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University of Southern California Dissertations and Theses
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The role of CD1d transmembrane and cytoplasmic tail domain in CD1d trafficking pathway
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The role of CD1d transmembrane and cytoplasmic tail domain in CD1d trafficking pathway
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Content
THE ROLE OF CD1d TRANSMEMBRANE AND CYTOPLASMIC TAIL DOMAIN IN
CD1d TRAFFICKING PATHW AY
by
Yang Yang
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
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2011
Copyright 2011 Yang Yang
ii
Acknowledgements
First, I would like to appreciate my supervisor, Dr.Weiming Yuan, for giving me the
professional idea about the project and valuable technical suggestions during my master’s
thesis project. Secondly, I also would like to thank Dr. Zoltan Tokes and Dr. Tobias
Ulmer for being my committee members. Finally I want to appreciate my lab colleagues,
Dr. Ping Rao, Dr. Hong Pham Thanh, Arpita Kulkarni and my friends for helping me
overcome some experimental problems and give me some technical advices.
iii
Table of Contents
Acknowledgements ............................................................................................................. ii
List of Tables ......................................................................................................................iv
List of Figures ...................................................................................................................... v
Abbreviations .................................................................................................................... vii
Abstract ...............................................................................................................................ix
CHAPTER 1 INTRODUCTION ......................................................................................... 1
1.1 Introduction ........................................................................................................ 1
1.2 CD1 molecules structure .................................................................................... 2
1.3 Endocytic and recycling pathway of CD1 molecules ........................................ 4
1.4 Lipid antigen uptake and presentation ................................................................ 7
CHAPTER 2 MATERIALS AND EXPERIMENTAL METHODS ................................. 10
2.1 Cell lines ............................................................................................................... 10
2.2 Antibodies and Reagents ....................................................................................... 10
2.3 Construction of the CD1d mutant and chimera plasmid ....................................... 11
2.4 Transient transfection of CD1d mutant and chimera construct into HeLa cell ..... 13
2.5 Immunofluorescence analysis of CD1d mutant and chimera constructions ......... 14
2.6 Fluorescence activated-cell sorting (FACS) analysis of CD1d mutant and
chimera constructs ................................................................................................ 16
2.7 Western blot analysis of CD1d mutant and chimera constructs ............................ 17
CHAPTER 3 RESULTS .................................................................................................... 20
3.1The cytoplasmic tail of CD1d is critical for its surface expression ....................... 20
3.2 The transmembrane domain of CD1d determines its cell surface expression ...... 31
CHAPTER 4 DISCUSSION AND FUTURE WORK ...................................................... 37
4.1 Discussion ............................................................................................................. 37
4.2 Future direction ..................................................................................................... 40
REFERENCES .................................................................................................................. 42
iv
List of Tables
Table 1 PCR reaction setting…………………………………………...……...….11
Table 2 Restrict enzyme digestion reaction……………………………...…….….12
Table 3 Fixative solution and permeabilization solution……………...…...…...…15
Table 4 Cytoplasmic tail sequence comparison of CD1d mutants………………..20
Table 5 Transmembrane domain sequence comparison of CD1d wild type and
CD1d-CD4-CD1d……………………………………………......………..33
v
List of Figures
Figure 1 the structure of CD1 molecules…………………………………...….….…2
Figure 2 Top and side views of crystal structure of CD1 molecule……….…..…..…3
Figure 3 Overview of CD1 molecules intracellular trafficking……………………..6
Figure 4 Dot plot pattern of CD1d wild type and CD1d TD13…………….…..…..21
Figure 5 Cell surface expression level comparison of CD1d wild type and
CD1d TD13 mutant…………………………..………………………..…22
Figure 6 Cell surface expression level comparison of CD1d wild type and
CD1d TD14 mutant…………….……………………………...…….…...24
Figure 7 Expression level comparison of CD1d wild type and CD1d TD14
mutant……………………………………………………...……..…........24
Figure 8 Immunofluorescence staining of CD1d TD13 and CD1d TD14 mutants
by CD1d 51.1.3 antibody…………………………………………....……25
Figure 9 Immunofluorescence staining of CD1d TD14 by anti CD1d D5
antibody……………………………………………….….…..…...….......26
Figure 10 CD1d T322N mutant cell surface expression dot plot pattern and
comparison…………………………………………………..……........…27
Figure 11 CD1d T322D mutant cell surface expression dot plot pattern and
comparison……………………………………………………….…….…28
Figure 12 Immunofluorescence staining of CD1d T322N and CD1d T322D
mutants by CD1d 51.1.3 antibody…………..……………………………30
Figure 13 CD1d-CD4-CD1d fusion mutant cell surface expression dot plot
pattern and comparison……………………………………………….…32
Figure 14 Immunofluorescence staining of CD1d-CD4-CD1d by
anti CD1d 51.1.3 antibody………………………………………...…..…34
vi
Figure 15 Immunofluorescence staining of CD1d-CD4-CD1d by anti CD1d D5
antibody……………………………………………………………...….34
Figure 16 Western blot pattern of CD1d constructs………………...…...…….….35
vii
Abbreviations
APC Allophycocyanin
BCS Bovine Calf Serum
BSA Bovine Serum Albumin
CD Cluster of Differentiation
CD1d Cluster of Differentiation 1 (class d)
EDTA Ethylenediaminetetraacetic acid
ER Endoplasmic Reticulum
FACS Fluorescence Activated Cell Sorting
FBS Fetal Bovine Serum
FITC Fluorescein Isothiocyanate
GRP94 Glucose-Regulated Protein 94
HRP Horseradish Peroxidase
IAA Iodoacetic Acid
MHC Major Histocompatibility Complex
PBS Phosphate Buffered Saline
PMSF Phenylmethanesulfonylfluoride
PVDF Polyvinylidene Fluoride
SDS PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
viii
T cell Thymus cell
TBST Tris-Buffered Saline Tween-20
ix
Abstract
CD1 molecules are a family of MHC class I-like lipid presenting molecules. They could
specifically recognize self lipid antigens and extracellular lipid antigens, and present
them to T lymphocytes. T lymphocytes are stimulated to secrete different cytokines to
execute their immunological functions. CD1d, a member of CD1 family, exists both in
human and in mouse. It can particularly activate invariant Natural Killer T (NKT) cells
which are mostly-studied non-conventional T cell types. CD1d molecule is delivered onto
cell surface membrane after synthesis in Endoplasmic Reticulum (ER) and is believed to
be internalized via AP-2 dependent endocytic pathway. During the trafficking route,
CD1d recognizes and assembles with lipid antigens to form antigen-CD1d complex in the
early endosome, the complex can be delivered onto the cell membrane after its formation.
So the intracellular trafficking pathway of CD1d molecule is important for its
immunological function. Based on the previous studies, tyrosine-based endosomal
targeting sequence, YXXZ (where Y is tyrosine, X is any amino acid and Z is bulky
hydrophobic amino acid) and some other amino acids on the cytoplasmic tail of CD1d are
the major functional sites for its endocytosis process. The purpose of the thesis is to study
the functions of CD1d cytoplasmic tail and transmembrane domain on its intracellular
trafficking routes. The results show that the 14
th
amino acid from the bottom on the
cytoplasmic tail may be the important site to determine CD1d molecule cell surface
x
expression and the entire transmembrane domain is required for CD1d normal expression
in cells.
1
CHAPTER 1 INTRODUCTION
1.1 Introduction
In order to execute its physiological function, immune system needs to uptake antigens in
the host cells and to present them to the specific T lymphocytes which can be efficiently
activated to eliminate the infected cells. Presenting of antigens to the T lymphocytes is
the most important step in the whole immune response reaction (Sugita, Grant et al.
1999). Immune system has three major types of antigen presenting molecules: major
histocompatibility complex (MHC) class I, MHC class II and CD1 molecules. MHC class
I and class II are capable of presenting antigen peptides to the CD4
+
CD8
+
T cells to
develop CD8 T cell and CD4 T cell separately, while CD1 molecules mainly present lipid
antigens to the T cells (Godfrey, Pellicci et al. 2010; Liu, Shaji et al. 2010). In the
endogenous antigen presenting pathway, intracellular peptides that are generated within
the target cells primarily associate with MHC class I molecule in the endoplasmic
reticulum (ER) and then are assembled to form the antigen-MHC complex. The complex
is recognized by T cell receptors (TCRs) on the CD8
+
T cells and activates the T cells to
kill the virus-infected cells (Heemels and Ploegh 1995). In contrast, MHC class II
molecules bind to the internalized exogenously derived peptides in late endosome and
present the peptides to CD4
+
helper T cells which are able to generate immunoregulatory
cytokines and to stimulate inflammatory reactions (Cresswell 1994). CD1 molecules are
2
the other major type of antigen-presenting molecules mainly recognizing the lipids and
glycolipids (Beckman, Porcelli et al. 1994).
1.2 CD1 molecules structure
CD1 molecules are a family of the transmembrane glycoproteins (Brigl and Brenner
2004), which are located on the surface of most antigen-presenting cells like dendritic
cells. CD1 molecules, which resemble MHC class I molecules, bind to lipid, glycolipids
and lipopeptides, and then present these antigens to T lymphocytes and a unique
subpopulation of nature killer (NK) cells called NKT cells that possess TCRs on its
surface (Brutkiewicz 2006). All the CD1 molecules contain three domains: extracellular
domain, transmembrane domain and
cytoplasmic tail domain. The
extracellular domain can be further
divided into three sub-domains located
on a transmembrane heavy peptide
chain: α1, α2 and α3. The α3 domain
can nonconvalently interact with
β
2
-microglobulins ( β
2
m) to form a
heterodimer in endoplasmic reticulum
(ER) after translation (Sugita, Porcelli
et al. 1997; Huttinger, Staffler et al.
Figure 1: the structure of CD1 molecules
(Gumperz 2006)
3
1999). Figure 1 shows the structure of CD1 molecules and the CD1 isoforms (Gumperz
2006). Figure 2 shows the crystal structure of CD1 molecule.
Five isoforms of CD1
molecule have been identified
till now: CD1a, CD1b, CD1c,
CD1d and CD1e. According to
the sequence homology, CD1a,
CD1b and CD1c appear to be
classified into a group,
whereas CD1d and CD1e
show divergence. CD1
molecules have been proved to
be evolutionarily conserved in
structure (Gumperz 2006). In all
mammals, at least one CD1
isoform is found, and in some
cases, CD1 genes have been
expanded by duplication events. Humans have all five isoforms of CD1 molecules on
human chromosome 1, while mouse only possess CD1d gene which is composed of two
copies, CD1d1 and CD1d2 (Dascher and Brenner 2003).
Figure 2: Top and side views of crystal
structure of CD1 molecule (Gumperz 2006).
The green part represents the CD1 chain, while
the blue part shows the β
2
micro globulins. The
lipid is shown with carbons drawn in white,
oxygen in red and nitrogen in blue
4
Human CD1d contains 335 amino acids in length. Most amino acids on the NH
2
group
side (295 amino acids) are defined as extracellular domain, which form a hydrophobic
cleft sequestering the alkyl chain of lipid antigens. The polar regions of antigens are more
accessible to the TCRs of T lymphocytes (Zeng, Castano et al. 1997; Gadola, Zaccai et al.
2002; Zajonc, Elsliger et al. 2003; Batuwangala, Shepherd et al. 2004). The middle 26
amino acids are defined as transmembrane domain, while the last 14 amino acids
compose the cytoplasmic tail domain including a typical tyrosine-based YXXZ endocytic
motif (where Y is tyrosine, X is any amino acid and Z is bulky hydrophobic amino acid)
(Liu, Shaji et al. 2010).
1.3 Endocytic and recycling pathway of CD1 molecules
CD1 heavy peptide chain is folded and the α3 domain is nonconvalently assembled with
β
2
m in the ER (Sugita, Porcelli et al. 1997; Huttinger, Staffler et al. 1999). During the
process, one or more chaperone molecules have been demonstrated to bind to the CD1
molecule hydrophobic pocket to help CD1 molecule finish folding process, for example,
phospholipids bind to CD1d and CD1b as the functional chaperone (Gumperz 2006).
CD1a, CD1b, CD1c and CD1d molecules are transferred to the Golgi complex from ER
and then directly located on the cell surface, while CD1e remains in the cytosol solution
after transported to the Golgi complex as a soluble or transmembrane protein (Angenieux,
Salamero et al. 2000). After they are expressed on the cell surface, CD1a, CD1b, CD1c
5
and CD1d could be internalized and trafficked through endosomal vesicle pathway and
then re-expressed on the cell surface. The intercellular pathways of different CD1
isoforms appear to be varied; even though, these routes have been proved to be
evolutionarily maintained based on the inspection of different compartments of
endosomal pathway (Dascher, Hiromatsu et al. 2002; Dascher and Brenner 2003;
Gumperz 2006). CD1 molecules are found in the sorting endosome, early endosome, late
endosome and lysosome compartments. However, the locations of particular CD1
isoforms may be various in different species and different tissue cells (Gumperz 2006).
Based on the previous research results, the intracellular pathway of CD1 molecules seem
to be largely dependent on the cytoplasmic tail amino acid sequence of the molecules
(Kang and Cresswell 2002). As mentioned above, most CD1 isoforms’ cytoplasmic tails
are composed of a tyrosine-based YXXZ motif which can be specifically recognized by
Adaptor protein (AP)-2. AP-2 protein could directly bind to the cytoplasmic tails of
CD1b, CD1c and CD1d molecules and guide their internalization process via the
formation of clathrin-coated pits (Sugita, Grant et al. 1999; Briken, Jackman et al. 2002;
Lawton, Prigozy et al. 2005) and then the clathrin-coated vesicles merge with early
endosome. CD1a molecule does not bind to AP-2, it is found to be internalized through
clathrin-coated vesicles by electron microscopy analysis. After that, the CD1a molecules
are directly transported to recycling endosome and then trafficked back to the cell surface
(Sugita, Grant et al. 1999). Majority of CD1 isoforms mainly stay in the early endocytic
6
system; however, the cytoplasmic tails of some CD1 isoforms contain the flanking
residues which can associate with AP-3 and they could be directed to late endosome
system (Sugita, Cernadas et al. 2004) such as human CD1b and mouse CD1d. In contrast,
human CD1c and CD1d cannot bind to AP-3; their recycling pathways are mostly
involved in the early endocytic pathways. Nevertheless, these molecules sometimes are
observed in the late endosomal pathways, indicating that there is some other mechanism
related. Suk-Jo Kang and Peter Cresswell have suggested that the human CD1d
molecules appear to be associated with MHC class II and are delivered to late endosome
and lysosome compartment. The endocytic and intracellular pathways of CD1 molecules
are summarized in Figure 3 (Gumperz 2006).
Figure 3: Overview of CD1 molecules intracellular trafficking (Gumperz 2006)
7
1.4 Lipid antigen uptake and presentation
By recognizing lipid antigens in the hydrophobic pocket, CD1 molecules have the ability
to present a variety of antigens to the T lymphocytes and NKT cells. These lipid antigens
include endogenous cellular lipids, foreign lipids produced by intracellular infective virus
or bacteria, and extracellular lipids of self or foreign lipids (Moody, Besra et al. 1999;
Vincent, Gumperz et al. 2003). The associated relationships between types of lipid
antigens and CD1 isoforms remain unclear; however, the recent researches have shown
that there are some overlapping capabilities of presenting lipid antigens in different CD1
isoforms (Gumperz 2006). Lipids with long alkyl chains were transported to the late
endosome where CD1b isoforms are capable to interact with them (Gadola, Zaccai et al.
2002; Batuwangala, Shepherd et al. 2004); lipids with unsaturated alkyl chains containing
repeating branched units traffick to the early endosome or recycling endosome which
contain CD1c (Moody, Ulrichs et al. 2000; Moody, Young et al. 2004). And the route of
intercellular traffic of CD1 isoform is significantly related to the type of lipid antigens
that CD1 isoforms bind to (Gumperz 2006).
When CD1 molecules associate with lipid antigens, the hydrophobic domains of lipid
antigens are inserted into the hydrophobic groove of CD1 molecules, making the polar
alkyl chains of the lipids much easier for being recognized and binding to T cell receptors.
Several chaperone proteins are involved in this process to increase the efficiency of
8
loading lipids to the CD1 molecules. At least three types of chaperone proteins have been
identified: glycosidase, lipid transfer proteins and proteins that bind and deliver
extracellular lipids to the intracellular sites (Gumperz 2006). Nevertheless, lipid loading
usually and efficiently occurs at intracellular portions of endosomal systems. Human and
mouse CD1d molecules are structurally related and share the similar presenting
characteristics (Koch, Stronge et al. 2005; Zajonc, Cantu et al. 2005). Both human and
mouse CD1d molecules can bind phospholipids once they are synthesized in the ER and
can associate with these self-lipids during the endosomal trafficking as well (Joyce,
Woods et al. 1998; Park, Kang et al. 2004; Gumperz 2006). The extracellular lipids are
usually delivered into the intracellular compartments and then bind to CD1 molecules
(Prigozy, Sieling et al. 1997; Gumperz 2006); however, a number of recent researchers
suggest that some other pattern recognition receptors could help extracellular lipids
interact and be presented by CD1 molecules. For example, apolipoprotein E (ApoE) is
the main serum factor that binds and regulates the uptake of extracellular lipids and then
loads them to CD1 molecules on myeloid dendritic cells (van den Elzen, Garg et al.
2005).
CD1d restricted-NKT cells are one subset of T lymphocytes that express evolutionarily
conserved and invariant TCR α-chain on their cell surface (Brutkiewicz 2006). They
could specially recognize self-lipid and extracellular lipids presented by CD1d isoform.
9
Among the many different types of non-conventional T lymphocytes, CD1d-restricted
NKT cell is the most studied and best understood (Godfrey, Pellicci et al. 2010). NKT
cells could autoreactively respond to the lipids presented by CD1d molecules, which may
be functionally critical to the immunological tolerance to the self-lipids (Gumperz 2006).
Due to evolutionarily conserved structure in human and mouse, CD1d could be the
representative of the study for CD1 molecule function. Previous studies have
demonstrated that the endocytic pathway of CD1d molecule plays a crucial role in its
lipid antigen uptake and presenting functions, thus it is a good point to investigate the
characteristics of the molecule that is related to the endocytic pathway.
The purpose of the project is to primarily investigate the influence of mutated
cytoplasmic tail and the one of replaced transmembrane domain of CD1d molecule in its
endocytosis pathway.
Aim 1: To determine how the amino acid mutants on the cytoplasmic tail of CD1d will
influence its cell surface delivery and endocytosis process.
Aim2: To determine which part of the cytoplasmic tail is critical for the trafficking of
CD1d molecule.
Aim3: To determine whether the transmembrane domain of CD1d is necessary for its cell
surface delivery and endocytosis.
10
CHAPTER 2 MATERIALS AND EXPERIMENTAL METHODS
Materials: cell lines, plasmids, antibodies and other reagents.
2.1 Cell lines
Human HeLa cell line and HeLa CD1d cell line which continuously expresses CD1d
molecule (provided by Dr. Weiming Yuan, University of Southern California, Los
Angeles, CA).
2.2 Antibodies and Reagents
Biotin-CD1d D5 antibody (From Dr. Weiming Yuan) is prepared at 1:200 dilution, and
anti GRP 94 rat antibody was prepared at 1:500 dilutions. Goat-anti-mouse HRP and
Goat-anti-rat HRP secondary antibody (Jackson Immune Research Laboratories, Inc West
Grove, PA USA) are prepared at 1:10000 dilutions in 10ml TBST (Tris-Buffered Saline
Tween-20). For flow cytometry staining, Alexa 647 conjugated anti CD1d 51.1.3 was
prepared at 1:20 dilution before using. For Immunofluorescence staining, primary
antibodies anti CD1d 51.1.3 (From Steven Porcelli Lab, Albert Einstein Medical College,
Bronx, NY USA), anti CD1d D5 (from Dr. Steve Balk, Harvard Medical School, Boston,
MA USA) and anti LAMP-1(Becton, Dickinson Franklin Lakes, NJ USA) were used at
1:1000, 1:1000 and 1:500 dilutions respectively; secondary antibodies Alexa 488
anti-IgG2b and Alexa 567 anti-IgG1(Invitrogen, Carlsbad, CA USA) were used at 1:2000
dilutions.
11
2.3 Construction of the CD1d mutant and chimera plasmid
Construction of CD1d TD13: the last 13 amino acids on the tail of CD1d were deleted.
The 5’ primer sequence of CD1d TD13 which includes Xho I enzyme site is
ATCTCGAGGTCCCACGCCGGGCGATATGG and the 3’ primer sequence including
Not I enzyme site is AGGCGGCCGCTCTAGAACTAGTGGATCCAGAGACACA
GATGTGGCAAGGCGATCACAGGACGCCCTGATAGGAAGTTTGCCTCTTAAACC
GGGAGTCAAA (reserved sequence).
For PCR reaction, experiment is set as the below table:
Table 1 PCR reaction setting
5x HF PCR buffer 10 μl
10mM dNTP 1 μl
10μM 5’ primer 2.5 μl
10μM 3’ primer 2.5 μl
pLPCX-CD1d 100ng
DNA polymerase 0.5 μl
H
2
O 33 μl
Total 50 μl
After PCR reaction, the PCR product was purified with Bioland extraction kit (Bioland
Scientific LLC, Cerritos, CA USA), and then the PCR product was directly cloned into
Zero-blunt Topo vector (Invitrogen, Carlsbad, CA USA), which could enhance the
12
following subcloning efficiency and clone accuracy, for the next subcloning steps. Topo
blunt reaction product 2 μL was transformed into Invitrogen One-shot competent E.coli
cell (Invitrogen, Carlsbad, CA USA). 4 colonies were randomly picked to extract Topo
blunt plasmid for next subcloning. After extracting plasmids from the E.coli cell,
restriction enzyme digestion was performed to Topo-PCR product and empty target
plasmid, pLPCX. Restriction enzyme digestion reaction was performed according to the
following table.
Table 2 Restriction enzyme digestion reaction
Topo blunt
product
Empty pLPCX
vector
NEB buffer 4 3 μl 3 μl
Xho I 1.5 μl 1.5 μl
Not I-HF 1.5 μl 1.5 μl
DNA 10 μl 5 μl
H
2
O 14 μl 19 μl
Total 30 μl 30 μl
Run all the digestion reaction products on 0.8% Agarose gel electrophoresis to purify the
DNA. Ligate the enzyme digested empty pLPCX vector and Topo blunt clone product
under 1:3 and 1:6 ratio by using Roche ligation Kit (Roche, Indianapolis, IN USA) and
transform 2 μl into Maxi-efficiency DH5 α competent cell (Invitrogen, Carlsbad, CA USA).
13
The next day 2 colonies of each ratio are picked and sent for sequencing verification.
After DNA sequencing verification, inoculate the correct colony into 200ml Lysogeny
broth (LB) medium for preparing large amount of plasmid DNA. Maxi-prep kit (Qiagen,
Valencia, CA USA) was used the next day to extract plasmid DNA.
For constructing CD1d TD14 (last 14 amino acids on the tail of CD1d are deleted), 5’
primer including Xho I enzyme site is ATCTCGAGGTCCCACGCCGGGCGATATGG,
and the 3’ primer including Not I enzyme site is
TGGCGGCCGCTCAAAAGCCCACAATGAGGAGGAA (reserved sequence).
The entire rest constructs were generated following the protocol as similar as CD1d
TD13.
To construct CD1d-CD4-CD1d chimera, 5’ primer sequence including Xho I enzyme site
is ATCTCGAGGTCCCACGCCGGGCGATATGG, and the 3’ primer sequence including
ClaI enzyme site is TTATCGATTCACAGGACGCCCTGATAGGAA
GTTTGCCTCTTAAACCGGGAGGTGAAGAAGATGCCTAGCCCAAT (reserved
sequence). For PCR reaction, I used CD1d-CD4 chimera plasmid (provided by Dr. Yuan)
as the template.
2.4 Transient transfection of CD1d mutant and chimera construct into HeLa cell
HeLa cells were split into 24-well plates containing coverslips one day before the
transfection with proper confluency to ensure that the cells reach 60-70% confluence on
the day of the experiment. On the day of the experiment, transfection complexes of
14
plasmid DNA and BioT reagent (Bioland Scientific LLC, Cerritos, CA USA) were
prepared. 0.5 μg plasmid DNA and 0.75 μl BioT reagent in 25 μl dPBS were added for each
well. Pipette up and down several times to mix well and incubate the mixture at room
temperature for 5 minutes. Add the entire mixture into the cells in 24-well plate. Tilt the
plate a few times to mix well. Incubate the cells at 37 degree Celsius in 5% CO
2
incubator for 16-24 hours. Replace the medium with fresh Dulbecco's Modified Eagle
Medium (DMEM) medium (Sigma, St. Louis, MO USA) containing 5% FBS and
incubate the transfected cells at 37 degree Celsius in 5% CO
2
one more day. The protein
expression should reach a high level after 48 hours post-transfection.
2.5 Immunofluorescence analysis of CD1d mutant and chimera constructions
Add about 25,000 cells per coverslip in 500 μl DMEM with 5% FBS and incubate 24
hours for cells to attach and spread.
Fixation of cells:
Aspirate the medium and quickly add 400 μl fixative solution (Table 4) and keep at room
temperature for 15 minutes; wash coverslips twice with 500 μl serum-free medium with
10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes).
Permeabilization and staining:
Aspirate the medium and add 500 μl permeabilization solution (PS) (Table 4) per well.
Incubate at room temperature for 15 minutes. Dilute the primary antibodies (anti CD1d at
1:1000 and anti lamp I at 1:500) in PS solution and vortex to mix well. Place 20 μl drop of
15
diluted antibodies on a flat piece of parafilm sheet (Pechiney plastic packaging, Chicago,
IL USA). Place the coverslips onto the drop of antibodies. Put moist paper towels around
the parafilm and cover everything to create a moist chamber and prevent antibody from
drying; Incubate at room temperature for 30 minutes in dark. Wash with 400 μl PS three
times. Dilute the secondary antibodies (1:2000 Alexa 488 anti-IgG2b and 1:2000 Alexa
568 anti-IgG1) in PS and vortex to mix. Place 20 μl drop of secondary antibodies on
parafilm and put moist paper towels around it. Incubate at room temperature for 30
minutes in dark. Wash with 400 μl PS three times. Wash coverslips quickly once with
water and aspirate the residual water from coverslips by vacuum aspirator. Turn the
coverslips (cell side down) onto 5 μl drop of Mowiol (thawed at 65 degree Celsius for 5
minutes) on a glass microscope slide. Let the slide dry 30 minutes at room temperature or
overnight at 4 degree Celsius before inspection.
Table 3 Fixative solution and permeabilization solution
89% volume serum-free DMEM
10% volume 37% formaldehyde
1% volume 1M Hepes, pH 7.4
16
Table 3, Continued
Permeabilization solution (PS) for 500mL:
10% BCS 50ml
0.05% saponin 0.25g
10mM glycine 0.375g
10mM Hepes, pH 7.4 5ml
0.02% sodium azide 5ml
Serum-free DMEM 440ml
2.6 Fluorescence activated-cell sorting (FACS) analysis of CD1d mutant and
chimera constructs
Cells preparation:
Split HeLa cells into 6-well plate at proper ratio to make sure cells reach 60-70%
confluence on the transfection day, including an untransfected control well. Co-transfect
CD1d mutant construct with peGFP vector as a transfection efficiency indicator. Add
DNA-BioT mixture into the cells and incubate 16-24 hours at 37 degree Celsius in CO
2
incubator. Change the medium after 16-24 hours post transfection. Harvest the cells
40-48 hours after transfection by incubating with 0.05% trypsin-EDTA in PBS solution at
37 degree Celsius for 5 minutes.
17
Antibody staining:
Spin the cell suspension at 1200rpm for 5 minutes at 4 degree Celsius. Aspirate the
supernatant and resuspend the cells in 400 μl dPBS+1% BSA. Count the cell number with
hematocytometer. Aliquot 62.5K cells per sample into 96-well V bottom micro plate
including an unstaining control sample. Spin the samples at 2500 rpm for 5 minutes at 4
degree Celsius and flip away the supernatant. Add 25 μl diluted primary antibodies (Alexa
647 conjugated anti CD1d 51.1.3) to the samples and wrap with aluminum foil to keep
away from light; Incubate on ice for 30 minutes. Spin down the cells at 2500 rpm at 4
degree Celsius. Flip away the supernatant. Wash the cells with 120 μl dPBS+1% BSA by
using multichannel pipette and spin down at 2500 rpm. Flip away the supernatant and
wash the cell pellets again. Wash the cells one more time with dPBS only. Resuspend the
cell pellets in 100μL 3.7% formaldehyde and keep on ice for 5 minutes. Transfer cells
into FACS tube and add another 200 μl dPBS. Apply stained samples on the Becton
Dickinson FACS machine and analyze.
2.7 Western blot analysis of CD1d mutant and chimera constructs
In order to test the expression of CD1d chimera mutants, we apply western-blot to
investigate whether the CD1d chimera plasmids could be efficiently expressed in the
transient transfection of HeLa cell.
18
Cell preparation and transfection:
Split HeLa cells into 10cm dish at proper ratios one day before transient transfection to
ensure that the cells reach 60-70% confluence on the day of the experiment. Mix 500 μl
dPBS, 10 μg DNA and 15 μl and incubate at room temperature for 5 minutes. Add CD1d
chimera DNA-BioT mixture directly into the cells. Incubate at 37 degree Celsius for
16-24 hours. Change the media after 16-24 hours post-transfection. Incubate at 37 degree
Celsius for 24 more hours.
Harvest the cells:
Add 5ml 0.05% trypsin-EDTA in PBS to the cells and incubate at 37 degree Celsius for 5
minutes to detach the cells. Transfer all the cells together with medium into 15ml tubes
and spin down the cells at 1200 rpm for 5 minutes at 4 degree Celsius. Resuspend the cell
pellets in 100 μl Triton X-100 lysis buffer which includes 100X IAA and 200X PMSF
protease inhibitors. Incubate on ice for 30 minutes. Pipette up and down to ensure cell
lysis. Spin down 1200 rpm 5 minutes to get rid of nuclei. Transfer the supernatant to a
new 1.5ml eppendorf tube. Use 2 μl cell lysate for Bio-Rad D
c
protein Assay kit (Bioland
Scientific LLC, Cerritos, CA USA) to measure protein concentration.
Western blot analysis:
Load 30 μg of each protein samples on mini SDS-PAGE gel. Run the samples under
20mA electricity for 90 minutes. Transfer the samples from SDS-PAGE gel to
Polyvinylidene Fluoride (PVDF) membrane (Millipore, Billerica, MA USA). Block the
19
membrane with 5% milk in TBST at room temperature for 30 minutes. Wash briefly with
TBST buffer one or two times. Incubate the membrane with 1
st
antibody (anti CD1d D5)
at 1:4000 dilution in 2% milk+0.02% NaN
3
at 4 degree Celsius overnight. Wash the
membrane with TBST twice and wash with TBST for 10 minutes. Incubate with 2
nd
antibody (Goat anti mouse) at 1:10000 dilution in TBST without NaN
3
. Wash briefly with
TBST. Apply HyGLO Quick spray Chemiluminescent HRP antigen detection reagent
(Denville Scientific, Inc Metuchen, NJ USA) to the membrane, incubate at room
temperature for 1 minute, and then expose the membrane from 4 seconds to 15seconds on
Fujifilm LAS 3000 detector.
20
CHAPTER 3 RESULTS
3.1The cytoplasmic tail of CD1d is critical for its surface expression
The YXXZ motif on the tail of CD1d molecule is considered to be functionally important
for CD1d molecules internalization where the lipid antigens are loaded onto them (Sugita,
Jackman et al. 1996). A previous experiment about Y331A mutant on the YXXZ motif of
CD1d molecule has been shown to repress its endocytic rate (Rodionov, Nordeng et al.
1999). To study the function of cytoplasmic tail of CD1d on its cell surface expression,
we generated different CD1d tail-truncated versions without YXXZ motif, CD1d TD13
and CD1d TD14 mutants (last 13 amino acids of tail deletion and last 14 amino acids of
tail deletion), and investigated the cell surface expression of each mutant by
Immunofluorescence and flow cytometry analysis. The purpose of co-transfection of
HeLa cell with GFP protein in FACS experiments is to use GFP to gate for positively
transfected cells. Table 4 shows the cytoplasmic tail comparison of different mutants.
Table 4 Cytoplasmic tail sequence comparison of CD1d mutants
322 323 324 325 326 327 328 329 330 331 332 333 334 335
CD1d WT T S R F K R Q T S Y Q G V L
CD1d TD13 T
CD1d TD14
CD1d T322N N S R F K R Q T S Y Q G V L
CD1d T322D D S R F K R Q T S Y Q G V L
21
In figure 4, flow cytometry result demonstrates the CD1d TD13 mutant cell surface
expression level. The gated population in figure 4 is the CD1d signal positive cell, and
we compare the expression level of CD1d wild type and CD1d TD13 mutant in the
selected area as showed in figure 5.
Figure 4: Dot plot pattern of CD1d wild type and CD1d TD13. The left upper pattern is the cell
surface CD1d expression of HeLa cell co-transfected by peGFP and CD1d wild type, while the
right upper pattern is the cell surface CD1d expression of HeLa cell co-transfected by peGFP and
CD1d TD13 mutant. The X-axis is the gradient of APC-H signal intensity which represents the
cell surface expression level of CD1d molecules. Y-axis is the gradient of FITC-H signal intensity
which represents the GFP protein expression. The lower patterns are positive FITC signal
population of CD1d wild type (left) and CD1d TD13 mutant (right).
22
From the histogram of APC-H signal (Figure 5), which represents the cell surface
expression level of CD1d, we could see that the CD1d TD13 expression is still detected
compared to the positive control, CD1d wild type. The experiments were repeated two
more times to confirm the result. Figure 6 and figure 7 show the comparison of the cell
surface expression level of CD1d wild type and CD1d TD14 mutant. From these two
figures, we could find that the CD1d TD14 mutant cell surface expression level is
significantly reduced compared to the CD1d wild type. The result is consistent with the
Figure 5: Cell surface expression level comparison of CD1d wild type and CD1d
TD13 mutant. APC-H represents the signal intensity of CD1d cell surface expression
level. The left peak is unstaining control and untransfected HeLa cell signals. The red
peak is the CD1d expression level of CD1d wild type transfected HeLa cells, while the
blue peak is the CD1d expression level of the positive transfected population of CD1d
TD13 mutant.
23
previously published research. (Liu, Shaji et al. 2010) There is about 85% difference
between the two samples; geometric mean of the APC-H peak area is 5886 of CD1d
TD14 mutant versus 41020 of CD1d wild type. From the Immunofluorescence staining
results, the expression signal is much easier to be displayed in these two mutant
constructs. In figure 8, we could clearly see that the green fluorescence in CD1d TD13
sample is bright and vivid on the cell surface, while it is less co-localized with LAMP-1
in lysosome. It means the CD1d tail without YXXZ motif could normally be expressed
on the cell surface like CD1d wild type but could not be internalized efficiently. In
contrast, the expression of CD1d TD14 on the cell surface is not detected, which is
consistent with our FACS results and the published paper (Liu, Shaji et al. 2010). In
figure 8, the CD1d TD14-green fluorescence signal accumulates in the cell. To identify
the location of CD1d TD14 molecule, we stained the CD1d TD14 sample with anti CD1d
D5 antibody which specifically recognizes the unfolding conformation of CD1d molecule.
In figure 9, we can see that CD1d TD14 does not co-localize with lysosome.
Over-expression of CD1d TD14 in the cell by transient transfection could lead
accumulation of target proteins in the cell rather than distribution at normal position. So
we need to use CD1d TD14 stable cells which continuously express CD1d TD14
molecule for investigation in the future.
24
Figure 7: Cell surface expression level comparison of CD1d wild type and CD1d TD14
mutant. APC-H represents the cell surface expression level. The left green and orange
peaks are unstaining control and untransfected HeLa cell signals. The red peak is the
expression level of HeLa cell co-transfected by peGFP and CD1d wild type, while the blue
peak is the expression level of the positive transfected population of CD1d TD14 mutant.
Figure 6: Dot plot patterns of CD1d wild type and CD1d TD14 mutant. The left
pattern is the flow cytometry dots pattern of CD1d wild type, while the right one is
the dots pattern of CD1d TD14 mutant. The X-axis is the gradient of APC-H signal
intensity which represents the cell surface expression level of CD1d molecules.
Y-axis is the gradient of FITC-H signal intensity which represents the GFP protein
expression.
25
Figure 8: Immunofluorescence staining of CD1d TD13 and CD1d TD14 mutants by
anti CD1d 51.1.3 antibody. The green signal represents cell surface expression of CD1d,
while the red signal represents the lampI which is the constant molecule in the lysosome
compartment of cell.
10μm
26
The difference between CD1d TD13 and CD1d TD14 indicates that the last 14th amino
acid on the CD1d tail may be a key site for CD1d molecule expression on cell surface. In
order to test the function of the specific site, further CD1d mutants are generated as CD1d
T322N and CD1d T322D to investigate the hypothesis.
Figure 10 and figure 11 separately show the FACS results of CD1d T322N and CD1d
T322D mutant constructs. CD1d T322N appears to express higher CD1d on cell surface
than CD1d wild type does, while CD1d T322D show similar expression levels in
transient transfected HeLa cell compared to CD1d wild type in transient transfected HeLa
cell.
Figure 9: Immunofluorescence staining of CD1d TD14 by anti CD1d D5 antibody. The
green signal represents intracellular expression of CD1d, while the red signal represents the
lampI which is the constant molecule in the lysosome compartment of cell.
10μm
27
Figure 10: CD1d T322N mutant cell surface expression dot plot pattern and
comparison. A) is co-transfection of CD1d wild type and GFP into HeLa cell, while B) is
co-transfection of CD1d T322N mutant and GFP into HeLa cell. C) The CD1d cells
surface expression level comparison between CD1d wild type and CD1d T322N mutant.
The red line means expression of CD1d wild type and the green line stands for the
expression of CD1d T322N mutant. The orange and blue peaks are unstaining and
untransfected control signals.
28
However, Liu and Shaji stated in their paper that CD1d T322D mutant could significantly
down-regulate the CD1d cell surface expression in the staining with the anti CD1d- β
2
m
Figure 11: The CD1d T322D mutant cell surface expression dot plot pattern and
comparison. A) is the cell surface expression level of co-transfection by CD1d wild type and
GFP. B) is the cell surface expression level of co-transfection by CD1d T322D mutant and
GFP. C) is the expression comparison between CD1d wild type and CD1d T322D mutant. The
red line is the CD1d wild type while the blue line is the CD1d T322D mutant.
29
monoclonal antibody. They proposed that T322D may be a signal residue for lysosomal
targeting and the other 13 amino acids on the tail contain the signal that traffic back to
cell surface (Liu, Shaji et al. 2010). Several flow cytometry experiments of CD1d T322D
mutant we did show the consistent results. The Immunofluorescence staining as well
provides an absolutely clear image that CD1d T322D mutant expresses on the cell surface
like CD1d wild type, while less co-localization with LAMP-1 is observed in confocal
microscopy image (Figure 12). We also did sequence verification of CD1d T322D mutant
plasmid to verify the mutant is correct. The Immunofluorescence staining of CD1d
T322N mutant shows less co-localization of CD1d and LAMP-1 in lysosome in figure 12.
The reason for the discrepancy between our result and the published paper is not well
understood and is under further investigation.
30
Figure 12: Immunofluorescence staining of CD1d T322N and CD1d T322D mutants by
CD1d 51.1.3 antibody. The green signal represents cell surface expression of CD1d, while
the red signal represents the lampI which indicates the lysosome compartment of cell.
25μm
31
3.2 The transmembrane domain of CD1d determines its cell surface expression
Based on the previous data, we could conclude that the cytoplasmic tail of CD1d is
functionally critical for its plasma membrane expression. The deletion of whole tail or
portion of it would reduce the surface expression and the T322 site mutant on the tail
domain might influence the endocytic process. So the results raise another question: Does
the transmembrane domain of CD1d molecules play an important role in its surface
expression or endocytic pathway? To investigate the function of transmembrane domain,
we generated CD1d-CD4-CD1d fusion mutant and analyzed via Immunofluorescence
and flow cytometry methods. Figure 13 shows the flow cytometry result of
CD1d-CD4-CD1d fusion mutant and table 5 shows the sequence comparison between
CD1d and CD4 molecule.
32
Figure 13: The CD1d-CD4-CD1d fusion mutant cell surface expression dot plot pattern
and comparison. A) is the cell surface expression level of co-transfection by CD1d wild type
and GFP. B) is the cell surface expression level of co-transfection by CD1d-CD4-CD1d
fusion mutant and GFP. C) is the expression comparison between CD1d wild type and
CD1d-CD4-CD1d mutant. The blue line is the CD1d wild type while the red line is the
CD1d-CD4-CD1d fusion mutant. The orange and blue peaks are unstaining and untransfected
control signals.
33
Table 5 Transmembrane domain sequence comparison of CD1d wild type and
CD1d-CD4-CD1d
Transmembrane domain
CD1d G G S Y T S M G L I A L A V L A C L L F L L I V G F
CD4 A L I V L G G V A G L L L F I G L G I F F
According to above flow cytometry analysis of CD1d-CD4-CD1d chimera mutant, we
could see that its cell surface expression is almost totally repressed and no signal is
detected by flow cytometry when compared to CD1d wild type as the peak signal of
CD1d-CD4-CD1d is overlapped with negative control, the unstained sample without
fluorescence-conjugated antibodies binding and untransfected sample without expression
of CD1d (Figure 13).
In figure 14, the immunofluorescence staining result of CD1d-CD4-CD1d fusion mutant
by anti CD1d 51.1.3 antibody is consistent with the flow cytometry result. No CD1d cell
surface expression is detected by confocal microscopy. In order to further investigate the
existence of CD1d-CD4-CD1d molecule, we stained the sample with anti CD1d D5
antibody (Figure 15). From the result, we can see CD1d-CD4-CD1d molecule in the cells,
however, the CD1d-CD4-CD1d fusion protein is not localized in lysosome compartment
either.
34
Figure 14: Immunofluorescence staining of CD1d-CD4-CD1d by anti CD1d
51.1.3 antibody.
Figure 15: Immunofluorescence staining of CD1d-CD4-CD1d by anti CD1d D5
antibody. The green signal represents intracellular expression of CD1d, while the red signal
represents the lampI which indicates the lysosome compartment of cell.
10μm
25μm
35
In order to test whether all the constructs we used in the project are normally transcript
and expressed, we performed western blot to detect the expression of all CD1d mutant
constructs.
Figure 16 showed all the CD1d constructs are transcript and expressed in the transient
transfection HeLa cell line even though some constructs had low CD1d expression level.
Figure 16: Western blot pattern of CD1d constructs. The patterns show the expression of
HeLa CD1d cell line (positive control), HeLa cell line (negative control) and CD1d TD14,
CD1d T322N, CD1d-CD4-CD1d, CD1d T322D and CD1d TD13 mutants.
36
The anti GRP 94 is an indicator of the protein sample loading amount. These data
indicate that the CD1d mutant constructs were normally transcripted, translated and
expressed in the cell as detected by the anti-CD1d D5 antibody in western blot analysis.
37
CHAPTER 4 DISCUSSION AND FUTURE WORK
4.1 Discussion
Of all the CD1 family members, only CD1a isoform does not possess YXXZ motif for
AP-2 protein recognition, the other members of CD1 family CD1b, CD1c and CD1d
could be expressed either on cell surface or in the endosomal compartments via
YXXZ-dependent internalization pathway (Sugita, Cao et al. 2002). This conservative
characteristic provides us an easy way to study the cell surface delivery and endocytic
pathways of most CD1 isoforms.
CD1d molecule mainly presents lipid antigens to invariant NKT cell and activates it to
secrete both Th1 (IFN- γ and GM-CSF) cytokines and Th2 (e.g. IL-4) cytokines which
play crucial roles during both innate and adaptive immune reaction process (Moody and
Porcelli 2003). Human and mouse CD1d could associate with lipid antigens once they are
biosynthesized in ER or later during the endocytic pathway (De Silva, Park et al. 2002;
Park, Kang et al. 2004). Some research results have proved that the mouse CD1d1
molecule could load endogenous antigens, such as glycolipids, onto CD1d molecule in an
endosomal compartment (Roberts, Sriram et al. 2002). They imply the potential
assembling process for CD1d1 molecule with antigens takes place during the cell surface
delivery and endocytic trafficking. Endogenous and internalized lipids are transported
to different endocytic vesicles depending on their structure and chemistry of their alkyl
38
chains, so the route of intracellular trafficking taken by a CD1 molecule seems to
determine what type of lipids it presents (Gumperz 2006). According to the published
papers, the cell surface delivery and endocytic pathway of CD1d are the major processes
that the molecule follows to fulfill its physiological function. Thus, the purpose of our
project is to investigate the different domains’ functions on the cell surface delivery and
endocytic pathway of human CD1d. Based on our experimental results, we suggest that
the entire cytoplasmic tail of human CD1d is functionally crucial for the proteins normal
expression on cell surface. The last 14th amino acid (T322) may be especially a key site
for the surface trafficking. The CD1d TD13 and CD1d TD14 mutants’ results indicate: 1)
human CD1d TD13 mutant could transport onto the cell surface compared to the CD1d
wild type; 2) cell surface delivery of human CD1d TD14 without the whole tail is
significantly reduced. According to the results of CD1d T322N, we speculate that CD1d
T322N mutant has partial influence on the YXXZ motif function because the mutant
CD1d cell surface expression is slightly higher than the wild type, implying that T322
site might disturb the recognition of YXXZ motif of AP-2 making it more difficult for
CD1d molecule to be internalized through the clathrin-dependent endocytosis. However,
more experiments are required to verify the speculation in the future. Liu, Shaji et al
stated that the amino acid T322 on the cytoplasmic tail of CD1d is a crucial major signal
site for lysosomal targeting; the phosphorylation-mimicking form (T322D) could directly
guide the CD1d to lysosome for degradation; the T322-based signal is prior to the YXXZ
39
signal (Liu, Shaji et al. 2010). However, our results are not consistent with their
conclusion. From our CD1d T322D mutant data, the mutant seems to perform as
normal as wild type in cell surface expression and endocytosis process. Even though we
repeated the experiments several times and adjusted the experimental conditions to the
ones they used in their paper including using the same cell line, no significant
down-regulation of CD1d cell surface expression is observed. The flow cytometry data is
consistent with the Immunofluorescence staining images as well, in which the CD1d
T322D molecules are mostly located on the cell surface rather than the late endosome or
lysosome. However, it is clear that the majority of CD1d’s cytoplasmic tail is not required
for its surface expression except the last amino acid (T322).
The CD1d molecule contains three functional domains including extracellular domain for
recognizing and binding the lipid antigen, the transmembrane domain and cytoplasmic
tail domain that directs CD1d’s internalization process and endocytic trafficking (Liu,
Shaji et al. 2010). The function of transmembrane domain on CD1d cell surface
expression delivery and endocytic pathway is an interesting point to study. In order to
deeply study the function of the transmembrane domains of CD1d molecule on the
molecule trafficking, we generated CD1d-CD4-CD1d fusion mutant in which the CD1d
transmembrane domain is replaced by the transmembrane domain of CD4 molecule. The
flow cytometry and immunofluorescence image results are consistent with each other.
40
Basically no CD1d signal either on the cell surface or in the endocytic compartments
could be observed by flow cytometry and immunofluorescence methods. The possible
explanation is that the chimera transmembrane domain interrupts the folding of the
protein after translation and cause the degradation by proteasome. The fusion mutant has
proved that the transmembrane domain is also crucial for the cell surface expression
delivery and/or endocytic pathway of CD1d. The western blot results provide the
evidence about general expression of all the CD1d mutant constructs in the cells.
4.2 Future direction
This project is a preliminary experiment to study how different domains of CD1d execute
their functions in the molecule’s cell surface delivery and its endocytosis pathway. We
have done the brief phenotype tests about the CD1d mutants via transiently transfecting
the plasmids into HeLa cell line. For further detailed research, we need to generate the
stable cell lines of all CD1d mutants that could continuously express these CD1d mutants
at a high level in order to eliminate the influence due to variable cell conditions. It is
interesting to test the location of CD1d mutants via immunofluorescence staining of ER
marker as well. We need to figure out a reasonable explanation about CD1d T322D
mutant results that is opposite to the proposal of Liu and Shaji(Liu, Shaji et al. 2010).
Based on our results, the 14
th
amino acid from the end of CD1d molecule is the key site
for its normal function. It is interesting to figure out that how modifications of this amino
acid affect the whole molecule’s behavior. We can mutate T322 to any other amino acids
41
to investigate potential effects. To figure out the detailed function of CD1d
transmembrane domain, comparison between the sequence of CD1d transmembrane
domain and the one of CD4 transmembrane domain is required to identify the
functionally and structurally critical site in the transmembrane domain as well.
42
REFERENCES
Angenieux, C., J. Salamero, et al. (2000). "Characterization of CD1e, a third type of CD1
molecule expressed in dendritic cells." J Biol Chem 275(48): 37757-37764.
Batuwangala, T., D. Shepherd, et al. (2004). "The crystal structure of human CD1b with a bound
bacterial glycolipid." J Immunol 172(4): 2382-2388.
Beckman, E. M., S. A. Porcelli, et al. (1994). "Recognition of a lipid antigen by CD1-restricted
alpha beta+ T cells." Nature 372(6507): 691-694.
Brigl, M. and M. B. Brenner (2004). "CD1: antigen presentation and T cell function." Annu Rev
Immunol 22: 817-890.
Briken, V., R. M. Jackman, et al. (2002). "Intracellular trafficking pathway of newly synthesized
CD1b molecules." EMBO J 21(4): 825-834.
Brutkiewicz, R. R. (2006). "CD1d ligands: the good, the bad, and the ugly." J Immunol 177(2):
769-775.
Cresswell, P. (1994). "Assembly, transport, and function of MHC class II molecules." Annu Rev
Immunol 12: 259-293.
Dascher, C. C. and M. B. Brenner (2003). "Evolutionary constraints on CD1 structure: insights
from comparative genomic analysis." Trends Immunol 24(8): 412-418.
Dascher, C. C., K. Hiromatsu, et al. (2002). "Conservation of CD1 intracellular trafficking
patterns between mammalian species." J Immunol 169(12): 6951-6958.
De Silva, A. D., J. J. Park, et al. (2002). "Lipid protein interactions: the assembly of CD1d1 with
cellular phospholipids occurs in the endoplasmic reticulum." J Immunol 168(2): 723-733.
Gadola, S. D., N. R. Zaccai, et al. (2002). "Structure of human CD1b with bound ligands at 2.3 A,
a maze for alkyl chains." Nat Immunol 3(8): 721-726.
Godfrey, D. I., D. G. Pellicci, et al. (2010). "Antigen recognition by CD1d-restricted NKT T cell
receptors." Semin Immunol 22(2): 61-67.
Gumperz, J. E. (2006). "The ins and outs of CD1 molecules: bringing lipids under immunological
surveillance." Traffic 7(1): 2-13.
43
Heemels, M. T. and H. Ploegh (1995). "Generation, translocation, and presentation of MHC class
I-restricted peptides." Annu Rev Biochem 64: 463-491.
Huttinger, R., G. Staffler, et al. (1999). "Analysis of the early biogenesis of CD1b: involvement of
the chaperones calnexin and calreticulin, the proteasome and beta(2)-microglobulin." Int
Immunol 11(10): 1615-1623.
Joyce, S., A. S. Woods, et al. (1998). "Natural ligand of mouse CD1d1: cellular
glycosylphosphatidylinositol." Science 279(5356): 1541-1544.
Kang, S. J. and P. Cresswell (2002). "Regulation of intracellular trafficking of human CD1d by
association with MHC class II molecules." EMBO J 21(7): 1650-1660.
Koch, M., V. S. Stronge, et al. (2005). "The crystal structure of human CD1d with and without
alpha-galactosylceramide." Nat Immunol 6(8): 819-826.
Lawton, A. P., T. I. Prigozy, et al. (2005). "The mouse CD1d cytoplasmic tail mediates CD1d
trafficking and antigen presentation by adaptor protein 3-dependent and -independent
mechanisms." J Immunol 174(6): 3179-3186.
Liu, J., D. Shaji, et al. (2010). "A threonine-based targeting signal in the human CD1d
cytoplasmic tail controls its functional expression." J Immunol 184(9): 4973-4981.
Moody, D. B., G. S. Besra, et al. (1999). "The molecular basis of CD1-mediated presentation of
lipid antigens." Immunol Rev 172: 285-296.
Moody, D. B. and S. A. Porcelli (2003). "Intracellular pathways of CD1 antigen presentation."
Nat Rev Immunol 3(1): 11-22.
Moody, D. B., T. Ulrichs, et al. (2000). "CD1c-mediated T-cell recognition of isoprenoid
glycolipids in Mycobacterium tuberculosis infection." Nature 404(6780): 884-888.
Moody, D. B., D. C. Young, et al. (2004). "T cell activation by lipopeptide antigens." Science
303(5657): 527-531.
Park, J. J., S. J. Kang, et al. (2004). "Lipid-protein interactions: biosynthetic assembly of CD1
with lipids in the endoplasmic reticulum is evolutionarily conserved." Proc Natl Acad Sci
U S A 101(4): 1022-1026.
Prigozy, T. I., P. A. Sieling, et al. (1997). "The mannose receptor delivers lipoglycan antigens to
endosomes for presentation to T cells by CD1b molecules." Immunity 6(2): 187-197.
44
Roberts, T. J., V . Sriram, et al. (2002). "Recycling CD1d1 molecules present endogenous antigens
processed in an endocytic compartment to NKT cells." J Immunol 168(11): 5409-5414.
Rodionov, D. G., T. W. Nordeng, et al. (1999). "A critical tyrosine residue in the cytoplasmic tail
is important for CD1d internalization but not for its basolateral sorting in MDCK cells." J
Immunol 162(3): 1488-1495.
Sugita, M., X. Cao, et al. (2002). "Failure of trafficking and antigen presentation by CD1 in
AP-3-deficient cells." Immunity 16(5): 697-706.
Sugita, M., M. Cernadas, et al. (2004). "New insights into pathways for CD1-mediated antigen
presentation." Curr Opin Immunol 16(1): 90-95.
Sugita, M., E. P. Grant, et al. (1999). "Separate pathways for antigen presentation by CD1
molecules." Immunity 11(6): 743-752.
Sugita, M., R. M. Jackman, et al. (1996). "Cytoplasmic tail-dependent localization of CD1b
antigen-presenting molecules to MIICs." Science 273(5273): 349-352.
Sugita, M., S. A. Porcelli, et al. (1997). "Assembly and retention of CD1b heavy chains in the
endoplasmic reticulum." J Immunol 159(5): 2358-2365.
van den Elzen, P., S. Garg, et al. (2005). "Apolipoprotein-mediated pathways of lipid antigen
presentation." Nature 437(7060): 906-910.
Vincent, M. S., J. E. Gumperz, et al. (2003). "Understanding the function of CD1-restricted T
cells." Nat Immunol 4(6): 517-523.
Zajonc, D. M., C. Cantu, 3rd, et al. (2005). "Structure and function of a potent agonist for the
semi-invariant natural killer T cell receptor." Nat Immunol 6(8): 810-818.
Zajonc, D. M., M. A. Elsliger, et al. (2003). "Crystal structure of CD1a in complex with a
sulfatide self antigen at a resolution of 2.15 A." Nat Immunol 4(8): 808-815.
Zeng, Z., A. R. Castano, et al. (1997). "Crystal structure of mouse CD1: An MHC-like fold with a
large hydrophobic binding groove." Science 277(5324): 339-345.
Abstract (if available)
Abstract
CD1 molecules are a family of MHC class I-like lipid presenting molecules. They could specifically recognize self lipid antigens and extracellular lipid antigens, and present them to T lymphocytes. T lymphocytes are stimulated to secrete different cytokines to execute their immunological functions. CD1d, a member of CD1 family, exists both in human and in mouse. It can particularly activate invariant Natural Killer T (NKT) cells which are mostly-studied non-conventional T cell types. CD1d molecule is delivered onto cell surface membrane after synthesis in Endoplasmic Reticulum (ER) and is believed to be internalized via AP-2 dependent endocytic pathway. During the trafficking route, CD1d recognizes and assembles with lipid antigens to form antigen-CD1d complex in the early endosome, the complex can be delivered onto the cell membrane after its formation. So the intracellular trafficking pathway of CD1d molecule is important for its immunological function. Based on the previous studies, tyrosine-based endosomal targeting sequence, YXXZ (where Y is tyrosine, X is any amino acid and Z is bulky hydrophobic amino acid) and some other amino acids on the cytoplasmic tail of CD1d are the major functional sites for its endocytosis process. The purpose of the thesis is to study the functions of CD1d cytoplasmic tail and transmembrane domain on its intracellular trafficking routes. The results show that the 14th amino acid from the bottom on the cytoplasmic tail may be the important site to determine CD1d molecule cell surface expression and the entire transmembrane domain is required for CD1d normal expression in cells.
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Asset Metadata
Creator
Yang, Yang
(author)
Core Title
The role of CD1d transmembrane and cytoplasmic tail domain in CD1d trafficking pathway
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2011-05
Publication Date
02/03/2011
Defense Date
01/24/2011
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
CD1d,OAI-PMH Harvest,tail domain,trafficking pathway,transmembrane domain
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Tokes, Zoltan A. (
committee chair
), Ulmer, Tobias (
committee member
), Yuan, Weiming (
committee member
)
Creator Email
bioyy2238@gmail.com,yang11@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m3642
Unique identifier
UC1232135
Identifier
etd-Yang-4319 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-426985 (legacy record id),usctheses-m3642 (legacy record id)
Legacy Identifier
etd-Yang-4319.pdf
Dmrecord
426985
Document Type
Thesis
Rights
Yang, Yang
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
Tags
CD1d
tail domain
trafficking pathway
transmembrane domain