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Effects of Rab3D deficiency on Paneth cell morphology and function

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Effects of Rab3D deficiency on Paneth cell morphology and function

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EFFECTS OF RAB3D DEFICIENCY ON PANETH CELL MORPHOLOGY AND
FUNCTION

BY

RAJALAKSHMI SUBRAMANI VENKATACHALAM

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
AUGUST 2013

COPYRIGHT 2013 RAJALAKSHMI SUBRAMANI VENKATACHALAM
ii

Acknowledgements
First and foremost, I would like to express my deepest gratitude to Dr. André J.
Ouellette for giving me an opportunity to work with him and for being such a
wonderful mentor. Without his constant support, guidance and enthusiasm, this
work wouldn't have been possible.
I would also like to express my appreciation to Dr. William Depaulo and Dr. Keigo
Machida for having served on my committee. Their thoughtful questions and
comments were valued greatly.
Secondly, I would like to take this opportunity to thank Dr. Ouellette and Dr.
Selsted lab members for providing technical assistance. In particular Dr. Paulina
Schmit, Dr. Dat Tran, Dr. Prasad Tongaonkar, Dr. Jennifer R. Mastroianni, Lili
Chen, Patti Tran, Justin Schall, Kevin Roberts and Katie Trihn for their constant
technical support and guidance all through my project.
Also, I would like to extend my gratitude to Dr Sarah F. Hamm-Alvarez and Dr.
Maria C. Edman-Woolcott for providing resources and being a wonderful
collaborators.
Last but not the least, I would like to thank my family for their constant support
and encouragement.

iii

Table of Contents
Acknowledgements ii
List of Figures iv
Abbreviations vi
Abstract viii
1. Introduction
1.1. Small Intestine - Structure and Function 1
1.2. Paneth Cell Development and Function 1
1.3. Paneth Cell α-defensin Processing 3
1.4. Microbicidal Activity of Paneth Cell Defensins 6
1.5. Defensins Modulate The Microbiota of The Gut 8
1.6. Paneth Cell Dysregulation 11
1.7. Rab3D - Structure and Function 13
2. Experimental Procedures
2.1. Animals 16
2.2. RNA Extraction and cDNA Synthesis 18
2.3. Quantitative RT-PCR 19
2.4. Protein Extraction 20
2.5. Protein Quantification 21
2.6. Acid-Urea Polyacrylamide Gel Electrophoresis 21
2.7. Cryptdin-1 Sandwich ELISA 22
2.8. Hematoxylin and Eosin Staining 23
2.9. Electron Microscopy 24

3. Results 25
4. Discussion 36
Bibiliography 40

iv

List of Figures
Figure 1: Parts of Small Intestine. 2
Figure 2: Luminal surface modifications of the small intestine. 4
Figure 3: Intestinal stem cell in small intestinal crypt development. 5
Figure 4: Electron microscopy of Paneth cell granule. 7
Figure 5: α-defensin processing by MMP-7. 9
Figure 6: Bactericidal activity of defensins. 10
Figure 7: Schematic representation of microbial composition. 11
Figure 8: Rab3D amino acid sequence. 14
Figure 9: Immunohistochemical analysis of Rab3D localization 15
in parotid acinar cells.
Figure 10: Pull down assay. 16
Figure 11: Gene targeting strategy for the rabphilin knockout. 17
Figure 12: Paneth cell mRNA expression levels in Rab3D WT and 28
KO mouse small intestine.
Figure 13: Paneth Cell mRNA expression levels in Rab3D WT and 29
KO mouse small intestine
v

.
Figure 14: Analysis of α-defensin peptides from mouse small 30
intestine by AU-PAGE

Figure 15: Cryptdin-1 Sandwich ELISA 32
Figure 16: H&E staining of Rab3D WT and KO small intesine 34
Figure 17: Electron microscopy of Paneth cell granules 35

vi

Abbreviations
ISC Intestinal Stem Cells
TA Transit Amplifying Cells
BMP Bone Morphogenetic Protein
Fz5 Frizzled Receptor 5
HHIP Hedgehog Interacting Protein
MMP-7 Matrix Metalloproteinase-7
AU-PAGE Acid-Urea Polyacrylamide Gel
Electrophoresis
HD-5 Human Defensin - 5
TG Transgenic
IBD Inflammatory Bowel Disease
UC Ulcerative Colitis
AGR2 Anterior Gradient 2
ER Endoplasmic Reticulum
CDR Complementary-determining region
vii

RabF Rab Family Motif
RabSF Rab SubFamily Motif
pIgR Polymeric Immunoglobulin Receptor
dIgA Dimeric IgA
Crp-1 Cryptdin-1
ACN Acetonitrile
H&E Hematoxylin and Eosin Staining
EM Electron Microscopy
TFA Trifluoroacetic acid

viii

Abstract
Paneth cells are located at the base of the small intestinal crypts and secrete
diverse range of antimicrobial peptides and proteins. They play a key role in
providing innate mucosal immunity and thereby maintain the homeostasis of the
gut. The Paneth cells secrete electron dense granules by sensing bacteria and
by cholinergic stimulation. Various defects leading to imbalances in the
homeostasis of the Paneth cell are associated with inflammatory bowel disease
and inflammation of the gastrointestinal tract. Rab3D, a member of the Ras
superfamily is involved in regulating exocytosis and secretion in varied secretory
glands. Rab3D is associated with the release of IgA from the lacrimal gland by
binding to pIgR, mediates amylase secretion from Parotid gland and regulates
exocytosis in Pancreatic acinar cells. However, its role in the secretory Paneth
cell has not yet been studied. In this paper, the α-defensin mRNA levels were
shown to be reduced in Rab3D deficient mice in initial studies, suggesting that
Rab3D deficiency is associated with the functioning of Paneth cells. However,
analyses of numerous individual knockout and wild-type mice showed extensive
variability between individual knockouts with close agreement among wild-type
mice. Also, morphological analyses showed defects in the Paneth cells within
crypts and dense core granule abnormalities, signifying the association of Rab3D
with granulogenesis.
ix

Further, AU-PAGE analysis of small intestinal Rab3D WT and KO protein
samples demonstrated unaltered protein levels. Cryptdin-1 ELISA results
provided more accurate results showing unaltered Crp-1 levels in WT and
Rab3D-null mice. These findings, taken together, suggest that Rab3D deficiency
affects the Paneth cell secretory pathway and the unaltered protein levels may
be associated with defects in Paneth cell homeostasis
1

1. Introduction
1.1. Small Intestine - Structure and Function
Small intestine is a part of the lower gastrointestinal tract involved in digestion of
food and absorption of nutrients. The small intestine is divided into three regions:
duodenum, jejunum and ileum (Figure 1). The duodenum starts after the pylorus
of the stomach and leads to the jejunum followed by the ileum. The ileocecal
junction separates the small intestine from the large intestine. The surface area
of the small intestinal lumen is enlarged by the formation of villi and microvilli
(Figure 2). The villus is surrounded by many crypts of Lieberkühn which house
the Paneth cells, surface absorptive cells, goblet cells and regenerative stem
cells.
1.2. Paneth Cell Development and Function
Paneth cells represent one of the four principal cell types of the small intestinal
epithelium. They provide mucosal immunity against pathogens and maintain gut
homeostasis by secreting dense core granules which are rich in various
antimicrobial peptides and proteins, including lysozyme and cryptdins (Figure 4)
(Ouellette 2010). Paneth cells reside adjacent to the intestinal stem cells (ISC)
located at the base of the crypts of Lieberkühn. These ISCs renew slowly and
give rise to transit amplifying (TA) cells. The TA cells migrate upwards towards
the villus and differentiate into absorptive cells or secretory cells while the Paneth
2

cells move to the bottom of the crypts (Figure 3) (Umar 2010). Three signaling
pathways define and localize the intestinal stem-cell niche - hedgehog, bone
morphogenetic protein (BMP), and the Wnt and Eph/ephrin pathways (Crosnier,
Stamataki et al. 2006). The differentiation of Paneth cells requires continuous
activation of Wnt signaling pathway through Frizzled receptor 5 (Fz5). In the
absence of Wnt signaling, Paneth cells are mispositioned and no longer
restricted to the base of the crypts (van Es, Jay et al. 2005), while hedgehog
signaling is involved in the formation of villi is restricted to the epithelium and its
inhibition by hedgehog-interacting protein (HHIP) causes absence of villi
completely (Crosnier, Stamataki et al. 2006).

Figure 1: Parts of small intestine. Red oval boxes indicates the three different
regions of the small intestine - duodenum, jejunum and ileum. The small intestine
leads to the large intestine - cecum and colon. The ileocecal junction separates
the ileum from the ascending colon.
3

1.3. Paneth Cell α-defensin Processing
Defensins are cysteine rich cationic peptides with characteristic β-sheet fold.
They posses antimicrobial, antiviral and immunomodulatory properties. The
mammalian defensins are classified into three different subfamilies namely α, β
and θ defensins (Ouellette and Selsted 1996). In humans, there are 6 different α-
defensins characterized by disulfide bonds between Cys
I
-Cys
VI
, Cys
II
-Cys
IV
, and
Cys
III
-Cys
V
. Human α-defensins 1-4 are expressed in bone marrow with the
peptides accumulating in neutrophils, while α-defensins 5 and 6 are expressed in
the Paneth cells located at the base of the crypts of the small intestine (Selsted
and Ouellette 2005). Mice are the only known mammals that lack neutrophil α-
defensins. They contain 5-7 abundant Paneth cell α-defensins. Mice contain
many additional, α-defensin mRNAs whose peptide levels have not yet been
measured. β-defensins are widely distributed on all mucosa and are produced by
the leukocytes in cattle and many different epithelial cells like the tongue, skin,
salivary glands. The third class of defensins known as θ-defensins is structurally
different from the other two defensins and has been found in the leukocytes of
rhesus macaques. They are the only macrocyclic proteins found in the animal
kingdom.
4

Figure 2 (Ganz 2000): Luminal surface modifications of the small intestine.
The lumen of the small intestine has numerous finger like projections called the
villi which increase the surface area of absorption. The lacteal is the lymphatic
channel. Muscularis mucosae causes shortening of the villus during digestion by
rhythmic contraction of the muscle fibers. Several crypts of Lieberkühn surround
the villus and house the Paneth cell.
In humans, the small intestinal Paneth cells secrete α-defensins as propeptides
that are processed luminally by trypsin. In mouse, activation of α-defensins
occurs intracellularly by matrix metalloproteinase-7 (MMP7). In the absence of
MMP-7, the α-defensins are secreted as propeptides and lack bactericidal
activity. The 9 acidic amino acids located near the N-terminal of the inactive
propeptides balances the positive charge of the arginine residues at the carboxy
terminal moiety, giving procryptdins a net neutral charge. This makes them
inactive against all bacteria. MMP-7 converts inactive propeptides to active
peptides. MMP-7 cleaves the propeptides at amino acid residue positions 44, 54
and 59 (Figure 5) (Wilson, Ouellette et al. 1999). MMP-7 processing of
propeptides activates the bactericidal activity of the cryptdins (crypt defensins).
5

Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) analysis of small
intestinal protein extracts from WT mice were shown to convert inactive
procryptdin to active cryptdin peptide, while the MMP7 KO mice were unable to
process the inactive procryptdin to mature peptides (Wilson, Ouellette et al.
1999). Studies have shown cleavage of procryptdins between Ser
43
and Ile
44
by
MMP-7 is sufficient to activate bactericidal peptide activity (Weeks, Tanabe et al.
2006). Processing of procryptdins by MMP-7 is independent of bacterial
exposure (Ayabe, Satchell et al. 2002).

Figure 3 (Umar 2010): ISC in small intestinal crypt development. (a) The
cartoon depicts small intestinal crypt. The ISCs reside at the bottom of the crypt,
either between the Paneth cells at +1 position called as crypt base columnar cells
(CBCs) (red) or above the Paneth cells at +4 position (brown).
6

The TA cells (yellow) arise from self-renewing CBCs. Goblet cells and
enterocytes are labeled purple and green, respectively. (b) Enlarged view of
small intestinal crypt showing different stem cell regions (+1 to +4 positions).
BMP signaling which inhibits proliferation has highest activity throughout the
villus and less activity within the crypt whereas the Wnt signaling stimulates
proliferation has highest activity at the base of the crypt and lowest as it
approaches the crypt-villus junction.
1.4. Microbicidal activity of Paneth Cell Defensins
Paneth cell α-defensins are microbicidal against gram positive and gram negative
bacteria, fungi, spirochetes, protozoa and viruses (Ganz 2003). The bactericidal
activity of defensins is mediated by membrane disruption. The human neutrophil
defensins develops a contact between cationic arginine groups and the
negatively charged head group of the target membrane phospholipids, creating
an electromotive force which pulls the hydrophobic part of the defensin dimer into
the membrane. Several dimers then come in contact with each other forming a
channel through the membrane leading to membrane disruption (Martin, Ganz et
al. 1995).
7

Figure 4 (Ouellette 2010): Diverse Paneth cell granule Constituents. Paneth
cell α-defensins have been labeled with antibody and detected using gold-
conjugated secondary antibody. Various Paneth cell secretions are listed to the
right of the EM.
All other α-defensins are known to cause transient defects in the membrane but
does not form stable pores. The mechanism of membrane disruption varies
among peptides. Some defensins kill bacteria by inhibiting bacterial cell wall
synthesis while the human neutrophil α-defensins mediate killing by non-
oxidative permeabilization of the membrane following phagolysosomal fusion
(Ouellette 2011). By disrupting bacterial membranes, defensins act as direct
effectors of innate immunity.
In the small intestine, cholinergic stimulation and bacterial antigen exposure
induces secretion of α-defensins at a concentration of 15-100 mg/ml per crypt
(Ayabe, Satchell et al. 2000). Paneth cells secrete abundant α-defensins
including human defensin-5 (HD-5).
8

In vitro studies have been performed to analyze the bactericidal activity of HD-5,
and its activity in vivo has been confirmed in vivo in mouse models. To perform in
vivo analysis of HD-5, transgenic mice expressing HD-5 was developed using a
2.9-kilobase HD-5 minigene containing two HD-5 exons and 1.4 kilobase of 5'
flanking sequence. The WT mice suffered illness, ruffled hair and diarrhea while
the TG mice expressing HD-5 resist oral challenge with virulent Salmonella
typhimurium [Figure 6]. The WT mice had 100% mortality rate in contrast to TG
HD5 mice which were immune to oral infection (Salzman, Ghosh et al. 2003).
Thus, the presence or absence of one α-defensin in the Paneth cell modulates
the bactericidal activity of Paneth cell secretions.

9

Figure 5 (Wilson, Ouellette et al. 1999): α-defensin processing by MMP-7.
(a) Complete sequence of procryptdin-4 peptide. The red arrows indicate the
cleavage site of MMP-7 at amino acid sequence 44, 54 and 59 of the
procryptdin-4 peptide. The 9 acid amino acids marked in red at the N-terminal
balance the cationic Arginine residues at the carboxy terminal. (b) AU-PAGE
analysis of α-defensin processing in MMP7 WT and KO mice. The two lanes in
the middle denotes α-defensins expression due to the presence of MMP7 and
the left most lane shows absence of defensins due to the lack of MMP-7.
Synthetic crp4 served as a control.
1.5. Defensins Modulate The Microbiota of The Gut
The gastrointestinal tract is colonized by a dynamic population of microbiota
spanning the length of the intestine. The composition of the microbial community
evolves throughout the individual's lifetime and is host specific. The presence or
absence of key species is important in maintaining the homeostasis of the gut.
The gut microbiota is predominantly composed of facultative anaerobes and
aerobes.
10

There are over 50 bacterial phyla described to date (Schloss and Handelsman
2004). The microbiota of the gut is dominated by Bacteroidetes and the
Firmicutes, while Proteobacteria, Actinobacteria and Cyanobacteria are present
at lower abundance (Figure 7) (Eckburg, Bik et al. 2005).

Figure 6 (Salzman, Ghosh et al. 2003): Bactericidal activity of defensins (a)
Analysis of HD5 mRNA in 35 day old mice by In situ Hybridization Analysis. TG
Defa5+/+ mice expresses HD5 while WT Defa5-/- does not express HD5 but has
other cryptdins. Black stain in TG Defa5+/+ mice shows the localization of HD5 at
the base of the crypt. (b) Survival curve showing TG mice expressing HD5 are
resistant to oral Salmonella typhimurim (n = 6). Two days after innoculation, WT
mice showed 100% mortality and the KO mice were able to recover from illness
and had no mortality.
The mucosal surface of the intestinal epithelium provides innate immunity by
secreting antimicrobial peptides (Selsted and Ouellette 2005). Paneth cell α-
defensins have effective bactericidal activity against bacterial pathogens. The
presence of a single new α-defensin (HD-5) provides immunity against orally
administrated Salmonella typhimurium (Ayabe, Satchell et al. 2000). The
composition of the small intestinal microbiota is by Paneth cell α-defensins.
11

The microbiota of transgenic HD-5 mice had 25.5% of Firmicutes when
compared 59% FvB controls. In contrast, the percentage of Bacteroidetes in TG
HD-5 mice were 69.3% but FvB controls had only 35% (Ayabe, Satchell et al.
2000). Thus, Paneth cell α-defensins are key regulators in maintaining the
microbiota of the gut.

Figure 7 (Eckburg, Bik et al. 2005). Schematic representation of microbial
composition. (a) Composition and number of microbiota varies across the
gastrointestinal tract. (b) Variation in microbial composition across the length of
the intestine.
1.6. Paneth cell dysregulation
Paneth cells are key contributors to innate mucosal immunity in the small
intestine, defects in Paneth cell homeostasis have shown to be associated with
various diseases such as gastrointestinal inflammation and inflammatory bowel
disease (IBD). IBD is an disease which causes chronic inflammation of the
intestine. It is of two types - Crohn's Disease (CD) and Ulcerative Colitis (UC).
12

Patients with CD are shown to have reduced levels of Paneth cell α-defensins
(Zhao, Edwards et al. 2010). Deficiency in Paneth cell α-defensins causes
reduced antimicrobial activity and has shown to change the levels of luminal
microbiota which has been studied using transgenic mice expressing HD-5. Mice
lacking MMP-7 have compromised immunity against oral infection due to the lack
of functional α-defensins as they are unable to process inactive procryptdins
(Wilson, Ouellette et al. 1999). Paneth cell defects leading to increased ER
stress and autophagy is found to be associated with IBD. Drastic expansion of
Paneth cell compartment is seen in mice lacking Anterior Gradient 2 (AGR2), a
protein disulfide isomerase which plays an important role as a developmental
regulator and survival factor for IBD, followed by loss of goblet cells,
inflammatory infiltration, increases endoplasmic reticulum (ER) stress and
apoptosis (Zhao, Edwards et al. 2010). Similarly, In mice hypomorphic for
autophagy gene Atg16l1, Paneth cells have been observed to be defective in
secretion (Cadwell, Liu et al. 2008). Paneth cell also provides essential survival
signals for the maintenance of the stem cell niche. For example, culture lacking
Lgr5+ stem cells or Paneth cells developed 0% organoids while in the
combination of both Paneth cells and Lgr5+ stem cells, the percentage of
organoids formed were 76.7 ± 8.8% (Sato, van Es et al. 2011). These studies
suggest that the dysregulation of Paneth cells is involved in the pathogenesis of
enteric diseases. The molecular mechanisms leading to these effects are yet to
be characterized in detail.
13

1.7. Rab3D - Structure and Function
Rab3D is a member of Ras protein superfamily, which are involved in exocytosis
and found to be abundant in cells with regulated secretory pathways. These Rab
GTPases (Rabs) are important regulators of membrane trafficking and constitute
the largest Ras superfamily with more than 50 mammalian and 11 yeast proteins
identified so far (Pereira-Leal and Seabra 2000). Members of the homologous
Rab3 subfamily are restricted to cells with regulated secretory function. Of the
Rab3 family, Rab3D was initially isolated from mouse adipocytes and is located
on chromosome 13 region A2-3 in humans. The structural gene of Rab3D is
located in exon 2 through 5 with a GTP-binding motif in each exon. Rab3D
contains functional domains, which include the effector region, the conserved
switch I and II domains, the Rab complementarity-determining region (CDR) and
the Cys-Ser-Cys prenylation motif. The effector region determines the functional
specificity of different GTPases while the variable RabCDR enables Rab proteins
to interact with various downstream effectors. Rab proteins must undergo post-
translation hydrophobic isoprenylation for membrane binding. In addition to the
functional domain, Rab3D contains three other motifs, a Rab family motif (RabF),
Rab subfamily motif (RabSF) and a canonical g-motif (Figure 8) (Millar, Pavios et
al. 2002). Rab3D has found to be localized in various secretory glands where it is
involved in regulating exocytosis and secretion. In parotid acinar cells, Rab3D
translocates from the cytosol to the membrane fraction (Nguyen, Jones et al.
2003).
14

Immunohistochemical analysis suggests the localization of Rab3D to parotid
acinar cells and its direct association with amylase secretion (Figure 9). qRT-
PCR analysis demonstrates the localization of Rab3D in pancreatic acinar cells
and western blot analysis has shown that Rab3D is associated with the zymogen
granules of the pancreatic acinar cells (Ohnishi, Ernst et al. 1996).

Figure 8 (Millar, Pavios et al. 2002). Rab3D amino acid sequence. Blue amino
acids denote the switch regions. The red amino acids denote the three CDRs
and the black box denotes the effector region. The Rab family sequence are
highlighted in yellow and the underlined sequence denotes the Rab subfamily
motifs. [Adapted from (Ohnishi, Ernst et al. 1996)]
The role of Rab3D in the lacrimal gland has been analyzed by studying its
interaction with Polymeric Immunoglobulin Receptor (pIgR) (Livak and
Schmittgen 2001). pIgR is a transmembrane domain receptor containing a ligand
binding extracellular domain. Binding of pIgR to dimeric IgA (dIgA) happens at
the basolateral surface, and the complex is proteolytically cleaved at the apical
surface leading to the apical release of secretory IgA, which contributes to
defense against pathogens at the mucosal surface.

15

Pull down assays suggested that Rab3D is involved in the transcytosis of pIgR
and the amount of pIgR pull down is directly proportional to the concentration of
Rab3D (Figure 10) (Livak and Schmittgen 2001). Thus, Rab3D is implicated in
exocytosis and secretion in diverse glands. Because Paneth cells have a highly
developed regulated secretory pathway, the Rab3D literature provided rationale
for examining the effect of Rab3D deficiency on the morphology and function of
Paneth cells.

Figure 9 (Nguyen, Jones et al. 2003). Immunohistochemical Analysis of
Rab3D localization in Parotid acinar cells. Immunohistochemical staining for
Rab3D (Red) and Amylase (green) were done on streptolysin O-permeabilized
acini. A, B and C denotes cells incubated with Phosphate Buffer Saline (PBS). D,
E and F denotes cells incubated with cyclic adenosine monophosphate (cAMP)
and calcium (Ca
2
+). C and F denotes the merged view showing Rab3D
association with amylase.
16

Figure 10 (Evans, Zhang et al. 2008). Pull down assay. pIgR interacts with
Rab3D in a concentration dependent manner. Lane A, B and C denotes pull
down assay with 40 μg, 80 μg and 0 μg of Rab3D. Screened for Rab3D and
pIgR. The amount of pIgR pull down is directly proportional to the concentration
of Rab3D present.
2. Experimental Procedures
2.1. Animals
Rab3D knockout mice were provided in collaboration with Dr. Sarah F. Hamm-
Alvarez, Department of Pharmacology and Pharmaceutical Sciences, University
of Southern California, Los Angeles, CA. Rab3D knockout mice were generated
originally by homologous recombination in embryonic E14.1 stem cells derived
from the mouse line 129Sv/J as described [(Riedel, Antonin et al. 2002),
(Schluter, Schnell et al. 1999)]. Gene targeting strategy was used to replace the
first exon of the Rab3D with a neomycin resistance cassette (Figure 1). Mice
were maintained, genotyped, and provided to us by Ms. Frances Yarber of the
Hamm-Alvarez lab.
17

Isolation of small intestinal tissue from Rab3D knockout mice were performed in
accordance with the policies for the use of animals for research and with
approval from the USC IACUC Protocol #11372. Rab3D KO mice originally
generated in 129Sv/J were bred onto the BL6 background. The small intestine
was removed from C57BL/6 and Rab3D knockout mice and flushed with
phosphate saline buffer to remove luminal contents, the whole organ was
preserved by immersion in in RNAlater, and stored at -20 ºC for further analysis.

Figure 11: Gene targeting strategy for the rabphilin knockout. Homologous
recombination replaces genomic sequences containing two exons encoding
residues 74- 146 with the neomycin gene. Black boxes indicate exons with their
corresponding residue markers. Gene cassettes for positive and negative
selection were Neomycin resistance gene and Thymidine Kinase gene marked
by open boxes. Arrows indicates positions of oligonucleotide primers for PCR
genotyping. (Adapted from Schluter et al. - Figure 2)

18

2.2. RNA Extraction and cDNA Synthesis.
The small intestine tissue samples from Rab3D WT and KO mice were
transferred from RNAlater to liquid nitrogen and pulverized into frozen tissue
powder using mortar and pestle. RNA was isolated using TRIzol reagent
(Ambion, Life Technologies) from 100 mg of powdered tissue according to
manufacturer protocol. Briefly, 100 mg of powdered tissue was incubated in 1 ml
of TRIzol reagent for 2 hours and homogenized using glass homogenizer.
Following homogenization, the solution was centrifuged at 12,000 x g for 10
minutes at 4 ˚C. The supernatant was transferred to a fresh eppendorf tube and
incubated at room temperature for 5 minutes to allow complete dissociation of
nucleoprotein complex. Following homogenization, the solution was incubated at
room temperature for 5 minutes with 200 µl of chloroform and centrifuged at
12,000 x g for 10 minutes at 4 ˚C.
The aqueous phase was then transferred to a fresh tube. RNA was precipitated
using 500 µl of 100 % isopropanol, incubated at room temperature for 10 minutes
followed by centrifugation at 12,000 x g for 10 minutes at 4 C. The resulting
RNA pellet was then washed with 75 % ethanol and centrifuged at 7500 x g for 5
minutes at 4 ˚C. The wash was discarded and pellet was vacuum dried. The
pellet was resuspended in 20 µl of RNase free water and incubated at 55 - 60 ˚C
for 10 minutes and stored at - 80 ˚C. Extracted RNA was treated with DNAse 1
(Invitrogen, Life Technologies) for 15 minutes at room temperature.
19

A second precipitation was performed with 0.3 M sodium acetate to eliminate
DNAse and degraded DNA. Isolated total RNA was quantified by ultraviolet
absorbance at 260nm using a Nanodrop 2000 spectrophotometer (Thermo
Scientific), and DNAse treated RNA was stored at -80 ⁰C. cDNA was generated
for each sample using 0.5 μl of SMART MMLV Reverse Transcriptase (Clontech)
using 0.5 μg oligo-(dT)12-18, 1 mM dNTPs, 10mM DTT and 1μg of total RNA in a
final reaction volume of 20 μl. cDNA was used immediately for qRT-PCR or
stored at -20 ⁰C.
2.3. Quantitative RT-PCR
Real-time PCR was performed for mouse α-defensins, lyzozyme and EF-1α
using a Roche light cycler 480 located at the Stem Cell Core of USC. Real-time
PCR was performed with tissue specific cDNA as a template to analyze the gene
expression of Paneth cell specific mRNA levels. Primers used for measuring the
levels of mRNA were: DEFA5 forward primer – 5’ GCT CAA CAA TTC TCC AGG
TGA CCC 3’ and reverse primer – 5’ AGC AGA CCC TTC TTG GCC TC 3’;
DEFA22 forward primer – 5’ GGC TGT GTC TGT CTC CTT TGG AG 3’ and
reverse primer – 5’ CAG CAT CAG TGG CCT CAG AG 3’; Pancrp forward primer
– 5’ AAG AGA CTA AAA CTG AGG AGC AGC 3’ and reverse primer – 5’ GGT
GAT CAT CAG ACC CCA GCA TCA GT 3’; lyzozyme 1 forward primer – 5’ AGC
CGA TAC TGG TGT AAT GAT GGC A 3’ and reverse primer – 5’ CCA TGC
CAC CCA TGC TCG AAT 3’; EF-1α forward primer – 5’ CTG CAT CCT ACC
20

ACC AAC TCG 3’ and reverse primer - 5’ TGA CTG GAG CAA AGG TGA CCA
3’. PCR was performed in triplicates using 1μl of 1:5 dilution of cDNA, 2.5 μl of
SYBR green master mix (Roche) and 5 μM of final primer concentrations. The
PCR conditions were (i) 50 ⁰C for 5 min (pre-incubation 1 cycle); (ii) 95 ⁰C for 10
sec, 62 ⁰C for 20 sec, 72 ⁰C for 20 sec (amplification 40 cycles); (iii) 95 ⁰C for 5
sec, 65 ⁰C for 1 min (melting curve 1 cycle) and (iv) 40 ⁰C for 30 sec (cooling 1
cycle). The relative changes in gene expression were determined using 2
-ΔΔC
T

method by the formula as described (Livak and Schmittgen 2001). Statistical test
to study the variance between two groups was analyzed by performing two-tailed
student t-test using Graphpad prism 5 software.
2.4. Protein Extraction
Small intestine tissues from Rab3D WT and KO mice were isolated and washed
with ice cold water to remove luminal contents. The tissue samples were
homogenized using Polytron homogenizer in 100 ml of ice-cold 60% acetonitrile
and 1% trifluoroacetic acid and incubated overnight at 4 ⁰C. Next morning, the
tissue samples were centrifuged at 15,000 x g for 60 min using a Sorvall RC-26
superspeed centrifuge. The supernatant was collected and concentrated using
speed vacuum concentrator to evaporate acetonitrile. The samples were shell
frozen and lyophilized. Lyophilized samples were then dissolved in 5 ml of 1%
acetic acid and stored at -20 ⁰ C.
21

2.5. Protein Quantitation
The fraction of total extracted protein was calculated determined by Bradford
Assay according to manufacturer protocol (Thermo-Scientific). Briefly, to
generate standard curve, Bovine Serum Albumin dilutions were prepared in 1%
acetic acid at the following concentrations: 750 μg/ml, 500 μg/ml, 250 μg/ml, 125
μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml and 0 μg/ml. Five μl of each protein
standard or unknown protein samples in 1:2 dilutions were added to appropriate
microtiter wells. 250 μl of Coomassie Reagent was added to each well and
incubated at room temperature for 10 minutes. Absorbance was measured at
595 nm using a plate reader. A Standard curve was generated by plotting
average blank subtracted absorbance vs. concentration. The Protein
concentrations of unknown samples were determined by extrapolation from using
the standard curve.
2.6. Acid-Urea Polyacrylamide Gel Electrophoresis (AU-PAGE)
AU-PAGE gels were prepared and pre-run as described (Figueredo, Mastroianni
et al. 2010). Two ml of lyophilized peptide samples dissolved in 5 ml of 1% acetic
acid were purified using Sep-Pak C18 reversed-phase HPLC cartridge (Waters)
using 5% and 80% acetonitrile. The 80% Acetonitrile (ACN) eluted samples were
concentrated using speed vacuum concentrator to evaporate acetonitrile. The
samples were lyophilized.
22

The lyophilized samples were dissolved in 15 μl of 1X sample loading buffer
(Figueredo, Mastroianni et al. 2010) and electrophoresed in 12.5% acid-urea
polyacrylamide gels for 5 hours at 250 V. The gel was viewed by staining with
Coomassie Blue R 250 for 1 hour and destained overnight in destaining solution
(74% water, 25% methanol, 1% formaldehyde) (Figueredo, Mastroianni et al.
2010). α-defensins were identified by their comigration with synthetic Crp4 which
served as control marker. Levels of α-defensins were determined by sandwich
ELISA.
2.7. Cryptdin-1 Sandwich ELISA
Cryptdin-1 (Crp-1) levels were quantified by sandwich ELISA in collaboration with
Dr. Tokiyoshi Ayabe, Hokkaido University, Sapporo, Japan. Lyophilized protein
samples of 20 µg each were analyzed for Crp-1 levels. Briefly, 10 different
monoclonal antibodies were developed against Crp-1 in rats. The antibody pair
was selected by performing sandwich ELISA for Crp-1 using all 10 antibodies
which served as both capture antibody and as biotinylated detection antibody.
Lyophilized samples were dissolved in 50 µl of Milli-Q water. Microtiter plates
were coated overnight with capture antibody (77-R5: Anti-Crp1) at 1 µg/ml at 4
ºC. The plates were then washed thrice with PBS and blocked with 200 µl of
Block Ace (DS Pharma Biomedical Co. Suita, Japan) for 2 hours. 100 µl of Crp1
or unknown protein samples were added and incubated for 2 hours at 25 ºC.
23

After incubation, the plates were washed with PBS and 100 µl biotinylated
detection antibody (77-R20: Anti-Crp1) was added at a concentration of 0.5 µg/ml
for 1 hour at 25 ºC. The wells were then incubated with 100 µl of Streptavidin-
HRP conjugate (GE Health Care Biosciences, Piscataway, NJ, USA) for 1 hour
at a dilution of 1:5000. The wells were washed and incubated for 30 min at 25 ºC
with 100 µl of TMB chromogenic substrate buffer. The reaction was stopped by
addition of 100 µl of 0.6 N sulphuric acid and absorbance was measured at 450
nm (absorption wavelength) and 620 nm (detection wavelength) using microtiter
plate reader (Multiscan FC, Thermo Scientific, Waltham, MA, USA). Standard
curve was generated using serial dilutions of Crp-1 in the range of 0.004 - 4
ng/ml. Crp-1 concentration was determined using the standard curve.
2.8. Hematoxylin and Eosin Staining
Freshly isolated small intestinal tissue were fixed in 4% paraformaldehyde
solution, dehydrated in ethanol and embedded in paraffin for sectioning. Sections
were prepared and hematoxylin and eosin (H&E) staining was performed by the
Pathology Core at USC using standard protocols. Briefly, the sections were
deparaffinized in xylene twice for 10 minutes each followed by rehydration using
absolute alcohol, 95% alcohol and 70% alcohol for 2-5 minutes. The tissues were
stained with Hematoxylin and Eosin stain for 5 minutes each with brief washing
between staining procedures and viewed under light microscopy.

24

2.9. Electron Microscopy
Electron microscopy of Paneth cell granules was performed in collaboration with
Dr. Susan J Hagen, Department of Surgery, Beth Israel Deaconess Medical
Center, Boston, Massachusetts. The electron microscopy was performed as
described previously (Ganz 2000). In short, the small intestinal tissues from
Rab3D WT and KO mice were fixed with 2% formaldehyde and 0.1%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in Unicryl at -
20º C. Thin sections were cut and counterstained with uranyl acetate and lead
citrate. The sections were viewed and photographed in JEOL 100 CX electron
microscope.

25

3. RESULTS
3.1. Rab3D Deficiency is Associated with Dysregulation in Enteric Paneth
Cell mRNA Levels
qRT-PCR results suggests that the deficiency of Rab3D is associated with the
reduction in the mRNA expression levels of α-defensins and lysozyme in the
Paneth cells. Paneth cells are known to secrete high levels of α-defensins (also
known as cryptdins in mice), lysozyme, phospholipase C and other enzymes.
Defensins have effective microbicidal action against bacteria [(Ouellette and
Selsted 1996), (Ganz 2003), (Ayabe, Satchell et al. 2000)] while the other Paneth
cell secretion have no evidence of microbicidal activity in vivo. Studies have
shown that Rab3D is involved in exocytosis and secretion in diverse secretory
glands such as the salivary gland, pancreas and lacrimal gland [(Nguyen, Jones
et al. 2003), (Ohnishi, Ernst et al. 1996)]. To study the effect of Rab3D deficiency
on the expression of secreted Paneth cell gene products, we examined the
enteric Paneth cell mRNA levels of lysozyme, defensin 5, defensin 22 and
pancryptdin by quantitative real time PCR. The experiment was performed by
optimizing the efficiency of primers using serial dilutions of cDNA (1, 0.5, 0.25,
0.125 and 0.062). EF-1α served as a control due to its stable expression in WT
and KO Rab3D small intestinal tissue. The qRT-PCR was performed using cDNA
in the dilution of 1:5 using Roche Light cycler 480 and analyzed by 2
-ΔΔC
T
method.
26

The reduced mRNA levels of Paneth cell gene products could possibly be due to
reduction in the number of Paneth cells in the KO compared to the WT small
intestine. To obtain more reliable statistical measurements, we performed
student t-test for various Paneth cell gene products and found that there is
extensive variation in defensin mRNA levels between the Rab3D KO's while the
WT showed mRNA levels in close agreement. Thus, the phenotype of the
Rab3D KO is highly variable between individuals, so variable that the KO group
variability prevents statistically valid comparisons with the wild type mice. Also,
possible defects in the granulogenesis caused by Rab3D deletion could also
influence the observed variability in Paneth cell mRNA levels.
3.2. α-Defensin Peptide Levels in Rab3D WT and KO Small Intestine
To further investigate if the reduction in mRNA levels of Paneth cell α-defensins
due to Rab3D deficiency affects the protein levels of α-defensins, we analyzed α-
defensin peptides in AU-PAGE. The levels of α-defensins increases from the
proximal to the distal small intestine in concert with higher numbers of Paneth
cells, with the highest levels of α-defensins being present at the distal end ileum.
AU-PAGE resolves proteins on the basis of both charge to mass ratio. In AU-
PAGE, the proteins are solubilized by urea and since defensins are more
compact than the other proteins migrate faster and gets separated.

27

Protein extracts from mouse Rab3D WT and KO small intestine were analyzed
for α-defensins following desalting using Sep-Pak C-18 reversed-phase HPLC
columns. In reversed-phase HPLC columns, molecules are separated based on
their hydrophobic character. Hydrophobic alkyl chain acts as the stationary phase
which interacts with the analyte. Length of the alkyl chain generally used for
peptides or small molecules are C-18. This is due to the fact that larger protein
molecule will likely have more hydrophobic moieties to interact with the column
and thus a shorter chain length is more appropriate. Since defensins are small
hydrophobic peptides, on passing through C-18 column, the hydrophobic moiety
binds to the column and gets eluted upon passing organic solvent. AU-PAGE is a
non-reducing denaturing gel system in which molecules migrate as fully
protonated forms. Since defensins are highly cationic and compact, they have
the highest mobility in AU-PAGE and get well resolved, while the non-defensin
proteins appear as smear at the top of the gel. Purified and lyophilized protein
extracts resolved in AU-PAGE showed similar peptide levels of α- defensins
(Figure 2). AU-PAGE analysis of protein extract suggests equivalent levels of
proteins in Rab3D WT and KO. Due to a technical difficulty, there was ambiguity
in the levels of defensins between the WT and KO Rab3D samples. In order to
determine the levels of defensin peptide levels more accurately, we performed
Crp-1 sandwich ELISA on Rab3D samples.
28

Figure 12: Paneth cell mRNA expression levels in Rab3D WT and KO
mouse small intestine. Quantitative real-time PCR analysis of mRNA
expression levels encoding mouse Paneth cell Defa5, Defa22, Pancrp, Lyz and
Rab3D was performed on RNA samples isolated from small intestine of Rab3D
KO and wild-type C57BL/6 mice ( N= 2 for WT and N= 4 for KO). EF-1α serves
as the control. Analysis shows 50% reduction in defensins expression and 30%
reduction in Lyzozyme expression levels in Rab3D KO relative to Rab3D WT.
29

Figure 13: Paneth cell mRNA expression levels in Rab3D WT and KO
mouse small intestine. Quantitative real-time PCR analysis of mRNA
expression levels encoding mouse Paneth cell Defa5, Defa22, Pancrp, Lyz and
Rab3D were performed on RNA samples isolated from small intestine of Rab3D
KO and wild-type C57BL/6 mice ( N= 7 for WT and KO). EF-1α serves as the
control. Analysis shows heterogeneous variability of mRNA levels between the
KO's while the WT showed close agreement in mRNA levels.
30

Figure 14: Analysis of α- defensin peptides from mouse small intestine by
AU-PAGE. Proteins extracted from mouse small intestine of Rab3D KO (1and 2)
and WT (3 and 4) mice were separated by AU-PAGE and analyzed for α-
defensin peptide after purifying by C-18 reversed-phase HPLC columns. The
gels were stained with Coomassie Blue stain. The boxed region indicated the
position of α-defensins. The α-defensin peptide levels were similar between WT
and KO Rab3D. Crp-4 loaded at lane 5 served as a positive control.
3.3. Quantification of Cryptdin-1 by Sandwich ELISA
The Crp-1 sandwich ELISA shows unaltered Crp-1 concentrations between the
WT and KO Rab3D small intestine. The Crp-1 sandwich ELISA was developed
by raising 10 different monoclonal antibodies against Crp-1 in rat, and was
developed by Dr. Tokiyoshi Ayabe. Optimization of mAb was done by performing
sandwich ELISA. For this purpose, 10 different antibody recognizing different
epitope was well established against Cryptdin-1 in rats.
31

Each of these 10 antibodies which recognizes different epitopes were arrayed as
both capture and detection antibodies and the antibody pair with the greatest
specificity and sensitivity was selected. Proteins were extracted from small
intestinal tissue of Rab3D WT and KO mice using 60% Acetonitrile (ACN) and
1% trifluoroacetic acid (TFA) from 3 mice in each group. The lyophilized protein
was dissolved in 1% acetic acid and the total protein concentration was
measured by Bradford's assay. 20 µg of protein extract was lyophilized and
measured for the amount of cryptdin-1 peptides by cryptdin-1 sandwich ELISA (
Dr. Tokiyoshi Ayabe, Hokkaido University, Japan) (Figure 3). The ELISA
determinations represent the first accurate measurements of Crp-1 in intestinal
protein extracts. ELISA was performed on six Rab3D protein extracts from
Rab3D WT and KO mice (WT n = 3 and KO n =3). The measurements were
done twice and the levels of Crp-1 were similar in both replicate experiments. We
speculate that the unaltered Crp-1 is associated with the defect in the mRNA
secretory pathway which may be associated due to the deletion of Rab3D.
32

Figure 15: Cryptdin-1 Sandwich ELISA. 20 µg of lyophilized protein extract
from Rab3D KO (n = 3) and Rab3D WT (n = 3) mice was dissolved in 50 µl of
Milli-Q water and the levels of cryptdin-1 was analyzed by sandwich ELISA using
capture antibody (77-R5: Anti-Crp1) and detection antibody (77-R20: Anti-Crp1)
by Dr. Tokiyoshi Ayabe, Hokkaido University, Japan. The concentration of
cryptdin-1 in WT and KO mice were found to be 272.13 ng/ml and 280.23 ng/ml
showing unaltered cryptdin-1 levels between WT and KO Rab3D small intestine.
The standard curve for cryptdin-1 measurement was generated using synthetic
Crp-1.

3.4. Rab3D Small Intestine Morphology
To examine the effects of Rab3D on Paneth cell morphology, we analyzed the
crypt and granule formation by H&E staining and electron microscopy. The small
intestinal epithelium has numerous finger like projections called the villi and
between the intervilli junction are numerous crypts called the Crypts of
Lieberkühn which house the Paneth cells.
33

The Paneth cells are located at the base of the crypts. The Paneth cells have
numerous dense core granules which are rich in antimicrobial peptides,
phospholipase C, lyzozyme and other enzymes. Normally, Paneth cells are well
formed and are packed with dense core granules. Dysregulation within Paneth
cells or genetic defects may lead to malfunction of Paneth cells, resulting dense
core granule deficiency, thereby affecting the mucosal immunity of the gut.
H&E staining involves the application of hemalum, a oxidation product of
haematoxylin and stains the nuclei blue in color based on the binding between
dye-metal complex to DNA. Eosin is used as a counter stain which stains
eosinophilic structures in red, pink or orange. The H&E staining revealed
distorted crypts. The dense core granules were fewer in KO compared to the WT
Rab3D, and the granule vesicles were enlarged and irregular. While the WT
tissues showed well-defined crypts and packed with dense core granules, KO
revealed distorted Paneth cell morphology. This was consistent with all the three
different KO subjected to H&E staining. The results suggests that Rab3D
deficiency is associated with the defect in the formation of Paneth cell
granulogenesis (Figure 4).The granule defects observed by H&E staining were
validated at high resolution by performing electron microscopy. The morphologic
defects of the Rab3D KO were more evident when examined by EM. EM
revealed KO containing granules with enlarged electro-lucent regions
surrounding them.
34

Some granules contained greater cytoplasmic area than the WT. We also found
inclusions in the KO Paneth cells, which may be due to induction of autophagy or
additional protein degradative mechanisms (Figure 5). The presence of electro-
lucent granules suggests that Rab3D deficiency affects the Paneth cell
morphology and granulogenesis.

Figure 16: H&E staining of Rab3D WT and KO small intesine (Pathology
Core at USC). A H&E staining of small intestinal tissue from WT Rab3D showing
well organized crypts and dense core granules. B, C and D are H&E staining of
Rab3D KO small intestine with poorly formed crypts and reduced granules. The
intensity of staining is less in KO compared to the WT Rab3D small intestinal
tissues. Arrows indicate improper crypt formation. Note A, B, C and D are small
intestinal tissues from individual Rab3D WT and KO mice.

A B
C D
35

Figure 17: Electron microscopy of Paneth cell granules. Thin sections of
Rab3D WT and KO small intestine was analyzed by EM using standard
procedures. A and C shows Rab3D WT small intestine with well formed electron
dense granules. C and D shows Rab3D deficient mice with less electron dense
granules and few electro-lucent granules indicating defects in the granulogenesis
caused due to Rab3D deficiency.

A
C
D
B
36

4. DISCUSSION
Paneth cells located in the small intestinal crypts of Leiberkühn provide mucosal
immunity against microbes by secreting granules rich in antimicrobial peptides
and proteins. Defects in the Paneth cell homeostasis leads to Inflammatory
bowel disease and gastrointestinal inflammation. In the present study, we
demonstrated that the deficiency of Rab3D causes changes in the morphology
and functioning of Paneth cells. H&E staining reveals poorly formed crypts and
dispersed Paneth cell granules. The EM of Rab3D KO small intestine reveals the
presence of numerous Paneth cell inclusion bodies which are absent in WT
Paneth cells. The presence of these inclusion bodies suggests that the induction
of autophagy here might be associated with Rab3D deficiency. Western blot
analysis for various autophagy markers could provide a test for this hypothesis
associated with Rab3D deficiency. Studies have shown the presence of Rab3D
in the cis-Golgi network, which is involved in the early stages of granulogenesis.
Based on these findings, we predict that the presence of electron-lucent granules
seen in Rab3D KO's could be associated with Rab3D deficiency and that Rab3D
is associated with granulogenesis. qRT-PCR analysis of ER stress markers such
as XBP 1, CHOP and others would provide insights into the role of Rab3D in
granulogenesis. Paneth cells constitute a smaller portion of the small intestine
which makes the analysis of ER stress difficult as the background levels of ER
stress from other normal cells in the small intestine would mask the ER stress
levels occurring in Paneth cells.
37

Also, the absence of Paneth cell line makes it difficult for analysis of ER stress
and autophagy specifically in Paneth cells. These problems could be overcome
by the recent development of enteroids which provide a system to assay for
Paneth cell ER stress if the effect of Rab3D deficiency on Paneth cell is a cell
autonomous effect or not. Future work would be the analysis of mRNA levels of
Paneth cell gene products from enteroids and to screen for ER stress and
autophagy in these enteroids. Therefore, analysis of Paneth cell function in
enteroids may provide a system for analyzing the effects of Rab3D deficiency on
Paneth cell function. The advantage of this system would be the ability to study
Paneth cell function in culture in the absence of circulatory mediators.
Gene expression analysis by qRT-PCR reveals extensive heterogeneity of
Paneth cell mRNA levels in Rab3D KO's compared relative to the control
samples. EM reveals the presence of intermediate cells with dense core granules
in Rab3D KO's while no intermediate cells were detected in BL6 control samples.
Intermediate cells are goblet cells with dense core granules and these granules
contain Paneth cell products. The heterogenous mRNA levels may be attributed
to the presence of variable levels of these intermediate cells among the Rab3D
KO cohort. qRT-PCR analysis of various enterocyte products would provide
evidence whether or not this difference in mRNA levels between the knockouts is
Paneth cell specific defect due to Rab3D deficiency. However, the mechanism by
which Rab3D gene deficiency alters the expression levels of α-defensin mRNA
levels is yet to be studied.
38

We speculate that the unaltered cryptdin-1 levels as reported from our ELISA
analysis is possibly caused due to the defects in mRNA secretory pathway.
These studies show that in the absence of Rab3D there is an obvious defects in
Paneth cell morphology specifically, in the morphology of Paneth cell secretory
granule.
Studies have shown the association of Rab3D in regulating exocytosis and
secretion of varied secretory glands. For example, In the lacrimal gland, Rab3D
mediates the transcytosis of dIgA in by its association with pIgR (Evans, Zhang
et al. 2008). It is found to be associated with the zymogen granules of the Parotid
acinar cells and also with Pancreatic acinar cells [(Nguyen, Jones et al. 2003),
(Ohnishi, Ernst et al. 1996)]. But the role of Rab3D and its association with
Paneth cell has not yet been studied so far. These findings provide insights for
better understanding of the Rab3D gene on Paneth cells.
Any change in the expression of Paneth cell α-defensin may have an impact on
enteric innate immunity. For example, transgenic mice expressing HD-5 are
resistant to oral infection by Salmonella while WT mice suffered severe illness
with 100% mortality (Salzman, Ghosh et al. 2003). Mutation in various proteins
indirectly affects the functioning of Paneth cells [(Wilson, Ouellette et al. 1999),
(Weeks, Tanabe et al. 2006), (Zhao, Edwards et al. 2010), (Cadwell, Liu et al.
2008)]. MMP-7 defective mice lack microbicidal activity due to the lack of
processed α-defensins (Weeks, Tanabe et al. 2006).
39

Similar studies could be performed on Rab3D deficient mice by oral Salmonella
infection and study how they respond to oral infection with Salmonella. Future
studies also could focus on the changes in the microbicidal properties of Paneth
cell secretion upon stimulation by carbamyl choline in Rab3D KO and WT small
intestine. Rab3D deficiency alters Paneth cell homeostasis in association with an
secretory pathway defect, which could induce changes in the composition of
microbiota by affecting delivery of -defensins to the small intestinal lumen. Also,
The effect of Paneth cell depletion in Rab3D KO and WT mice by injecting
dithizone could provide interesting insights about the role of Rab3D in Paneth
cells. For example, this strong chelating agent depletes Paneth cells of secretory
granules. Because Rab3D knockout implicates it in Paneth cell granulogenesis,
one result of Rab3D deficiency may be to reduce the ability of Paneth cells to
recover from induced granule depletion.

40

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mice." Dev Biol 338(2): 270-279.

Abstract (if available)

Abstract Paneth cells are located at the base of the small intestinal crypts and secrete diverse range of antimicrobial peptides and proteins. They play a key role in providing innate mucosal immunity and thereby maintain the homeostasis of the gut. The Paneth cells secrete electron dense granules by sensing bacteria and by cholinergic stimulation. Various defects leading to imbalances in the homeostasis of the Paneth cell are associated with inflammatory bowel disease and inflammation of the gastrointestinal tract. Rab3D, a member of the Ras superfamily is involved in regulating exocytosis and secretion in varied secretory glands. Rab3D is associated with the release of IgA from the lacrimal gland by binding to pIgR, mediates amylase secretion from Parotid gland and regulates exocytosis in Pancreatic acinar cells. However, its role in the secretory Paneth cell has not yet been studied. In this paper, the α-defensin mRNA levels were shown to be reduced in Rab3D deficient mice in initial studies, suggesting that Rab3D deficiency is associated with the functioning of Paneth cells. However, analyses of numerous individual knockout and wild-type mice showed extensive variability between individual knockouts with close agreement among wild-type mice. Also, morphological analyses showed defects in the Paneth cells within crypts and dense core granule abnormalities, signifying the association of Rab3D with granulogenesis.

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Asset Metadata

Creator Subramani Venkatachalam, Rajalakshmi (author)

Core Title Effects of Rab3D deficiency on Paneth cell morphology and function

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 07/30/2013

Defense Date 06/03/2013

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

Tag gut homeostasis,microbiota,OAI-PMH Harvest,Paneth cell,Pathology,Rab3D,small intestine

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ouellette, Andre J. (committee chair), DePaolo, R. William (committee member), Machida, Keigo (committee member)

Creator Email rajalaks@usc.edu,raji26venkat@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-307339

Unique identifier UC11294713

Identifier etd-SubramaniV-1887.pdf (filename),usctheses-c3-307339 (legacy record id)

Legacy Identifier etd-SubramaniV-1887-0.pdf

Dmrecord 307339

Document Type Thesis

Format application/pdf (imt)

Rights Subramani Venkatachalam, Rajalakshmi

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA