies, respectively. (B) The ratio of pIgR to rab3D was calculated and
normalized to that in resting LGAC (% of binding at 0 min). Results shown are
representative of at least three independent experiments.

63
64
E. Discussion
We report that rab3D directly interacts with the cytoplasmic and/or
transmembrane domain of the pIgR and may regulate its trafficking in LGAC.
Our immunofluorescence data showed that rab3D, the secretory vesicle
marker, was co-localized with pIgR at vesicles subjacent to the APM surrounding
the lumenal regions in LGAC (figure 3.1). This is consistent with our previous
data that a substantial fraction of pIgR is localized in rab3D-containing
compartments of rabbit LGAC by subcellular fractionation [Qian et al., 2003]. In
polarized cultured hepatocytes, rab3D-positive vesicles localize near the APM
and in the apical cytoplasm, and immunoisolation of rab3D-containing vesicles
was found to be enriched in the transcytosed form of pIgR [Larkin et al., 2000].
In pIgR-transfected MDCK cells, rab3B was found associated with vesicles
containing transcytosing pIgR that were concentrated in the apical cytoplasm and
near the centrosomes [van IJzendoorn et al., 2002]. This led us to investigate the
relationship between rab3D and pIgR, and it is likely that rab3D is utilized to
regulate pIgR trafficking in lacrimal gland.
The significant co-localization of pIgR and rab3D in LGAC implies that
they may interact. The pull-down assay was able to detect the binding of rab3D
and pIgR not only in LGAC (figure 3.2) but also in pIgR-transfected MDCK cells
(figure 3.4), suggesting that the interaction of rab3D and pIgR a general
phenomenon. A number of rab proteins, including rab1, rab2, rab6, rab3B, rab3D,
rab11, rab13, rab17, and rab25 in hepatocytes have been implicated as possible
65
transcytotic regulators of pIgR [Jin et al., 1996; Tuma et al., 2001; Larkin et al.,
2000]. Detailed information about a role of rab3B in pIgR trafficking and
transcytosis was described in MDCK cells [van IJzendoorn et al., 2002]. Our
results are consistent with these observations. Quantitative analysis showed that
pIgR pull-down signals were proportional to the amount of recombinant
rab3DWT protein (40 μg, 80 μg) added to lysates from LGAC, indicating a linear
binding range (figure 3.2). This result is to some extent in agreement with the
findings that glutathione S-transferase (GST)-rab3-interacting molecule (RIM)
pulled down rab3D, and pull-down signals corresponded to the quantity of mouse
pancreatic acinar lysates loaded [Chen et al., 2003].
The activity of rab proteins is controlled by GTP/GDP cycling [Boguski et
al., 1993]. To address how the guanine nucleotide binding status of rab3D
affected rab3D-pIgR interaction, rab3DWT and mutated rab3D were used in pull-
down assays. In figure 3.3A & 3.3B, pIgR interacted differently with different
rab3D mutants. The pIgR interacts most strongly with dominant-negative
rab3DT36N (136% ± 7%), less strongly with rab3DWT (100%), but did not
interact with constitutively-active rab3DQ81L in LGAC (4% ± 5%). When GTP
or GTP γS was included in the pull-down assays, no interaction was detected (9%
± 9% or 1% ± 0%, respectively) (figure 3.3A & 3.3B). These results were
reproduced in pIgR-transfected MDCK cells (figure 3.4). Pull-down data
collected from LGAC and pIgR-transfected MDCK cells indicate that, in vivo,
pIgR predominantly interacts with the inactive GDP-bound form of rab3D and,
66
the GTP-bound form of rab3D impairs rab3D-pIgR interaction. Thus, GDP- or
GTP- binding state of rab3D determines interaction of rab3D and pIgR. Given
that the presence of interaction between rab3D and pIgR may be the obstacle for
SC release, our data are consistent with some previous studies in different cell
types. For example, in AtT-20 cells, dominant-negative rab3DN135I blocks
regulated secretion of ACTH [Baldini et al., 1998]; in mouse pancreatic acinar
cells, two dominant-negative mutants of rab3D, T36N and N135I, inhibits
regulated amylase secretion [Chen et al., 2002], and CCK can stimulate
pancreatic exocytosis partially by enhancing GTP incorporation into rab3D
[Ohnishi et al., 1997].
To examine further the physical interactions of rab3D and pIgR, overlay
assays were performed to test whether rab3D and pIgR interact directly. As
shown in figure 3.5 left lane, rab3D bound to a protein migrating at ~120 kDa
which corresponds exactly to the position of pIgR. However, rab3D did not bind
to immunoprecipitated SC, the extracellular domain of pIgR, at ~80 kDa (figure
3.5, right lane), suggesting that rab3D-pIgR interaction most likely occurs at the
cytoplasmic and/or membrane spanning domain. In concert with our observations,
van Ijzendoorn et al., found direct interaction between the cytoplasmic tail of
pIgR and rab3B in transfected MDCK cells [van IJzendoorn et al., 2002], and
Seachrist et al., demonstrated direct interaction between rab5A and the
cytoplasmic tail of angiotensin II type 1A receptor [Seachrist et al., 2002].
Therefore, our results demonstrate that the interaction between rab3D and pIgR is
67
direct, specific, and does not require any adaptors. This is the first evidence for
the existence of direct interaction between rab3D and pIgR in any cell type.
Cholinergic agonist is the major stimulus of lacrimal gland secretion
[Dartt, 2004]. Previous research revealed that 100 μM carbachol (CCH), a
cholinergic agonist, stimulated the release of SC from lacrimal acini [Jerdeva et
al., 2005; Schechter et al., 2002]. We next investigated the functional correlation
of rab3D and pIgR, that is, what is the relationship between stimulated SC
secretion and rab3D-pIgR interaction. As shown in figure 3.6A & 3.6B, treatment
of LGAC with 100 μM CCH for 30 min or 60 min leads to a loss of pIgR binding
to rab3DWT in pull-down assays, from 100% (0 min) to 7% ± 10% (30 min) or
1% ± 0% (60 min), suggesting that the cabachol-dependent physiological
signaling pathway is involved in the modulation of the interaction between rab3D
and pIgR in LGAC. Surprisingly, we also observed that carbachol-stimulated
release of SC showed an initial rapid burst of secretion (0-10 min) very similar to
that of the exogenously expressed SV marker, syncollin-GFP [Jerdeva et al.,
2005], and that of the endogenous secretory enzyme, β-hexosaminidase
[Schechter et al., 2002; Evans et al., 2006], which is quite distinct from the
sustained transcytotic release of SC. It is important to note that all these
experiments were done without dIgA, which may substantially stimulate pIgR
transcytosis, and consequently increase total SC release by enhancing sIgA
secretion. Thus, it appears that the carbachol-stimulated loss of interaction
between rab3D and pIgR could provide an explanation for the observed
68
stimulation of SC secretion in lacrimal acinar cells, particularly if the rab3D-pIgR
interaction negatively regulates the terminal steps in SC release. In addition,
considering that the GDP- or GTP- binding status of rab3D determines rab3D-
pIgR interaction (figure 3.3), whether CCH-activated intracellular signaling
pathway in LGAC promotes GTP binding to rab3D, resulting in the disruption of
rab3D-pIgR interaction followed by the rapid SC secretion, is still a question that
needs to be addressed.
There are two types of protein secretion from the lacrimal gland,
constitutive and regulated secretion [Hodges et al., 2003]. Secretion of sIgA and
SC are considered as the best-studied examples of constitutive secretion [Sullivan
et al., 1984]. This means that SC or sIgA is synthesized at a particular rate and
secreted without storage in SV. As opposed to constitutive secretory proteins,
regulated secretory proteins are stored in SV after synthesis and secreted rapidly
upon stimulation. Rab3 family members are typically involved in the process of
regulated secretion, and they are expressed predominantly in cells with
specialized secretory functions [Lledo et al., 1994; Geppert et al., 1998]. Rab3D
is localized to secretory granules of various exocrine secretory cells including
lacrimal gland. In this study, we provide evidence for the colocalization of rab3D
and pIgR in SV subjacent to the APM (figure 3.1), and for direct interaction
between rab3D and pIgR (figure 3.2, 3.3, 3.4, 3.5). This localization and
interaction imply that rab3D may be involved in the regulated secretion of SC.
Combined with the observation that acute treatment of lacrimal acinar cells with
69
CCH disrupted the interaction of rab3D and pIgR, correlating to the rapid initial
phase of SC release, we hypothesize that rab3D sequesters merocrine stores of
pIgR for secretagogue-dependent release of free SC. That is, the intracellular
trafficking of pIgR in LGAC includes two pathways (figure 3.7): a constitutive
transcytotic pathway (red arrows), and a regulated merocrine pathway (blue
arrows) in which the merocine effector rab3D is proposed to be the major
regulator of formation of SV, sorting pIgR to accumulate in SV, and fusion of SV
with the APM to release SC upon stimulation. However, after pIgR synthesis in
ER and exit from TGN, how rab3D works to recruit pIgR to merocrine pathway
still needs to be determined. We propose two possibilities (figure 3.7): i) the
pIgR is segregated into two groups in the TGN, one is sorted to regulated vesicles
by rab3D for the merocrine pathway (blue arrows), the other to constitutive
vesicles for the transcytotic pathway (red arrows); ii) the pIgR is first subjected to
transcytotic pathway (i.e., delivered to the basolateral cell surface followed by
endocytosis and transported through a series of endosomal compartments) (red
arrows), then some pIgR is recruited from the AE to regulated vesicles by rab3D
(green arrows), while the rest of the pIgR continues their constitutive transcytotic
pathway (red arrows). So far, we do not have enough evidence to determine
whether only one or both possibilities may happen in vivo.
Therefore, the regulated merocrine pathway of pIgR transport is an
innovative model which challenges the existing dogma that free SC in tears is
solely delivered by a constitutive transcytotic pathway in the lacrimal gland.
70
Although the principles of vesicle-mediated trafficking via rab protein regulation
are unclear to date, our analysis suggests a novel mechanism of rab3D-regulated
secretion of pIgR transport. Some independent reports support our hypothesis.
Identification of rab3D in rat hepatocytes, traditionally viewed as constitutive
secretory cells, generated speculation of the existence of a regulated secretory
pathway in these cells [Larkin et al., 2000]. Furthermore, rab11 has been shown
to be associated with both the constitutive and the regulated secretory pathway in
PC12 cells [Urbe et al., 1993]. Since the association of rab3D and pIgR may be
independent of cell type, which has been tested in both LGAC and MDCK cells
(figure 3.3 & 3.4), our findings are very likely to yield new information regarding
an important physiologic process and may become a paradigm for the study of
other proteins in human lacrimal glands and other epithelial cells.
In conclusion, our results are the first to identify a direct interaction
between rab3D and pIgR, initiating a novel insight into the molecular mechanism
of how rab3D may regulate pIgR trafficking and therefore SC release by LGAC
in a stimulation-dependent manner. Although our study provides an intriguing
start, there is still much work to completely characterize the precise steps in
rab3D-regulated pIgR trafficking.

pIgR
dIgA
SC
sIgA
AE
SV
TGN
BE
Apical
Basolateral
rab3D
ER
rab3D
rab3D

Figure 3.7 Regulated merocrine and constitutive transcytotic pathways involved in the
regulation of pIgR trafficking and SC secretion in LGAC. After pIgR synthesis in
endoplasmic reticulum (ER) and exit from trans-Golgi network (TGN), we hypothesize two
possibilities of how rab3D recruits pIgR to merocrine pathway: i) the pIgR is segregated
into two groups in the TGN, one is sorted to regulated vesicles by rab3D for the merocrine
pathway (blue arrows), the other to constitutive vesicles for the transcytotic pathway (red
arrows); ii) the pIgR is first subjected to the transcytotic pathway (red arrows) (i.e.,
delivered to the basolateral cell surface followed by endocytosis and transported through a
series of endosomal compartments), then some pIgR is recruited from the AE to regulated
vesicles by rab3D (green arrows), while the rest of the pIgR continues their constitutive
transcytotic pathway (red arrows).
71
72
CHAPTER IV - CROSS-TALK BETWEEN RAB11 AND RAB3D IN
REGULATION OF THE POLYMERIC IMMUNOGLOBULIN
RECEPTOR TRAFFICKING MAY REFLECT COMMUNICATION
BETWEEN REGULATED AND CONSTITUTIVE SECRETORY
PATHWAYS IN LACRIMAL GLAND ACINAR CELLS
A. Abstract
Purpose: Rab11, a member of the ras-like small GTP-binding protein
superfamily, is considered to regulate various cellular functions, including plasma
membrane recycling, phagocytosis, and cytokinesis. In this study, we
preliminarily characterize the role of rab11 in the regulation of polymeric
immunoglobulin receptor (pIgR) trafficking in primary cultured rabbit lacrimal
gland acinar cells (LGAC). Methods: Primary rabbit LGAC cultured for 3 days
were processed for confocal fluorescence microscopy utilizing appropriate
primary and secondary antibodies to rab11 and pIgR. Co-immunoprecipitation
and subcellular fractionation experiments were performed using resting or 100
µM carbachol-stimulated LGAC according to established procedures. Rab11 and
pIgR were pulled down from resting LGAC lysates with recombinant (His)
6
-
tagged wild-type rab3D (rab3DWT) or mutated rab3D proteins. Results:
Confocal fluorescence microscopy revealed that the apical endosomal marker
rab11 exhibited significant colocalization with the pIgR beneath the apical plasma
membrane in LGAC, suggesting that rab11 participates in pIgR transcytosis. The
pIgR can be co-immunoprecipitated from lysates of resting LGAC with either
73
monoclonal or polyclonal anti-rab11 antibodies, indicating that rab11 interacts
with pIgR. When co-immunoprecipitation was conducted using lysates from
LGAC stimulated with 100 µM carbachol, a cholinergic agonist, for 30min, the
co-immunoprecipitated amount of pIgR appeared to increase. This is the first
demonstration of co-immunoprecipitation of pIgR with rab11 from any cell type.
Subcellular fractionation demonstrated that rab11 and rab3D are mainly
associated with the membrane fraction. Upon 100 µM CCH stimulation for 30
min, the total amount of rab11 or rab3D has an increased redistribution from
membrane to cytosol, separately. Pull-down of rab11 and pIgR from resting
LGAC lysates with recombinant (His)
6
-tagged wild-type rab3D (rab3DWT) or
mutated rab3D proteins showed that rab3DWT and a dominant-negative GDP-
locked mutant rab3D, rab3DT36N, bind to rab11 and pIgR; however, the
constitutively-active GTP-locked rab3D mutant, rab3DQ81L, does not bind to
rab11 nor pIgR. Furthermore, when GTP γS or GTP is included in the pull-down
assays, binding of rab3DWT to both rab11 and pIgR is significantly inhibited.
Conclusions: These findings suggest that rab11 and rab3D may cross-talk in
regulating pIgR trafficking in LGAC through constitutive and regulated secretory
pathways, respectively.

B. Introduction
The lacrimal gland, the effector of the secretory immune system of the
ocular surface, has many of its secretory proteins involved in immunological
74
protection of the eye. The lacrimal gland is densely innervated with
parasympathetic (cholinergic) nerves and less densely innervated by sympathetic
(adrenergic) nerves [Ding et al., 2001]. Cholinergic agonists and norepinephrine
are major stimuli of lacrimal gland secretion [Dartt et al., 2004].
The polymeric immunoglobulin receptor (pIgR) is responsible for the
basal-to-apical transcytosis of dimeric IgA (dIgA) across epithelial cells. The
extracellular dIgA binding domain of pIgR, called secretory component (SC), in
association with dIgA, is known as secretory IgA (sIgA), it represents the primary
defense against pathogens at mucosal surfaces. Tear fluid is relatively rich in
sIgA and SC [Gundmundsson et al., 1985]. Sullivan et al., identified the exorbital
lacrimal gland as the major source of SC in rat ocular surface fluid [Sullivan et al.,
1984], and the pIgR has been identified in lysates from primary cultures of rabbit
lacrimal gland acinar cells (LGAC) [Qian et al., 2003].
The rab11 family of small molecular weight ras-like GTP-binding proteins
is critical regulators of the plasma membrane vesicle recycling system [Hobdy-
Henderson et al., 2003]. There are currently three identified members of the
mammalian rab11 family, rab11a, rab11b, and rab25 [Bhartur et al., 2000].
Among three rab11 family members, rab11a was the first of the family cloned and
so most of the work done on this family was focused on rab11a. Rab11a, rab11b,
and rab25 are closely related, evolutionarily conserved rab proteins that are
differentially expressed. Rab11a is ubiquitously expressed [Sakurada et al., 1991],
rab11b is enriched in brain and heart [Lai et al., 1994], and rab25 is found only in
75
certain epithelial cells of tissues, such as gastrointestinal mucosa, kidney and lung
[Goldenring et al., 1993]. Although ubiquitously expressed, rab11 has been
reported to be more abundant in tissues with a high level of secretion [Sakurada et
al., 1991]. As a marker for plasma membrane recycling systems, rab11a is
associated with the plasma membrane recycling system in nonpolarized cells
[Green et al., 1997; Ullrich et al., 1996]. In polarized cells, the analogous
structure, the apical recycling system, is responsible for processing of apical
recycling and basolateral-to-apical transcytosis [Apodaca, Katz et al., 1994].
While rab11a is known to regulate transferrin trafficking in nonpolarized cells and
IgA trafficking in polarized cells, rab11b exhibits little colocalization with either
of these cargoes [Lapierre et al., 2003].
Transcytosis is a multistep trafficking pathway, and each step is likely to
be regulated. A number of rab proteins, including rab1, rab2, rab6, rab3B, 3D,
rab11, rab13, rab17, and rab25 have been implicated as possible transcytotic
regulators [Jin et al., 1996; Tuma et al., 2001; Larkin et al., 2000]. Although the
association of rab11 with the apical recycling system in polarized epithelial cells
is well established, still little is known about its function involved in pIgR
trafficking in LGAC. In this study, we present data suggesting that rab11
interacts with pIgR and rab3D, and these two rab proteins, rab11 and rab3D, may
cross-talk in regulating pIgR trafficking through the regulated and constitutive
secretory pathways, respectively, in LGAC.

76
C. Materials and methods
1. Reagents
CCH, GTP γS, GTP, Ni
2+
-nitriloacetate (NTA) beads, and other chemical
reagents were obtained from Sigma-Aldrich (St. Louis, MO). Protein G-
Sepharose was from Pharmacia LKB Biotechnologies Inc. (Alameda, CA).
Sheep anti-rabbit SC polyclonal antiserum was generated by a commercial
vendor (Capralogics, Hardwick, MA) against SC purified from rabbit bile (Pel-
Freeze, Rogers, AK) by preparative gel electrophoresis. The antiserum was of
sufficient titer to use diluted for Western blotting, immunoprecipitation and
immunofluoresecnce.
Plasmid encoding (His)
6
epitope-tagged forms of wild-type rab3D
(rab3DWT), the constitutively-active mutant Q81L (rab3DQ81L), and the
dominant-negative T36N (rab3DT36N) mutant were gifts from Dr. John A
Williams (University of Michigan, Ann Arbor, Michigan). They were expressed
in Escherichia coli (E. Coli) and purified on Ni
2+
-nitriloacetate (NTA) beads.
Anti-rab3D polyclonal antibodies were generated in rabbits against recombinant
(His)
6
epitope-tagged wild-type rab3D expressed in E. coli and purified by
chromatography over protein A/G agarose (Antibodies Inc., Davis, CA).
Anti-rab11 monoclonal antibodies were obtained from Becton-Dickinson
(Franklin Lakes, NJ), and anti-rab11 polyclonal antibodies were purchased from
Zymed Laboratories (S. San Francisco, CA). These two types of anti-rab11
antibodies can recognize both rab11a and rab11b.
77
ProLong antifade mounting kit, goat anti-rabbit secondary antibody
conjugated to Alexa Fluor-568 were from Molecular Probes (Eugene, OR).
Donkey anti-sheep secondary antibody conjugated to FITC was from Jackson
ImmunoResearch Laboratories Inc. (West Grove, PA). Goat anti-rabbit
IRDye800-, goat anti-mouse IRDye800-, and donkey anti-sheep IRDye700-
conjugated secondary antibodies were purchased from Rockland (Gilbertsville,
PA). Cell culture reagents were from Invitrogen Inc. (Carlsbad, CA).

2. Acinar cell isolation and primary culture
Isolation of lacrimal acini from female New Zealand white rabbits (1.8-2.2
kg) obtained from Irish Farms (Norco, CA) was in accordance with the Guiding
Principles for Use of Animals in Research. Lacrimal acini were isolated as
described [da Costa et al., 1998; Gierow et al., 1995] and cultured for 2-3 days.
Cells prepared in this way aggregate into acinus-like structures while individual
cells within these structures display distinct apical and basolateral domains and
maintain a robust secretory response [da Costa et al., 1998, 2003; Wang et al.,
2003]. Cell treatment with carbachol (CCH) was used at 100 µM.

3. Confocal fluorescence microscopy
Reconstituted rabbit lacrimal acini cultured on Matrigel-coated coverslips
were fixed and processed as described [da Costa et al., 1998, 2003; Wang et al.,
2003]. Acini were incubated with appropriate primary and fluorophore-
78
conjugated secondary antibodies. Most confocal images were obtained with a
Zeiss LSM 510 Meta NLO imaging system (Germany) equipped with Argon, red
and green HeNe lasers mounted on a vibration-free table and attached to an
incubation chamber controlling temperature, humidity and CO2. The ability of
this system to acquire fluorescence emission signals resolved within narrow
ranges in multitrack mode, and the use of singly-labeled control samples ensured
the validity of co-localization studies by assessing lack of signal bleed-through.
Panels were compiled in Adobe Photoshop 7.0 (Adobe Systems Inc, Mountain
View, CA).

4. Co-immunoprecipitation of pIgR with anti-rab11 mAb
Resting and CCH-stimulated LGAC were solubilized in a Triton dilution
buffer containing 2.5% Triton X-100, 100 mM triethanolamine, pH 8.6, 100 mM
NaCl, 5 mM EDTA, and 0.02% NaN
3
, and rab11 was immunoprecipitated at 4 °C
overnight with an anti-rab11 mAb and protein G-sepharose. After washing, the
immunoprecipitate was run on SDS-PAGE, transferred to nitrocellulose
membrane and analyzed by Western blot with primary anti-SC, monoclonal anti-
rab11 antibodies and appropriate secondary antibodies. To confirm the binding,
the nitrocellulose membrane was stripped and reprobed with polyclonal anti-
rab11 antibodies and appropriate secondary antibodies.

79
5. Nitrocellulose membrane stripping
Nitrocellulose membrane was soaked in a buffer containing 100 mM 2-
mercaptoethanol, 62.5 mM Tris-HCl, pH 6.8, and 2% SDS at 50 °C for 30 min to
remove previously bound primary and secondary antibodies. After washing and
blocking, the membrane was reprobed with appropriate primary and secondary
antibodies and analyzed by Western blot.

6. Subcellular fractionation
Resting and CCH-stimulated LGAC were scraped, pelleted and
resuspended in PMEE buffer (35 mM PIPES, 5 mM MgSO
4
, 1 mM EGTA, and
0.5 mM EDTA, pH 7.4) supplemented with a protease inhibitor cocktail (1 mM
dithiothreitol (DTT), 174 mg/ml phenylmethanesulfonyl fluoride (PMSF), 1
mg/ml pepstatin A, 10 mg/ml N-tosyl-L-phenylalanine chloromethyl ketone
(TPCK), 1 mg/ml leupeptin, 10 mg/ml α-chymotrypsin (TLCK), and 10 mg/ml p-
toluenesulfonyl-L-arginine methyl ester (TAME)). The suspension was
homogenized by 40 passages through a Balch cell press (H&Y Enterprises,
Redwood City, CA) and centrifuged at 9,200 х g for 10 min at 4 °C. Following a
subsequent centrifugation of the supernatant at 112,817 х g for 30 min at 4 °C in a
Sorvall S120-AT2 rotor, the resulting cytosolic supernatants were concentrated by
Microcon YM-10 (Millipore, Billerica, MA) at 4 °C until reaching the appropriate
volume, and the membrane pellets were resuspended to the same volume as
80
cytosolic supernatants. The distribution of proteins of interest was determined by
SDS-PAGE and Western blot.

7. Recombinant rab3D pull-down of rab11 and pIgR
(His)
6
epitope-tagged forms of wild-type rab3D (rab3DWT), the
constitutively-active Q81L (rab3DQ81L) mutant, and the dominant-negative
T36N (rab3DT36N) mutant were expressed in E. coli and purified on Ni
2+
-NTA
bead columns. 3.6×10
7
resting LGAC were solubilized in pull-down buffer (PDB)
containing 1% Triton X-100, 20mM Na-HEPES, pH 7.4, and 50mM KCl, and
incubated overnight at 4 °C with 40 μg of recombinant wild-type or mutant rab3D.
In some cases, the lysate was supplemented with 10 μM nonhydrolyzable GTP γS
or 0.5 mM GTP. Rab3D was recovered from the lysates by incubation with Ni-
NTA beads for 1 hour at room temperature and washed by PDB. Rab3D and any
interacting proteins were eluted from the beads with SDS-PAGE sample buffer,
and analyzed by Western blots.

8. Western blots
Western blots were processed utilizing appropriate primary antibodies and
secondary antibodies conjugated to either IRDye-800 or IRDye-700. Blots were
quantified using Li-Cor Odyssey Scanning Infrared Fluorescence Imaging System
(Lincoln, NE). For display, fluorescent signals were converted digitally to black
and white images.
81
D. Results
1. Rab11 is colocalized with pIgR in lacrimal gland acinar cells
To investigate whether rab11 was involved in the trafficking of pIgR, we
compared the intracellular localization of rab11 with that of pIgR in LGAC.
From confocal fluorescence microscopic analysis of the immunofluorescence
associated with pIgR (figure 4.1, green), pIgR immunoreactivity was distributed
on the basolateral membrane (BLM), and additional pIgR immunoreactivity was
detected in vesicles or organelles localized beneath the apical plasma membrane
(APM) (figure 4.1, arrows) surrounding the lumenal regions (figure 4.1, *). The
distribution of the apical endosome marker, rab11 (figure 4.1, red) also was
localized to APM region in similar structures. In the merged image, there
appeared to be significant colocalization of the pIgR and rab11 signals near the
apical membrane (figure 4.1, yellow). In fact, the quantification of the
colocalization between the signals from two proteins revealed that about 40% of
the rab11 was co-localized with pIgR/SC, and vice versa (data not shown).

2. The pIgR is co-immunoprecipitaed with rab11 from lacrimal gland
acinar cell lysates
The significant co-localization of pIgR and rab11 in LGAC suggests that
they may interact. Therefore, co-immunoprecipitation was performed to test
whether rab11 interacts with pIgR. Monoclonal rab11 antibody and protein G-
sepharose beads were used in co-immunoprecipitation with lysates from resting

pIgR rab11 Merge

Figure 4.1 Colocalization of rab11 and pIgR. The LGAC grown on Matrigel-coated
coverslips were fixed and permeabilized by 100% cold ethanol, followed by blocking with
1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Then cells were
treated with primary anti-SC and anti-rab11 antibodies, and secondary FITC-conjugated
donkey anti-sheep and Alexa Fluor-568-conjugated goat anti-rabbit antibodies, respectively.
Immunofluorescence was observed by confocal microscopy (Zeiss, Germany) to detect rab11
and pIgR localization in LGAC. Green, pIgR; red, rab11; arrows, regions of co-localization;
*, lumena; bars, 5 μm. Results shown are representative of at least three independent
experiments.

and 100 μM carbachol treated (30 min) LGAC. As shown in figure 4.2, pIgR was
co-immunoprecipitated with rab11 from lysates of LGAC (left lane), indicating
that pIgR interacts with rab11. Furthermore, the amount of pIgR appeared to be
increased in co-immunoprecipitation from CCH-treated cells (figure 4.2, middle
lane of top panel). To confirm that rab11 was immunoprecipitated, we used both
monoclonal and polyclonal anti-rab11 antibodies to detect rab11. Comparison of
the middle panel and the bottom panel in figure 4.2 suggests that rab11 from
lysates of LGAC can be recognized by either type of antibodies and that rab11
was successfully immunoprecipitated. This is the first demonstration of pIgR co-
immunoprecipitation with rab11 in any cell type. Together with the
82
immunofluorescence data, these results reveal that rab11 may interact with the
pIgR and regulate its trafficking.

CCH (100 μM)
– pIgR (WB: SC)
- + Control
– rab11 (WB: rab11 mAb)
– rab11 (WB: rab11 pAb)
25 kDa –

20 kDa –
25 kDa –

20 kDa –
150 kDa –

100 kDa –

Figure 4.2 Co-immunoprecipitation of pIgR with anti-rab11 mAb. Resting and 100 μM
CCH-stimulated (30 min) LGAC were solubilized in 2.5% Triton dilution buffer, and pIgR
was immunoprecipitated with an anti-rab11 mAb. The immunoprecipitate was run on SDS-
PAGE, transferred to nitrocellulose membrane and analyzed by Western blot using primary
anti-SC (top panel) and anti-rab11 monoclonal antibodies (mAb, middle panel), and
secondary donkey anti-sheep IRDye700- and goat anti-mouse IRDye800-conjugated
antibodies, respectively. We also stripped and re-probed the nitrocellulose membrane with
primary anti-rab11 polyclonal antibodies (pAb, bottom panel) and secondary goat anti-
rabbit IRDye800-conjugated antibodies. LGAC lysate was used as a blotting control in the
far right lane. pIgR does not precipitate with beads in the absence of anti-rab11 mAb (data
not shown). Results shown are representative of at least three independent experiments.

3. CCH induces rab11 and rab3D redistribution in lacrimal gland acinar
cells
In our earlier work, we found that 30 min incubation of 100 µM CCH with
LGAC disrupted the rab3D and pIgR interaction [Zhang et al., 2005]. In contrast
83
84
to rab3D, the rab11 and pIgR interaction appeared to be reinforced upon the same
stimulation (figure 4.2). Thus, we next examined the distribution of rab11 and
rab3D in LGAC, and the possible movement of these two rab proteins upon CCH
stimulation between the membrane and cytosolic fractions. After incubation with
CCH for 0 min and 30 min, LGAC were rapidly homogenized, and membrane
pellets and cytosolic supernatants were separated by subcellular fractionation,
then analyzed for rab11 and rab3D contents by Western blots to determine their
distributions.
As shown in figure 4.3, in the resting state, rab11 and rab3D could be
found either associated with membrane (M
0
), or in the cytosol (C
0
), and most of
the rab11 and rab3D is associated with the membrane fraction (M
0
). After CCH
treatment, the total amount of rab11 or rab3D (membrane-associated plus
cytosolic, i.e. M+C) appears to increase. In addition, comparing with the steady
state (M
0
, C
0
), there is an increased redistribution of rab11 and rab3D from
membrane to cytosol (M
30
, C
30
).

4. Rab11 and pIgR from lacrimal gland acinar cell lysates bind to rab3D
in pull-down assays
We have reported that rab3D interacts directly with pIgR, and rab3D
regulates pIgR trafficking through regulated secretory vesicles by LGAC in a
stimulation-dependent manner [Zhang W, et al., 2005]. Immunofluorescence and
co-immunoprecipitation studies suggest that rab11 may interact with the pIgR and

– rab3D
– rab11
M
0
C
0
M
30
C
30
Control
CCH (100 μM)
28 kDa -
24 kDa -

Figure 4.3 CCH induces a movement of rab11 and rab3D from membrane to cytosol. 100
μM CCH-stimulated (0 min and 30 min) LGAC were homogenized and centrifuged at 9,200
х g for 10 min at 4 °C. Following a subsequent centrifugation of the supernatant at 112,817
х g for 30 min at 4 °C, the resulting membrane pellets (M
0,
M
30
) were resuspended to the
same volume as concentrated cytosolic supernatants (C
0,
C
30
). Then samples were analyzed
by Western blot with primary anti-rab3D and anti-rab11 antibodies, and secondary goat
anti-rabbit IRDye800- and goat anti-mouse IRDye800-conjugated antibodies. LGAC lysate
was used as a blotting control in the far right lane. Results shown are representative of at
least three independent experiments.

regulate pIgR trafficking via the constitutive secretory pathway. In addition, the
quantification of the colocalization between the signals from two rab proteins
showed that about 60% of the rab11 was co-localized with rab3D, and vice versa
(data not shown). These observations led us to explore whether there is cross-talk
between rab11 and rab3D in the regulation of pIgR trafficking, that is, what is the
relationship between rab11 involved in the transcytotic pathway and rab3D-
dependent merocrine pathway in regulating pIgR trafficking.
Attempts at co-immunoprecipitation of rab11 and rab3D were not as
successful as pull-down assays. Thus, pull-down assays with recombinant (His)
6
-
tagged wild-type rab3D (rab3DWT) were applied. Different amounts (40 μg, 80
85
μg) of recombinant rab3D protein were used in pull-down assays with lysates
from resting (untreated) LGAC. Rab3DWT could successfully pull down rab11
and pIgR from these lysates, indicating that rab3D interacts with rab11 and pIgR,
separately or as a complex (figure 4.4). In addition, the amounts of rab11 and
pIgR pulled down with rab3DWT were proportional to the amount of rab3DWT
protein added (figure 4.4). To some extent, these data support our hypothesis that
rab11 and rab3D may cross-talk in the modulation of pIgR trafficking.

– rab3D
– rab11
WT 40 μg
WT 80 μg
Beads alone
Control
– pIgR
150 kDa –
100 kDa –
37 kDa –
25 kDa –
25 kDa –
20 kDa –

Figure 4.4 Recombinant rab3DWT pull-down of rab11 and pIgR. WT rab3D expressed as
(His)
6
-tagged proteins in E. coli were purified on Ni
2+
-nitriloacetate (NTA) beads, and 40µg
or 80µg of recombinant rab3D were used in pull-down assays with lysates from resting
LGAC. After rab3D was recovered from lysates by incubation with Ni-NTA beads, rab3D
and any interacting proteins were eluted from the beads with SDS-PAGE sample buffer and
analyzed by Western blot using primary anti-SC, anti-rab3D and anti-rab11 antibodies, and
secondary donkey anti-sheep IRDye700-, goat anti-rabbit IRDye800-, and goat anti-mouse
IRDye800-conjugated antibodies, respectively. LGAC lystate was used as a blotting control
in the far right lane. Results shown are representative of at least three independent
experiments.

86
87
5. Interaction between rab11-rab3D, and pIgR-rab3D in pull-down
assays is dependent upon GDP/GTP bound state of rab3D
To examine how the guanine nucleotide binding state of rab3D affects
rab3D-rab11 and rab3D-pIgR interactions, the binding of rab11 or pIgR to
rab3DWT or mutated rab3D proteins was tested in pull-down assays. Pull-down
of rab11 and pIgR from resting LGAC lysates with either recombinant WT or
mutant rab3D proteins demonstrated that rab3DWT and a putative dominant-
negative, GDP-locked rab3D mutant T36N, bind to rab11 and pIgR (figure 4.5).
Interestingly, however, the constitutively-active rab3DQ81L, a GTP-locked
rab3D mutant, does not bind to rab11 nor pIgR (figure 4.5). Furthermore, as
shown in figure 4.5, when 10 μM GTP γS or 0.5 mM GTP is included in the pull-
down assays with rab3DWT, binding of rab11 or pIgR to rab3D are significantly
inhibited. These data suggest that rab11 and pIgR can interact with rab3D when
rab3D is in the GDP-bound form. The GTP-bound form of rab3D, whether
induced by a mutation or by addition of GTP or GTP γS, appears to be incapable
of interacting with rab11 and pIgR. These results further confirm our speculation
that rab11 and rab3D may co-regulate pIgR trafficking via communication
between different secretory pathways.

E. Discussion
Rab11 exists in both nonpolarized and polarized cells. In nonpolarized
cells, including fibroblasts [Ullrich et al., 1996] and erythroleukemia cells [Green

WT+GTP γS
Beads alone
T36N
Q81L
WT+GTP
37 kDa –
25 kDa –
– rab3D
20 kDa –
25 kDa –
– rab11
WT
Control
150 kDa –
100 kDa –
– pIgR

Figure 4.5 Recombinant wild-type and mutant rab3D pull-down of rab11 and pIgR. WT,
mutant constitutively-active Q81L and dominant-negative T36N rab3D expressed as (His)
6
-
tagged proteins in E. coli were purified and 40 µg of each used in pull-down assays with
lysates from resting LGAC. In some cases, the lysate was supplemented with 10 μM GTP γS
or 0.5 mM GTP. Western blot utilized primary anti-SC, anti-rab3D and anti-rab11
antibodies, and secondary donkey anti-sheep IRDye700-, goat anti-rabbit IRDye800-, and
goat anti-mouse IRDye800-conjugated antibodies, respectively. LGAC lysate was used as a
blotting control in the far right lane. Results shown are representative of at least three
independent experiments.

et al., 1997], rab11 has been localized to the recycling endosome and trans-Golgi
network [Ullrich et al., 1996; Green et al., 1997; Ren et al., 1998]. In polarized
epithelial cells, rab11a was observed in the subapical region of a number of cell
types [Goldenring et al., 1996]. In polarized LGAC, rab11 can be detected
beneath the APM from our immunofluoresecence image (figure 4.1, red). The
apical recycling endosome (ARE) is a major station in the transcytotic pathway
[Apodaca, Katz et al., 1994]. In transfected MDCK cells, electron microscopic
88
89
analysis indicated that the ARE, to which rab11 predominantly localizes, is
enriched in the transcytotic marker, pIgR [Gibson et al., 1998; Hoekstra et al.,
2004]. Thus, the significant co-localization of rab11 and pIgR close to the apical
membrane in LGAC (figure 4.1, yellow) is consistent with that observed in other
cell systems.
The pIgR is an apical recycling system cargo in transfected MDCK cells
[Hobdy-Henderson et al., 2003]. Investigations over the past decade have
established the important role of rab11a in the regulation of cargo processing
through plasma membrane recycling systems in both polarized and nonpolarized
cells [Wang et al., 2000; Ullrich et al., 1996]. In polarized MDCK cells, rab11a
is present within the apical recycling system, regulating both transcytosis of pIgR
from the basolateral plasma membrane to the apical plasma membrane as well as
apical recycling [Wang et al., 2000; Apodaca, Katz et al., 1994]. However, to
date, there is no evidence for the direct interaction between rab11 and pIgR. Co-
immunoprecipitation of rab11 and pIgR (figure 4.2) not only demonstrates an
association between rab11 and pIgR, but also suggests that rab11 is involved in
pIgR trafficking in LGAC. To our knowledge, this is the first report of an
interaction between rab11 and pIgR in any cell type.
Subcellular fractionation demonstrates that rab11 and rab3D have similar
membrane and cytosolic distributions. Both of them are mainly in the membrane-
associated fraction rather than the cytosolic fraction (figure 4.3), which is in
agreement with distribution of rab11 in transfected-MDCK cells [Henderson-
90
Hobdy et al., 2003] and rab3D in rat pancreatic acinar cells [Chen et al., 2003],
respectively. Since CCH has apparent opposite effects on rab11-pIgR
(reinforcement, figure 4.2) and rab3D-pIgR (disruption) interactions [Zhang W, et
al., 2005], the influences of secretagogues on rab11 and rab3D distribution were
examined. CCH appears to cause a consistent increase in the total amount of
rab11 and rab3D, as well as an increased redistribution of rab11 and rab3D from
membrane to cytosol. The redistribution of rab3D and other rab proteins has been
tested in other secretory cells. For example, in isolated nerve terminals following
stimulation of neurotransmitter release, synaptic vesicle-associated rab3A and
rab3C were observed to redistribute from membrane to cytosol [Fischer von
Mollard et al., 1991, 1994]. In rat adipocytes, insulin treatment resulted in a 50%
decrease in rab4 content, concomitantly with a redistribution of rab4 from
membrane to cytosol, and its movement was reversed by insulin withdrawal,
whereas rab3B, rab3C and rab8 distributions were not affected [Cormont et al.,
1993]. In carbachol-treated pancreatic acini, an increase in immunoreactivity of a
rab3-like protein on the Golgi complex was detected by immunogold electron
microscopy, but cytosolic levels of the rab3-like protein were not altered [Jena et
al., 1994]. In rat parotid acini, isoproterenol induced redistribution of rab3D from
the cytosol to the membrane [Raffaniello et al, 1999]. It is apparent that the
nature of rab redistribution varies with respect to different rab proteins present in
different cells. The molecular mechanisms involved with rab11 and rab3D
redistribution in LGAC are presently unclear. According to this study,
91
redistribution of rab11 and rab3D may involve changing their interaction with
their cargo proteins (e. g. pIgR), and/or effector proteins in the modulation of
pIgR trafficking.
The detection of rab11-pIgR interaction and of similar distribution
properties of rab11 and rab3D in LGAC led us to investigate the relationship
between rab11 and rab3D in regulating pIgR trafficking. Rab11a was initially
characterized as a 24-kDa GTP-binding protein from bovine brain membranes
[Kikuchi et al., 1988]. The molecular weight of native rab3D from rat pancreas
zymogen granule membranes is 28-kDa [Ohnishi et al., 1996]. In figure 4.4, the
band positions of rab11 and rab3D are consistent with those results,
demonstrating that rab11 and rab3D are separated distinctly by SDS-PAGE and
do not overlap with each other. Rab11 and rab3D can thereby be specifically
recognized by their respective antibodies. In addition, rab11 and pIgR bind to
rab3D in a linear range.
Many studies of rab protein function have been facilitated by the use of
dominant- negative and constitutively-active mutants, which are deficient in GTP
binding or GTPase activity, respectively. Like all GTPases of the ras family,
rab11 cycles through GTP- and GDP-bound forms, and this cycle is related to its
function. We therefore applied (His)
6
epitope-tagged forms of wild-type rab3D
(rab3DWT), the constitutively-active Q81L (rab3DQ81L) mutant, and the
dominant-negative T36N (rab3DT36N) mutant proteins in pull-down assays. We
found that both rab11 and pIgR have similar binding characteristics in binding to
92
rab3D (figure 4.5). Rab3D binds to rab11 and pIgR when rab3D is in the GDP-
bound form. The GTP-bound form of rab3D, whether induced by a mutation or
by addition of GTP or GTP γS, appears to be incapable of interacting with either
rab11 or pIgR, suggesting that rab11 and rab3D co-regulate pIgR trafficking in
LGAC. Several independent reports support this possibility. In rat heptocytes,
pIgR and putative trafficking regulators, including rab3D, rab11a and syntaxin2
were found in a cellular fraction enriched in transcytotic compartments,
implicating these proteins in the regulation of basolateral-to-apical transcytosis
[Larkin et al., 2003]. Jin et al., found that multiple small GTP-binding proteins
were present on transcytotic vesicles in rat liver, including rab1a, rab1b, rab2,
rab6, and rab17 [Jin et al., 1996].
Rab11a functions differently in nonpolarized and polarized cells. Studies
in nonpolarized cells have established that rab11a is a marker of the plasma
membrane recycling system involved in trafficking of the transferrin receptor
[Ullrich et al., 1996; Green et al., 1997; Ren et al., 1998]. In polarized MDCK
cells both rab11a and rab25 are associated with the apical recycling endosome and
the trafficking of polymeric IgA, but not transferrin [Casanove et al., 1999; Wang
et al., 2000; Leung et al., 2000]. Unlike the rab11 isoforms which serve general
roles in transcytosis and endocytosis in most cells, rab3D is specific to secretory
cells. Rab3 isoforms are expressed in cells with regulated secretory pathways
[Larkin et al., 2000]. Rab3D, a regulated secretory vesicle marker, has been
found to interact directly with the pIgR and to regulate pIgR trafficking through a
93
regulated merocrine pathway in LGAC [Zhang et al., 2005]. In this study, we
provide evidence for rab11-pIgR, rab3D-pIgR, and rab11-rab3D interactions,
indicating that rab11 and rab3D may cross-talk in regulating pIgR trafficking in a
stimulation dependent manner by cholinergic agonists in LGAC. We therefore
propose a model of the two major pathways involved in the regulation of pIgR
trafficking in LGAC, a constitutive transcytotic pathway and a regulated
merocrine pathway (figure 4.6). For the constitutive transcytotic pathway (figure
4.6, red arrows), rab11 is proposed to be a major regulator of pIgR trafficking at
the apical endosome (AE), similar to its modulatory roles in transcytosis as
observed served in other cell types. For the regulated merocrine pathway (figure
4.6, blue arrows), rab3D is proposed to be the major regulator of formation of
secretory vesicles (SV) from the trans-Golgi network (TGN) and /or the fusion of
SV with the apical plasma membrane. Acute treatment of CCH may disrupt the
interaction of rab3D and pIgR, leading to the rapid initial phase of SC release in
regulated merocrine pathway. In contrast, CCH stimulation will accelerate pIgR
transcytosis, resulting in pIgR accumulating in AE to reinforce its binding to
rab11 in the constitutive transcytotic pathway. Significant communication may
exist between two pathways (figure 4.6, dashed green arrow) to regulate pIgR
trafficking in LGAC.
Our data have suggested an interaction between rab3D- and rab11-
dependent trafficking pathways in the regulation of pIgR trafficking in LGAC. It
is possible that each transport step requires one major rab. The interactions
among multiple rabs may control the specificity and directionality of vesicular
transport. The important question to be addressed in future studies is how rab11
and rab3D function together to regulate pIgR trafficking in LGAC.

94

pIgR
dIgA
SC
sIgA
AE
SV
TGN
BE
Apical
Basolateral
rab3D
ER
rab3D
rab3D
+
rab11

Figure 4.6 Two major pathways involved in the regulation of pIgR trafficking in LGAC are
the constitutive transcytotic pathway (red arrow) and regulated merocrine pathway (blue
arrows). For the constitutive transcytotic pathway, rab11 is proposed to be a major
regulator of pIgR trafficking at the apical endosome (AE). For the regulated merocrine
pathway, rab3D is proposed to be the major regulator of formation of secretory vesicles (SV)
from the trans-Golgi network (TGN) and /or the fusion of SV with the APM. Significant
cross-talk may exist between the two pathways (dashed green arrow).
rab11 ?
95
CHAPTER V – CONCLUSIONS AND FUTURE DIRECTIONS
A. Conclusions
In conclusion, we developed two pIgR-related models in ocular tissues.
One is a preliminary demonstration of pIgR in conjunctival epithelial cells that
may lead to a model for the role of the conjunctiva in ocular mucosal immunity.
The other one is to characterize the molecular mechanisms of regulated merocrine
secretion and constitutive transcytotic traffic of pIgR in lacrimal gland acinar cells.
To our knowledge, we are the first to identify pIgR in the superficial layer
of multilayered rabbit conjunctival epithelial cells, suggesting an important role of
the conjunctiva in ocular mucosal immune protection via the pIgR. Therefore, the
potential of utilizing endogenously expressed pIgR in conjunctiva for prevention
of ocular surface-related diseases can be explored, and a pIgR-based model of
ocular mucosal immunity can be developed.
In lacrimal gland acinar cells, we provide evidence for rab3D-pIgR, rab11-
pIgR, and rab3D-rab11 interactions, indicating that rab11 and rab3D may cross-
talk in regulating pIgR trafficking in a stimulation dependent manner by
cholinergic agonists in LGAC. We therefore propose a model of the two major
pathways involved in the regulation of pIgR trafficking in LGAC, a constitutive
transcytotic pathway and a regulated merocrine secretory pathway (figure 4.6).
For the constitutive transcytotic pathway (figure 4.6, red arrows), rab11 is
proposed to be a major regulator of pIgR trafficking at the apical endosome (AE),
consistent with its modulatory roles in transcytosis and endocytosis in most other
96
polarized cell types. For the regulated merocrine pathway (figure 4.6, blue
arrows), rab3D is proposed to be the major regulator of formation of secretory
vesicles (SV) from the trans-Golgi network (TGN), sorting pIgR to accumulate in
SV, and fusion of SV with the apical plasma membrane to release SC upon
stimulation. Acute treatment of CCH may disrupt the interaction of rab3D and
pIgR, leading to the rapid initial phase of SC release by the regulated merocrine
pathway. In contrast, CCH stimulation will accelerate pIgR transcytosis, resulting
in pIgR accumulating in AE to reinforce its binding to rab11 in the constitutive
transcytotic pathway. Significant communication may exist between two
pathways (figure 4.6, dashed green arrow) to regulate pIgR trafficking in LGAC.
Therefore, the regulated merocrine pathway of pIgR transport is an
innovative model that challenges existing dogma of production of free SC in tears
solely by a constitutive transcytotic pathway in the lacrimal gland. Although the
principles of vesicle-mediated trafficking via rab protein regulation are unclear to
date, our analysis suggests a novel mechanism of rab3D-regulated secretion of
pIgR transport. Since the association of rab3D and pIgR may be independent of
cell system, which has been tested in both LGAC and MDCK cells (figure 3.3 &
3.4), our findings are very likely to yield new information regarding an important
physiologic process and may become a paradigm for the study of other secreted
proteins in human lacrimal gland and other epithelial cells.

97
B. Future directions
Our study has identified pIgR in RCEC and implicated specific rab
proteins in the regulation of pIgR trafficking in LGAC. It is possible that similar
rab proteins are present in RCEC to regulate pIgR trafficking, and each transport
step requires one major rab. The interactions among multiple rabs may control
the specificity and directionality of vesicular transport. The important questions
to be addressed in future studies are whether such rab proteins exist in RCEC,
how rab3D works to recruit pIgR to the merocrine pathway, how rab11 and rab3D
function together to regulate pIgR trafficking both in RCEC and in LGAC, and
whether pIgR is essential for maintenance of ocular surface integrity and
immunity.
1. To determine whether rab proteins exist in RCEC
Since the association of rab3D and pIgR may be independent of cell type,
which has been tested in both LGAC and MDCK cells (figure 3.3 & 3.4), it is
very likely this important physiologic process presents in other epithelial cells,
such as RCEC. We will perform the corresponding experiments in RCEC as we
did in LGAC to examine whether similar rabs, including rab3D and rab11, exist
in RCEC to regulate pIgR trafficking.

2. To determine whether rab11 directly interact with pIgR or rab3D
Co-immunoprecipitation in figure 4.2 shows that rab11 interacts with pIgR.
Pull-down assays in figure 4.4 and 4.5 implicates that rab11 associates with rab3D.
98
However, the co-immunoprecipitation and pull-down assays do not distinguish
whether rab11-pIgR interaction or rab11-rab3D association is a direct or indirect
one. We will perform overlay assays using recombinant rab11 or rab3D proteins
to test whether rab11-pIgR or rab11-rab3D interact directly. Therefore, how
rab11 and rab3D function together to regulate pIgR trafficking in LGAC can be
explored.

3. To determine the region of pIgR that binds to rab3D or rab11
The site of pIgR required for binding to rab3D or rab11 may control its
specificity and directionality of vesicular transport. Figure 3.5 suggests that
rab3D directly interacts with the cytoplasmic and/or membrane spanning domain
of pIgR. Figure 4.2 demonstrates that rab11 is associated with pIgR. If we can
also prove that rab11 and pIgR interact directly, then we will map and
characterize the region of pIgR responsible for binding to rab3D or rab11 utilizing
several well-characterized MDCK cell lines stably transfected with WT or mutant
pIgR as a source of rabbit pIgR. Using a similar approach in transfected MDCK
cells [van IJzendoorn et al., 2003], van IJzendoorn et al., mapped the site of pIgR
required for binding to rab3B to a membrane-proximal 14 amino acids residue of
its cytosolic domain that has been previously shown to be essential for dIgA-
stimulated transcytosis of the pIgR [Luton, Mostov et al., 1999].

99
4. To determine whether dIgA regulates rab3D-pIgR or rab11-pIgR or
rab3D-rab11 interaction
The pIgR transcytoses dIgA from the basolateral to the apical plasma
membrane in epithelial cells. Transcytosis of pIgR is stimulated upon its binding
to dIgA [Luton, Mostov et al., 1999]. We will examine whether dIgA affects the
interaction of rab3D-pIgR, rab11-pIgR or rab11-rab3D, GTP bound state of
rab3D/rab11, and subsequent pIgR trafficking in regulated merocrine pathway
and constitutive transcytotic pathway. In transfected MDCK cells, van
IJzendoorn et al., reported that binding of dIgA to pIgR abolishes the interaction
between GTP-bound rab3B and pIgR [van IJzendoorn et al., 2003].

5. To determine whether rab11 is involved in both constitutive
transcytotic pathway and the regulated merocrine pathway
Figure 4.4 and 4.5 implies that rab11 and rab3D regulate pIgR trafficking
in the constitutive transcytotic pathway and the regulated merocrine pathway,
respectively. Other investigators have found that rab11 and rab3 proteins were
colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on
mature synaptic vesicles in brain [Khvotchev et al., 2003]. Urbe et al., identified
that rab11 is associated with the trans-Golgi network (TGN) and with TGN-
derived vesicles of both the constitutive and the regulated secretory pathway in
PC12 cells [Urbe et al., 1993]. Khvotchev et al., provided evidence that rab11b
participates in Ca
2+
-regulated exocytosis of dense core and synaptic vesicles and
100
integrates regulated and constitutive exocytosis in neurons and neuroendocrine
cells [Khvotchev et al., 2003]. We will perform pulse-chase, subcellular
fractionation and immunofluorescence experiments to study rab11’s traffic
between different membrane compartments upon stimulation, and therefore to
explore whether rab11 is also involved in the regulated merocrine pathway in
LGAC and in RCEC.

6. To determine how pIgR expressed in different ocular tissues including
conjunctiva and lacrimal gland is correlated to the protection of
ocular surface from pathogen infection
It has been proposed that SC and sIgA are essential components of tears,
while their alterations are correlated with a number of ocular surface disorders
[Lan et al., 1998; Lan et al., 1999; Leher et al., 1998; Masinick et al., 1997]. We
will use pIgR-knockout mouse to test whether pIgR is important for the formation
and maintenance of SV to sequester correct cargo proteins, and for the alteration
of tear protein composition. We will also investigate how loss of SC and sIgA
influences the susceptibility of ocular surface to bacterial pathogens using
adenovirus conjunctivitis and Pseudomonas aeruginosa corneal keratitis model.
We therefore can explore whether pIgR is essential for maintenance of
conjunctiva and lacrimal gland functions, and ocular surface integrity and
immunity.
101
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Abstract (if available)

Abstract Conjunctiva and lacrimal gland are two major ocular epithelial tissues included in the mucosal immunity system. The polymeric immunoglobulin receptor (pIgR) is expressed in a wide variety of secretory epithelial cells. The pIgR is the precursor to secretory component (SC) and secretory IgA (sIgA). It regulates secretion of SC and sIgA through its transcytosis across epithelial cells. The pIgR therefore plays an important role in mucosal immunity. To explore its role in ocular surface immunity, we sought to identify and characterize pIgR in ocular tissues.

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Asset Metadata

Creator Zhang, Wenzheng (author)

Core Title The polymeric immunoglobulin receptor in ocular tissues

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 10/12/2008

Defense Date 08/24/2006

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag conjunctival epithelium,lacrimal gland,OAI-PMH Harvest,polymeric immunoglobulin receptor,rab11,Rab3D,secretory component

Language English

Advisor Okamoto, Curtis T. (committee chair), Kim, Kwang-Jin (committee member), Mircheff, Austin K. (committee member), Shen, Wei-Chiang (committee member), Trousdale, Melvin D. (committee member)

Creator Email zhangsunshine@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m92

Unique identifier UC1164303

Identifier etd-Zhang-20061012 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-19062 (legacy record id),usctheses-m92 (legacy record id)

Legacy Identifier etd-Zhang-20061012.pdf

Dmrecord 19062

Document Type Dissertation

Rights Zhang, Wenzheng

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu