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Lipid Protein Interactions: The Assembly of
CD1d1 with Cellular Phospholipids Occurs in
the Endoplasmic Reticulum
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of June 18, 2017.
A. Dharshan De Silva, J.-June Park, Naoto Matsuki,
Aleksandar K. Stanic, Randy R. Brutkiewicz, M. Edward
Medof and Sebastian Joyce
J Immunol 2002; 168:723-733; ;
doi: 10.4049/jimmunol.168.2.723
http://www.jimmunol.org/content/168/2/723
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References
Lipid Protein Interactions: The Assembly of CD1d1 with
Cellular Phospholipids Occurs in the Endoplasmic Reticulum1
A. Dharshan De Silva,2,3* J.-June Park,3* Naoto Matsuki,* Aleksandar K. Stanic,*
Randy R. Brutkiewicz,† M. Edward Medof,‡ and Sebastian Joyce4*
A
member of the evolutionarily conserved lipid Ag-presenting CD1 family of proteins, CD1d1 controls the development and function of a subset of thymus-derived
lymphocytes called NKT cells. NKT cells are characterized by the
expression of cell surface markers typical of NK cells and T lymphocytes. Among the CD1d-restricted NKT cells are those that
express the highly conserved V␣14J␣15 Ag receptor ␣-chain and
those that express diverse TCRs. The CD1d1-restricted V␣14J␣15
NKT cells are conserved in both humans and mice, and hence are
predicted to impart an evolutionarily conserved immune function.
This immune function, albeit elusive, is thought to be immunoregulatory in nature. Upon activation in vivo through their Ag
receptors, NKT cells promptly elicit large amounts of immunoregulatory cytokines such as IL-4, IFN-␥, TNF-␣, and GM-CSF.
Furthermore, V␣14J␣15 NKT cell dysfunction in autoimmuneprone mice, as well as in individuals afflicted with autoimmune
diabetes, underscore their importance in the physiology of normal
immune responses (reviewed in Ref. 1). Thus, delineating the
structure and function of CD1d is essential to understanding the
biology of NKT cells.
*Department of Microbiology and Immunology, Vanderbilt University School of
Medicine, Nashville, TN 37232; †Department of Microbiology and Immunology, Indiana University School of Medicine, Walther Oncology Center, Indianapolis, IN
46202; and ‡Institute of Pathology, Case Western Reserve University, Cleveland, OH
44106
Received for publication August 24, 2001. Accepted for publication November
1, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health (AI42284
to S.J., AI46455 to R.R.B., and DK56309 to M.E.M.), Juvenile Diabetes Research
Foundation International, and the Human Frontiers in Science Program.
2
Current address: Department of Microbiology and Immunology, Albert Einstein
College of Medicine, Yeshiva University, Bronx, NY 10461.
3
Humans express group I CD1a, CD1b, and CD1c and group II
CD1d molecules (2– 4). Mice and rats express only CD1d (5, 6).
Topologically, CD1 resembles the classical MHC-encoded Agpresenting molecules. Its domain organization and its association
with ␤2-microglobulin (␤2m)5 for complete assembly are similar
to MHC class I molecules. A distinguishing feature of CD1d1 is its
exclusively nonpolar hydrophobic Ag-binding groove. The groove
consists of a large pocket A⬘ that is almost completely covered
from all sides except for a narrow lateral opening connecting it to
a short apically exposed pocket F⬘ (7). Thus, CD1 molecules have
evolved to present hydrophobic Ags to the mammalian immune
system. Indeed, structure function studies pioneered by Brenner
and colleagues (reviewed in Refs. 8 and 9 –11) have revealed that
human CD1b and CD1c present lipid and glycolipid Ags to specific T cells reactive to mycobacterial Ags. Ags presented by CD1b
include mycolic acid, glucosylmonomycolate, phosphatidylinositol (PI)-mannans, and lipoarabinomannans (9 –11). CD1b also presents self glycolipids such as GM1 to autoreactive T cells (12).
CD1c, in contrast, presents mycobacterial dolichylphosphorylmannose (DPM) to specific T cells (13). Current evidence suggests
that mouse and human CD1d present ␣-galactosylceramide (␣GalCer) and potently activate mouse V␣14J␣15 and human V␣24J␣Q
NKT cells, respectively (14 –17). Additionally, CD1d1 also presents PI and activates a small proportion of V␣14J␣15 NKT cells
(18). Thus, self and nonself lipids presented by CD1 are T
cell Ags.
The ability to study lipid CD1 interactions in vitro has illuminated several physico-chemical aspects of lipid presentation and
recognition (reviewed in Ref. 1). Notwithstanding, all of the above
studies have relied on purification of mycobacterial or cellular lipids and their ability to reconstitute functional CD1 molecules in
vitro, either on live cells or using a cell-free system, recognizable
Abbreviations used in this paper: ␤2m, ␤2-microglobulin; PI, phosphatidylinositol;
DPM, dolichylphosphorylmannose; ER, endoplasmic reticulum; hAs, heteroantiserum; endo H, endoglycosidase H; C:M, chloroform:methanol; PI-PLC, PI-specific
phospholipase C; PLA2, phospholipase A2; ␣GalCer, ␣-galactosylceramide; Rf, relative migration.
5
A.D.D. and J.-J.P. contributed equally to this research work.
4
Address correspondence and reprint requests to Dr. Sebastian Joyce, Department of
Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232. E-mail address: [email protected]
Copyright © 2002 by The American Association of Immunologists
0022-1767/02/$02.00
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CD1d1 is a member of a family of lipid Ag-presenting molecules. The cellular ligands associated with CD1d1 were isolated and
characterized by biochemical means as an approach to elucidate the mechanism by which CD1 molecules assemble in vivo. Natural
ligands of mouse CD1d1 included cellular phosphatidylinositol and phosphatidylinositol-glycans that are synthesized in the endoplasmic reticulum. Further biochemical data revealed that the two CD1d1 mutants, one defective in recycling from-and-to the
plasma membrane and the other in efficiently negotiating the secretory pathway, associated with phosphatidylinositol. Thus
phosphatidylinositol associated with CD1d1 in the early secretory pathway. Phosphatidylinositol also associated with CD1d1 in
Pig-A-deficient cells that are defective in the first glycosylation step of glycosylphosphatidylinositol biosynthesis. Moreover, cellular
phosphatidylinositol-glycans are not V␣14J␣15 natural T cell Ags. Therefore, we predict that cellular lipids occlude the hydrophobic Ag-binding groove of CD1 during assembly until they are exchanged for a glycolipid Ag(s) within the recycling compartment for display on the plasma membrane. In this manner, cellular lipids might play a chaperone-like role in the assembly of
CD1d1 in vivo, akin to the function of invariant chain in MHC class II assembly. The Journal of Immunology, 2002, 168: 723–733.
724
by specific T lymphocytes. To elucidate the basis for CD1d1 function, our approach has been to isolate and characterize the associated ligand by biochemical methods. The data revealed that CD1d1
expressed in mammalian cells assembled with a phospholipid,
which was identified as GPI (19). Consistent with this data, a recent study demonstrated that a V␣14J␣15 NKT cell hybridoma
recognized PI presented by CD1d1 as its Ag (18). Notwithstanding, PI does not represent a major NKT cell Ag (20) and, hence,
the role of phospholipid(s) in CD1d1 function remains elusive.
Thus, to clarify the role of CD1d1-associated PI and PI-glycans in
CD1 function, we have studied the assembly of this molecule in
vivo. The data are consistent with the idea that the natural CD1d1associated ligands play a chaperone-like role during its assembly
in vivo akin to the function of invariant chain in MHC class II
assembly.
ASSEMBLING A CD1 MOLECULE IN VIVO
Table I. Description of cell lines generated and used in this study
Cell Lines
O␤
mCD1d1
sCD1d1
sCD1d1-er
sCD1d1-Kb tail
K562
K562-mCD1d1
IA
IA-mCD1d1
Description
NS0 plasmacytoma expressing mouse ␤2ma
O␤ expressing wild-type mouse CD1d1
O␤ expressing soluble mouse CD1d1;
hexahistidine-tagged
O␤ expressing sCD1d1 fused to c-Myc tag
and KDEL ER retention sequence at the
carboxyl terminus
O␤ expressing sCD1d1 fused to the
transmembrane and cytosolic tail
sequence of H2Kb at the carboxyl
terminus
Human erythroleukemia cell line
K562 expressing wild-type mouse CD1d1
Pig-A-deficient derivative of K562
IA expressing wild-type mouse CD1d1
Materials and Methods
Expression constructs
Cell lines
See Table I for a description of CD1- and H2 class I-expressing cell lines.
O␤ that expresses mouse ␤2ma was generated by gene transfer into NS0
plasmacytoma as described (21). Transfected cells were selected 24 h later
in L-glutamine-free medium. O␤ cell lines expressing soluble H2Db as well
as wild-type and mutant CD1d1 were generated by electroporation of the
respective cDNA constructs. Transfected cells were selected in the absence
of L-glutamine but in the presence of 0.8 mg/ml of geneticin (G418; Life
Technologies, Rockville, MD). O␤ and derived lines were maintained in
DMEM (Media-Tech, Herndon, VA) containing
10% dialysed FBS (HyClone Laboratories, Deer Park, PA, or Life Technologies) and 0.5 mg/ml G418. Kb-high and Db-high cells were maintained as described (21). Pig A- (S49a) and Pig E- (BW5147e) mutants (22)
were from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 (Media-Tech) containing 5–10% FBS.
The human erythroblastoid cell line K562 and its derived mutant IA (23)
were transfected with cDNA encoding either the wild-type CD1d1. IA cells
are PigA-deficient and hence are unable to synthesize N-acetylglucosaminyl-PI (23), the first glycosylated product in GPI biosynthesis. Electroporation of cDNA into K562 and IA was performed as described (24). The
transfectants were selected in RPMI 1640 containing 0.8 mg/ml G418;
stable lines were maintained in 0.5 mg/ml G418.
All cell lines used in this study express wild-type and mutant CD1d1 as
well as H2Kb and H2Db molecules in the appropriate cell lines, which was
confirmed by flow cytometry and/or immune precipitation methods using
specific Abs (data not shown).
NKT cell hybridomas DN32.D3 and 431.A11 (gifts from A. Bendelac of
Princeton University, Princeton, NJ) as well as N38-2C12 and N37-1A12
(generously provided by K. Hayakawa of Fox Chase Cancer Institute, Philadelphia, PA) were maintained in RPMI 1640 containing 10% FBS.
L-glutamine-deficient
Flow cytometric analysis
To determine CD1d1 expression, ⬃5 ⫻ 105 cells were reacted with ⬃0.5
␮g of biotinylated 1B1, a CD1d1-reactive mAb, and biotinylated AF6, a
H2Kb-reactive mAb, and were detected with streptavidin-CyChrome using
a FACScan flow cytometer (BD Biosciences, San Jose, CA). All reagents
were from BD PharMingen (San Diego, CA).
Labeling CD1d1-associated ligand with [3H]mannose,
[32P]orthophosphate, [3H]inositol, [3H]ethanolamine, and
[3H]mevalonic acid
At least ⬃2 ⫻ 107 CD1d-expressing cells or control cells were tritium
labeled with 250 ␮Ci of [3H]2-mannose or [3H]ethanolamine (American
Radiolabeled Chemicals, St. Louis, MO) as described (19). Cells were
labeled with 250 ␮Ci of [3H]inositol (NEN, Boston, MA) in inositol-free
DMEM (Life Technologies) or with 250 ␮Ci of [3H]mevalonic acid
(American Radiolabeled Chemicals) in DMEM. [32P]Orthophosphate labeling was accomplished using phosphate-free DMEM (Life Technologies) as described (25). Culture supernatants from tritium-labeled cells
were collected for the purification of soluble molecules. The harvested cells
were solubilized in 2.5–3 ml of PBS containing 1% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (Sigma-Aldrich, St. Louis, MO). Cell lysates were clarified by centrifugation. Membrane-bound CD1d1 and class I molecules were isolated from the
postnuclear fraction, whereas total cellular lipids were extracted from the
nuclear and membrane pellet.
Mouse CD1d1-specific hAs
Polyclonal heteroantiserum (hAs) against CD1d1 was generated in a rabbit
immunized i.m. with ⬃0.1 mg of Ni-affinity purified sCD1d1 (this Ag was
⬎95% pure) emulsified in Ribi adjuvant. The sCD1d1 immune rabbit was
boosted three times with 50 ␮g of sCD1d1 at 3- to 4-wk intervals. The
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The full-length ␤2ma cDNA from pEE6-␤2m (21) was digested with
HindIII and BamHI, and the resulting fragment was subcloned into
HindIII-BamHI-digested pEE12 (CellTech, Slough, England). The resulting
pEE12-␤2m was checked for integrity by restriction mapping. Full-length
CD1d1 cDNA (pBluescript-mCD1d1; kindly provided by Dr. S. Balk, Harvard Medical School, Boston, MA), was subcloned into the pVL1393 vector (kindly provided by Dr. M. D. Summers, Texas A&M University, College Station, TX) using the XhoI-NotI restriction sites. The EcoRI-EcoRV
fragment from pVL1393-CD1d1 containing the first four exons of CD1d1
was subcloned into pCR3 (Invitrogen, Carlsbad, CA). Finally, the EcoRVNotI fragment containing exons 5 and 6 of CD1d1 was subcloned into
pCR3 containing exons 1– 4 of CD1d1 resulting in pCR3-mCD1d1; thus,
the full-length cDNA encoding wild-type CD1d1 was generated. The
cDNA encoding soluble CD1d1 (pBluescript-sCD1d1; also provided by
Dr. S. Balk) was digested with XhoI and blunted with Klenow polymerase.
The XhoI blunt-NotI fragment containing the sCD1d1 cDNA was subcloned into pCR3, resulting in pCR3-sCD1d1. For cloning, the pCR3 vector was prepared by digesting with HindIII and then was blunted with
Klenow polymerase and digested with NotI. The cDNA for an endoplasmic
reticulum (ER)-retained CD1d1 molecule was constructed by appending
the ER retention signal KDEL to the carboxyl terminus of sCD1d1. This
construct also contained a c-Myc tag between sCD1d1 and the ER retention
signal to facilitate detection of the expressed CD1d1. The sCD1d1-er
cDNA was constructed using a 70-bp-long DNA encoding the c-Myc tag
and KDEL. It entailed the annealing of the following four oligonucleotides:
a, 5⬘-AAGACTGAAATGGAGCAAAAGCTCATTTCTGAA-3⬘; b, 5⬘GAGGACCTGAATTCGGAGAAGGATGAGCTCTGAAGATCTGC-3⬘;
c, 5⬘-GCGGCCGCAGATCTTCAGAGCTCATCCTTCTCCGAATTCAG-3⬘;
and d, 5⬘-TCCTCTTCAGAAATGAGCTTTTGCTCCATTTCAGTCTT-3⬘.
This resulted in a DNA fragment with a 5⬘ blunt end and a 3⬘ NotI overhang. The oligonucleotides were phosphorylated and ligated together to
form the ⬃70-bp fragment. The resulting fragment was cloned into the
EcoRV-NotI site of pCR3-mCD1d1, resulting in pCR3-sCD1d1-er. A
CD1d1 mutant lacking its internalization signal was constructed by substituting the transmembrane and cytosolic region of CD1d1 with that of
H2Kb, resulting in CD1d1-Kbtail. The CD1d1-Kbtail cDNA was constructed by subcloning an EcoRV-NotI fragment containing exons 5– 8 of
H2Kb into the EcoRV-NotI site of pCR3-sCD1d1. In sCD1d1 cDNA, the
EcoRV site lies downstream of exon 4, which encodes the ␣3 domain of
the mature protein. cDNA encoding the soluble H2Db was constructed by
PCR amplification of exons 1– 4 using 5⬘-CACAAGCTTGGGAATTC
CGGGGGCGATGGCTCCGCG-3⬘ forward and 5⬘-CGGGATCCCGTCA
CCATCTCAGGGTGAGGGG-3⬘ reverse primers. The resulting product
was cleaved with HindIII and BamHI and cloned into the HindIII and
BamHI site of pCR3. An authentic pCR3-Db-sol determined by Sanger
dideoxynucleotide sequence analysis was used for gene transfer.
The Journal of Immunology
immune rabbit was terminally bled 2 wk after the last boost. The specificity
of the resulting hAs was determined using CD1d1-positive cell lines by
flow cytometry as well as by immune precipitation of [35S]cysteine/
[35S]methionine-labeled proteins. It only precipitates CD1d1 from cell
lines and no other molecule. In fact, it does not even cross-react with the
human homolog CD1d or paralog CD1b (data not shown). Therefore, it is
less likely to cross-react directly with a lipid, lipoprotein, or another lipid
binding protein.
Biosynthetic labeling of proteins
Affinity chromatography
CD1d1 molecules and the control class I molecules from each sample were
sequentially purified by immune affinity chromatography as previously described (28). CD1d1-specific and class I-specific affinity columns were
prepared by prebinding ⬃0.1– 0.15 mg of purified Ab to 1 ml of 50%
protein A-Sepharose slurry (Repligen, Needham, MA). Unbound Ab and
nonspecifically bound proteins were washed away with PBS and resuspended in an equal volume of the same buffer. Each Ab-bound protein
A-Sepharose was packed into a 5-ml disposable column (Pierce, Rockford,
IL) and used to purify CD1d1 and class I molecules. Immune affinity chromatography was performed as described earlier (28). Secreted CD1d1 from
the tritium labeling supernatants was purified using Hi-Trap metal chelating columns (Amersham Pharmacia Biotech, Piscataway, NJ) according to
the manufacturer’s instructions. Ten 0.5-ml fractions were collected; onetenth of each fraction was dissolved in Econo-safe scintillation fluid (RPI,
Mount Prospect, IL) to measure radioactivity using a scintillation counter
(LS6500; Beckman Coulter, Fullerton, CA).
Lipid extraction from affinity-purified proteins
Radioactive fractions from each affinity-purified sample were pooled. An
equal number of fractions not containing radioactivity were pooled separately. Lipids were extracted by Bligh-Dyer method (29) with two volumes
of chloroform:methanol (2:1 C:M). After thorough mixing, the top aqueous
and the bottom organic phases were allowed to separate. The organic phase
was carefully transferred into another tube. The aqueous phase was extracted two or three more times. The resulting organic phase was pooled
with the first and dried under vacuum or a gentle stream of N2 gas. The
dried extract was redissolved in 0.5 ml of C:M. Radioactivity was monitored by scintillation counting using ⬃10 –20 ␮l of the extracted lipids.
cilitated by an FLA 2000 Fluorescent Image Analyzer (Fuji Medical
Systems).
NKT cell stimulation and ELISA
Equal numbers (⬃5 ⫻ 104 cells per well) of stimulator cells and responder
NKT cell hybridomas were cocultured for 18 –20 h at 37°C. Stimulation of
hybridomas was measured by monitoring IL-2 secretion by ELISA. ELISA
was performed using JES6-1A12 and JES5-5H4 (BD PharMingen) as IL-2
capture and detection mAbs, respectively, according to the manufacturer’s
instructions.
Results
A mannosylated phospholipid associates with mouse CD1d1
Initial characterization of the CD1-associated ligand(s) relied on
determining the mass of the compounds eluted from CD1d1. Interpretation of the mass spectral data suggested the natural ligand
of CD1d1 to be GPI (19). To study CD1-lipid interactions in vivo
as well as to characterize the natural CD1-associated ligand(s), a
biochemical method was established. For this purpose, cells that
express the soluble form of CD1d1 and H2Db class I molecules,
sCD1d1 and Db-sol, respectively, were generated. Soluble molecules were used to establish the biochemical method because it
provides an abundant source of CD1d1 and, hence, the associated
ligand(s). Additionally, in a previous study we had demonstrated
that [3H]2-mannose-labeled ligands specifically associated with
sCD1d1 (19).
Thus, to characterize the natural CD1d1-associated ligand(s),
both sCD1d1 and Db-sol were radiolabeled with [3H]2-mannose.
[3H]2-mannose was used in this experiment because 1) its label is
seldom lost to another sugar but fucose (31) and 2) it labels GPI as
well as DPM-glycans. sCD1d1 and control Db-sol were affinity
purified from the supernatant of [3H]2-mannose-labeled cells. The
associated ligand(s) were extracted by the Bligh-Dyer method,
separated by TLC in a neutral mobile phase along with nonradioactive mammalian PI as a standard, and visualized by fluorography. The data revealed that [3H]2-mannose was incorporated into
a dominant lipid species along with two minor ones that were
associated with sCD1d1 (Fig. 1, lane 1) but not with Db-sol (Fig.
1, lane 2). Interestingly, the three CD1d1-associated lipid species
have a migratory pattern on the TLC that was distinct from the
nonradioactive PI standard (Fig. 1, arrow) with a relative migration
(Rf) suggestive of a polar compound.
One possible explanation of the above analysis is that [3H]labeled lipids spilled into the tissue culture medium from cells
Enzymatic digestions of lipids
C:M in two equal aliquots of the lipid extracts were evaporated under
vacuum or N2 gas. One aliquot was dissolved in 50 ␮l of PBS (pH 7–7.4)
and digested with ⬃55 mU of PI-specific phospholipase C (PI-PLC; Glyko,
Novato, CA) at 37°C for 1 h. Likewise, the second aliquot was dissolved
in 50 ␮l of buffer containing 4.9 mM CaCl2 and 147 mM NaCl (pH 8.9)
and digested with ⬃1 U of phospholipase A2 (PLA2) (Sigma-Aldrich) at
25°C for 1 h. The enzymatic reactions were stopped by C:M extraction.
The organic phase was evaporated, resuspended in 20 – 40 ␮l of C:M, and
spotted onto a 20-cm ⫻ 20-cm K6 silica gel TLC plate (Whatman, Clifton,
NJ). Approximately 25 ␮g of PI was spotted and served as a standard.
Additionally, ⬃0.025 ␮Ci of [3H]PI or [3H]DPM (American Radiolabeled
Chemicals) was used as radioactive standard.
TLC
TLC was performed in a chamber saturated with 100 –150 ml of neutral
mobile phase containing chloroform:methanol:water in 10:10:3 ratio (30).
The plate was dried for at least 1 h, sprayed with EN[3H]ANCE (NEN Life
Sciences) according to the manufacturer’s directions, and exposed to autoradiographic film or a phosphoimager TR plate specifically sensitive to
tritium (Fuji Medical Systems, Stamford, CT). Phosphorimaging was fa-
FIGURE 1. Mannose-containing lipids associate with CD1d1. Lipids
were extracted from affinity-purified soluble CD1d1 and soluble H2Db expressed by [3H]2-mannose-labeled sCD1d1 (lane 1) and Db-sol (lane 2)
cells. They were separated by TLC using a neutral mobile phase (C:M:
HOH, 10:10:3 ratio). Tritium signal was amplified with EN[3H]ANCE
spray and detected by phosphorimaging. Nonradioactive PI was used as the
standard in this experiment; its position after TLC is indicated by an arrow.
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Steady-state as well as pulse labeling of cells with [35S]cysteine/[35S]methionine and the chase of labeled proteins were performed as described
(21). After preclearing with normal mouse serum, samples were immune
precipitated with the anti-CD1d1 hAs or an appropriate control Ab as described. An H2Kb-specific (Y3) (26) or H2Db-specific (B22-249) (26) mAb
was used when experiments were performed with mouse cell lines. W6/32
(generously provided by J. Yewdell of National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD), an antiHLA class I-specific mAb (27), was used when experiments were performed in human cell lines. Immune precipitates were separated by 15%
SDS-PAGE and visualized by autoradiography. In pulse-chase experiments, the immune precipitates were subjected to 1–2 mU or varying concentrations of endoglycosidase H (endo H) for 14 –16 h and were processed
as above.
725
726
FIGURE 2. A natural ligand of CD1d1 is mannosylated-PI glycan. A,
Lipids were extracted from affinity-purified soluble CD1d1 and soluble
H2Db secreted by [3H]2-mannose-labeled sCD1d1 and Db-sol cells. Lipids
extracted from sCD1d1 were divided in two aliquots. One aliquot was
subjected to PI-PLC digestion (lane 1) and the other to PLA2 digestion
(lane 2), and then they were re-extracted and separated by TLC using a
neutral mobile phase (C:M:HOH, 10:10:3 ratio) along with untreated lipid
extract of Db-sol (lane 3). About 0.025 ␮Ci of [3H]PI and [3H]DPM were
used as standards (lanes 4 and 5, respectively). Tritium signal was amplified with EN[3H]ANCE spray and detected by autoradiography. B, Total
lipids were extracted from the postnuclear fraction of [3H]2-mannose-labeled Db-sol (lane 1) and sCD1d1 (lane 2) cells solubilized in detergent.
The two lipid samples along with [3H]PI and [3H]DPM were separated by
TLC and visualized as in A.
added to proteins contains three sialic acid units, it is less likely to
migrate to the center of the TLC as did the [3H]2-mannose-labeled
ligand extracted from sCD1d1. Therefore, we conclude that the
[3H]2-mannose-labeled compound associated with CD1d1 is not
its own glycan.
To determine the chemical nature of the [3H]2-mannose-labeled
lipid(s) associated with CD1d1, in the second experiment the
Bligh-Dyer extracted ligand(s) was subjected to PI-PLC or PLA2
digestion. PI-PLC specifically cleaves inositol-containing phospholipids; its enzymatic activity is sensitive to acyl-modification of
the inositol head group (32). In contrast, PLA2 specifically cleaves
fatty acyl modification at C-atom 2 of glycerolipids; it is sensitive
to the stereochemistry of the asymmetric C-atom 2 (33). The enzymatic reaction was stopped by lipid extraction; the products
were separated by TLC in a neutral gradient along with PI as well
as [3H]PI and [3H]DPM standards and were detected by autoradiography. The data revealed that cleavage with PI-PLC resulted in
the loss of the [3H]2-mannose label from the sCD1d1-associated
ligand (Fig. 2A, lane 1). This result is consistent with release of the
water-soluble [3H]2-mannose-labeled phosphoinositol-glycan
from mannosylated PI because the glycan does not partition into
the organic phase. In contrast, cleavage with PLA2 did not result in
the loss of the [3H]2-mannose label (Fig. 2A, lane 2). Both the
PLA2-cleaved and uncleaved [3H]2-mannose-labeled PI partition
into the organic phase during Bligh-Dyer extraction. Therefore, it
is unclear whether the sCD1d1-associated mannosylated PI is sensitive to PLA2 or not. Thus, together with the previously published
mass spectral data, we conclude that GPI is a major [3H]2-mannose-labeled lipid associated with CD1d1.
To determine the diversity of cellular lipids that incorporate
[3H]2-mannose, one-tenth of the postnuclear detergent lysate of
[3H]2-mannose-labeled sCD1d1 and Db-sol cell lines was subjected to Bligh-Dyer extraction. The organic phase of this extract
was separated by TLC in a neutral mobile phase and visualized by
autoradiography. The data revealed that both sCD1d1 and Db-sol
cell lines incorporated [3H]2-mannose into several glycolipids
(Fig. 2B) whose identities, because they are not essential to this
study, were not determined. Thus CD1d1 selects a single dominant
ligand from a cellular pool of several [3H]2-mannose-labeled
glycolipids.
Stoichiometry of CD1d1-GPI association. Previously, we reported that ⬎90% of CD1d1 molecules are occupied by GPI. Being a glycoprotein, [3H]2-mannose labels the glycan moiety of
CD1d1 ␣-chain as well. Therefore, the ratio of the [3H]2-mannose
label associated with CD1d1 to that of the extracted ligands provides a measure of what percentage of CD1d1 molecules is associated with GPI. To determine the number of glycan groups on
CD1d1, it was immune precipitated from cells pulse labeled with
[35S]cysteine/[35S]methionine and either not chased or chased for
2 h. The immune precipitates were then subjected to digestion with
varying concentrations of endo H. H2Kb, which has one N-glycan
modification (34), was used as the control. The data revealed five
endo H-sensitive radioactive bands in the case of CD1d1 (Fig. 3A)
and one, as expected, in the case of H2Kb (Fig. 3B). Thus, all
predicted glycosylation sites of CD1d1 are modified by N-linked
glycans.
To determine the stoichiometry of CD1d1 and GPI, the amount
of radioactivity in the aqueous and the organic phases of the BlighDyer extract were monitored. Because CD1d1 is modified by five
glycans, it would contain ⬃15 mannose residues. GPI, in contrast,
contains a minimum of three mannose residues. Therefore, a 1:1
stoichiometry of CD1d1 to GPI should yield five times as much
radioactivity in the aqueous phase compared with the organic
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dying during the labeling period could nonspecifically bind
CD1d1. However, this is less likely because of the following observations. First, such lipids would be captured by BSA used in the
tissue culture medium. BSA binds nonspecifically to numerous
lipids and glycolipids that were tested in an in vitro binding assay
(S. Joyce, unpublished data). It copurifies during affinity chromatography with Ni-Sepharose, especially when hexahistidine-tagged
proteins in the culture supernatant are in low concentrations (S.
Joyce, unpublished data). Despite nonspecific purification, [3H]labeled lipids were not found in Db-sol supernatants passed over
Ni-Sepharose columns (Ref. 19 and data not shown). Second,
CD1d1 should bind all the [3H]2-mannose-labeled lipids synthesized by the cell (Fig. 2B) were it nonspecifically “mopping-up”
the numerous [3H]-labeled lipids spilled into the tissue culture medium. This it does not, and hence the [3H]-labeled lipids shown in
Fig. 2A are specifically bound to CD1d1.
PI-PLC-sensitive ligand associates with CD1d1. Mannose can
randomize to glucose, galactose, and fucose. However, because the
label in [3H]2-mannose is on the hydrogen of C-atom 2, the label
would be predominantly found in mannose and fucose. In rare
instances, the label from [3H]2-mannose can be found in glucose
and lactic acid, suggesting that during oxidation and reduction reaction resulting in the epimerization of mannose to glucose, the
label may be transferred from C-atom 2 to C-atom 1 (see Ref. 31).
Thus, the major labeled spot in Fig. 1 could be due to a glycan
modifying a lipid and/or a protein. For three reasons we believe
that the [3H]2-mannose-labeled compound is a glycolipid and not
a glycopeptide or glycoprotein. First, the control Db-sol, also a
glycoprotein with two N-linked glycans, processed in the same
manner as sCD1d1, does not contain the tritiated spots seen with
the CD1 sample (Fig. 1). Second, the CD1d1-associated [3H]2mannose-labeled ligands are PI-PLC sensitive as described below.
Third, sialic acid-containing glycans have poor mobility on TLC
and hence remain at or a bit above the origin in the mobile phase
used. For example, ceramide migrates to the front of the TLC,
whereas sialic acid containing glycoceramides remains close to the
origin (S. Joyce, unpublished data). Because each glycan moiety
ASSEMBLING A CD1 MOLECULE IN VIVO
The Journal of Immunology
phase. In over three separate experiments, approximately seven to
10 times the radioactivity was found in the aqueous phase compared with that in the organic phase (data not shown). Thus,
greater than half the total CD1d1 molecules were associated with
[3H]2-mannose-labeled GPI. Considering 70–80% efficiency of
Bligh-Dyer extraction, consistent with the previous report (19),
⬎90% of CD1d1 molecules are associated with GPI.
Calculations of stoichiometry require steady-state labeling of
constituents being compared. Note that DPM and dolichylphosphorylglycans that contain mannose are precursors that feed into
the biosynthesis of glycans added onto PI as well as glycoproteins. Their rates of synthesis are about the same within cells and,
hence, the precursor pools should be the same after pulse with
[3H]2-mannose, which in our experiments was for 24 h. Therefore,
the results presented herein from our calculations will not differ
from the actual stoichiometry of CD1d1 and the associated
compound.
phospholipids. The data revealed that three [3H]inositol-labeled
lipids were associated with CD1d1 (Fig. 4A, lane 2). Two of the
[3H]inositol-labeled lipids were specifically extracted from
CD1d1, but the third faster migrating lipid was also found in extracts of H2Kb (Fig. 4A, lanes 1 and 2). The two [3H]inositollabeled lipids were selected by CD1d1 among several 8–10 biosynthetically labeled lipids synthesized by the cell (Fig. 4C, lane
1). Of the two [3H]inositol-labeled lipids associated with CD1d1,
one has the same Rf as the [3H]PI standard, suggesting that this
lipid may be PI. The [3H]PI standard consists of stearic and arachidonic acid at C-atoms 1 and 2 (according to the supplier), the
two fatty acids predicted from the mass spectral data of CD1d1associated ligand(s) reported previously (19).
To determine whether the [3H]inositol-labeled lipids were PI,
they were subjected to PI-PLC digestion. The lipid spot that comigrates with the [3H]PI standard was specifically lost upon digestion
with PI-PLC (Fig. 4A, lanes 2 and 4). However, the slow migrating
lipid spot was partially resistant to PI-PLC (Fig. 4A, lane 4). PIPLC activity on CD1d1-associated [3H]inositol-labeled ligand is
specific because it cleaves [3H]PI standard (Fig. 4B, lane 2) but not
[3H]DPM (data not shown). Also note that PI-PLC digestion of
[3H]PI results in a slow migrating spot (Fig. 4B, arrow) similar to
that observed upon cleavage of CD1d1-associated ligand with PIPLC (Fig. 4A, arrow). This new species could be [3H]inositolphosphate. Together, the data suggest that the faster migrating CD1d1associated lipid that comigrates with the [3H]PI standard is
mammalian PI, whereas the identity of the second lipid species is
unknown. Thus, PI is another CD1d1-associated natural ligand.
The extent to which it occupies CD1d1 was not measurable in the
current experiment.
Of the two CD1d1-associated lipid spots, only the slowest migrating species was partially susceptible to PLA2 (Fig. 4A, lanes 2
and 3). Resistance to PLA2 was not due to inactivity of the enzyme, because a larger quantity of [3H]PI standard compared with
Select few cellular lipids associate with CD1d1
As noted above, the majority of sCD1d1 assemble with GPI. However, because [3H]2-mannose does not label all cellular lipids, it
does not reveal the repertoire of ligands that associate with CD1d1.
Additionally, our previous study revealed an ion pertaining to free
PI and several unidentified ions. These observations raised the
question regarding the diversity of the repertoire of CD1d1-associated cellular lipids. To address this question, cell lines expressing
wild-type CD1d1 and control H2Kb were generated. These were
radiolabeled with [32P]orthophosphate (a precursor of phospholipids), [3H]inositol (a precursor of PI and GPI), [3H]ethanolamine (a
precursor of phosphatidylethanolamine and, to a lesser extent, of
phosphatidylserine and phosphatidylcholine), or [3H]mevalonic
acid (a precursor of dolichol and cholesterol) (35, 36). The associated ligand(s) was isolated and characterized as described above.
Because the same lipid spots of very similar intensity were extracted from CD1d1 and the control H2Kb (data not shown), it was
difficult to ascertain the phospholipid repertoire specifically associated with CD1d1 by [32P]orthophosphate labeling method.
CD1d1 associates with PI. Therefore, the above experiments
were repeated using labels that are precursors of specific classes of
FIGURE 4. CD1d1 assembles with PI. A, [3H]Inositol-labeled lipids
were extracted from affinity-purified CD1d1 (lanes 2– 4) as well as control
class I H2Kb (lane 1), and the CD1-derived lipids were divided into three
aliquots. One aliquot was treated with buffer alone (lane 2), the second with
PLA2 (lane 3), and the third with PI-PLC (lane 4). After enzymatic digestion, the lipids were extracted and separated by TLC as in Fig. 2B. [3H]Labeled lipids were detected as in Fig. 1. B, [3H]PI standard was undigested (lane 1) or digested with either PI-PLC (lane 2) or PLA2 (lane 3),
extracted, separated, and visualized as described in Fig. 2B. C, Total lipids
were extracted from the postnuclear fraction of [3H]inositol-labeled
mCD1d1 (lane 1) and Kb-high (lane 2) cells solubilized in detergent. The
two lipid samples were separated by TLC and visualized as in Fig. 2B.
Standards included ⬃0.025 ␮Ci of [3H]PI and [3H]DPM (lanes 5 and 6 in
A, lanes 4 and 5 in B, and lanes 3 and 4 in C). Note that a slow migrating
polar compound, presumably [3H]inositol-1-phosphate, results from the digestion of CD1d1-associated PI as well as the [3H]PI standard (see Fig. 4,
A and C). F, Front; O, origin; a, [3H]DPM standard; b, [3H]PI standard.
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FIGURE 3. CD1d1 is modified at all predicted N-linked glycosylation
sites. Cells expressing wild-type CD1d1 and H2Kb were pulse-labeled for
30 min with [35S]cysteine/[35S]methionine, washed with PBS, and divided
in two equal aliquots. One aliquot was maintained on ice, whereas the other
was chased for 2 h. Both were solubilized in detergent, and the postnuclear
fraction was divided into six equal aliquots. CD1d1 (A) and H2Kb (B) were
immune precipitated from the respective postnuclear fractions. The immune precipitates were subjected to cleavage of N-linked glycans with
varying concentrations of endo H, separated by 10% SDS-PAGE under
reducing conditions and detected by autoradiography. The arrows show the
various N-glycosylated forms of CD1d1 H chain. Data are representative of
two similar experiments.
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CD1d1 assembles with PI in the ER
It is clear that neither GPI nor PI is the natural Ag of NKT cells
(20). However, it is possible that GPI and PI have a chaperone-like
function in the assembly of CD1d1. If they do indeed function as
chaperones, then GPI and/or PI would assemble with CD1d1 in the
ER. Thus, two cell lines were generated. One line retains the expressed CD1d1 in the ER by virtue of containing the KDEL signal
(37, 38) at its carboxyl terminus (sCD1d1-er). The second line
expresses a mutant CD1d1 that had its transmembrane and cytosolic tail replaced with that from the nonrecycling H2Kb (sCD1d1Kbtail) and, hence, has lost its endosome/lysosome-targeting signal. The expression and intracellular trafficking patterns of the
wild-type CD1d1 as well as sCD1d1-er and sCD1d1-Kbtail mutants were determined. For this purpose, cell lines expressing the
wild-type and the mutant CD1d1 molecules were pulse labeled
with [35S]cysteine/[35S]methionine and chased for the indicated
time periods. Cells were solubilized with detergent, and the postnuclear fraction was subjected to immune precipitation with
CD1d1-specific hAs. The sCD1d1-er mutant was also immune precipitated from the labeling supernatant. Immune precipitates were
subjected to endo H digestion. Both CD1d1 and sCD1d1-Kbtail
traffic through the secretory pathway with similar kinetics; their
t1/2 in the ER is between 1 and 2 h (Fig. 6A, upper two panels).
Thus, replacement of the transmembrane and the cytosolic regions
of CD1d1 with those of H2Kb did not significantly alter the intracellular traffic of the mutant CD1d1 molecule.
In contrast, the traffic pattern assessed by pulse-chase experiments described above is significantly altered in the case of
sCD1d1-er. Its t1/2 in the ER is ⬃4 – 8 h (Fig. 6A). The delayed
egress of the sCD1d1-er mutant from the ER is consistent with the
function of the KDEL ER-retention signal (37, 38). A reason that
the ER retention signal was appended to the secreted form of
CD1d1 is that, if it did egress from the ER, it would not be retrieved but secreted into the growth medium so that it will not
confound the experiment described in Fig. 6C. The sCD1d1-er
mutant is indeed secreted into the labeling supernatant upon
achieving endo H resistance (Fig. 6A).
Whether sCD1d1-Kbtail had lost its ability to traffic through the
endosome/lysosome compartment was determined using CD1d1restricted NKT cell hybridomas as probes. One V␣14J␣15 NKT
cell hybridoma, DN32.D3, recognizes an endosomal/lysosomal Ag
presented by CD1d1 (39) and, hence, should not be activated by
sCD1d1-Kbtail. A second CD1d1-restricted NKT cell hybridoma
431.A11, which expresses the V␣3 receptor, recognizes an Ag
present in the anterograde secretory pathway (39). It should be
activated by sCD1d1-Kbtail. Thus, both DN32.D3 and 431.A11
recognize mCD1d1 (Fig. 6B). Moreover, as expected, DN32.D3
did not recognize, whereas 431.A11 did recognize, sCD1d1-Kbtail
(Fig. 6B). Thus, sCD1d1-Kbtail inefficiently, if at all, traffics
through the endosomal/lysosomal compartments.
Thus, the wild-type and the two mutant CD1d1 molecules provide a good system to determine the site of CD1d1 assembly with
cellular lipids. Cell lines expressing the three forms of CD1d1 as
well as control H2Kb were metabolically labeled with [3H]inositol
and solubilized in detergent, and CD1d1 as well as H2Kb were
sequentially affinity purified from their postnuclear fractions. The
eluted fractions were collected and the amount of radioactivity
in each of 10 fractions was measured. The data revealed that
[3H]inositol was incorporated into molecules associated with
CD1d1 but not significantly into H2Kb (Fig. 6C). The parent cell
line used for ectopic expression of CD1d1 and H2Kb expresses a
small amount of endogenous CD1d1 (data not shown). Because
[3H]inositol is incorporated into PI, assembly of CD1d1 with PI
occurs in the ER. Moreover, GPI is associated with sCD1d1, a
form that does not enter the endosome/lysosome compartment.
Thus, we conclude that the assembly of PI and GPI with CD1d1
occurs in the ER.
PI is sufficient for complete assembly and intracellular traffic of
CD1d1
FIGURE 5. Dolichylphosphorylglycans are not natural CD1d1 ligands.
A, Lipids were extracted from affinity-purified CD1d1 (lanes 4 and 6) and
H2Kb (lanes 3 and 5) from [3H]mevalonic acid-labeled mCD1d1 (lanes 5
and 6) and Kb-high (lanes 3 and 4) cells. Data are representative of two
similar experiments. B, Total lipids were extracted from the postnuclear
fraction of [3H]mevalonic acid-labeled mCD1d1 (lane 1) and Kb-high
(lane 2) cells solubilized in detergent. The lipids were separated by TLC
and detected as in Fig. 2B. Standards included ⬃0.025 ␮Ci of [3H]PI and
[3H]DPM (lanes 1 and 2 in A; lanes 3 and 4 in B).
Because GPI is the major natural ligand of CD1d1 and PI assembles with CD1d1 in the ER, it is possible that these cellular phospholipids play a chaperone-like role in the assembly of CD1d1.
Self-peptides derived from the cytosol play a critical role in the
assembly of MHC class I molecules in the ER. Proper assembly of
class I molecules is inhibited by the lack of peptides in the ER (40).
Thus, we reasoned that the absence of GPI and PI in cells would
adversely affect CD1d1 assembly in GPI-deficient cell lines.
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the CD1d1-associated ligand was sensitive to PLA2 (Fig. 4B, lane
3). Because PLA2 is sensitive to the stereochemistry of C-atom 2
of glycerophosphatides, the configuration of this atom in CD1d1associated PI may be distinct from the standard mammalian PI.
It was puzzling that the [3H]inositol label was not incorporated
into GPI. If GPI was labeled, it, being more polar, would migrate
more slowly than PI upon TLC. Such a slow migrating lipid with
an Rf similar to the mannosylated-PI-glycan observed in Fig. 1 was
not present among CD1d1-associated [3H]inositol-labeled lipids
(Fig. 4A). The reason for this is currently unknown.
Dolichylphosphorylglycans and phosphatidylethanolamine do
not assemble with CD1d1. Because diolichylphosphorylglycans
and phoshatidylethanolamine are indigenous to the ER, whether they
also assemble with CD1d1 was determined. The data revealed that
neither CD1d1 nor H2Kb contained any [3H]mevalonic acid-labeled
lipid (Fig. 5A), despite the fact that the cell lines that express them
contained numerous biosynthetically labeled lipids (Fig. 5B). The fast
migrating predominant spot extracted from CD1d1 has an Rf predicted for cholesterol; it was not found consistently. Additionally, this
lipid was also found in extracts of the control class I molecules in the
repeat of this experiment (data not shown). Additional data revealed
that, similar to the [32P]orthophosphate labeling experiments, the
[3H]ethanolamine label was nonspecifically associated with lipids extracted from CD1d1 as well as H2Kb. Thus, dolichylphosphorylglycans and phosphatidylethanolamine may be minor, if at all, ligands of
CD1d1.
ASSEMBLING A CD1 MOLECULE IN VIVO
The Journal of Immunology
729
CD1d1 assembles with PI in GPI-deficient cells
FIGURE 6. CD1d1 assembles with PI in the ER. A, Cell lines expressing wild-type mCD1d1, as well as the two mutants sCD1d1-Kbtail and
sCD1d1-er, were pulse labeled with [35S]cysteine/[35S]methionine and
chased for the indicated times. CD1d1 was immune precipitated with
CD1d1-specific hAs; the immune complexes were digested with endo H,
separated by 15% SDS-PAGE, and detected by autoradiography. In the
case of sCD1d1-er, secreted CD1d1 was also immune precipitated from the
labeling and the chase supernatants to detect any sCD1d1-er that escaped
ER retention. r, Endo H resistant; s, endo H sensitive. Data are representative of three similar experiments. B, V␣14J␣15-positive DN32.D3 and
the V␣14-negative 431A.11 (V␣3) NKT cell hybridomas were individually
cocultured with wild-type or mutant CD1d1. The supernatants were collected after 12–18 h of coculture, and secreted IL-2, a measure of NKT cell
activation, was detected by ELISA. Data are representative of at least five
similar experiments. C, CD1d1-positive cell lines, mCD1d1, sCD1d1Kbtail, and sCD1d1-er, as well as Kb-high, were labeled with [3H]inositol.
CD1d1 and H2Kb were affinity purified from the labeled cells, and the
radioactivity within each of the ten eluted fractions was monitored by scintillation counting. Data are representative of two similar experiments.
To determine whether CD1d1 expressed in GPI-negative cells assembles with a cellular lipid, K562-mCD1d1 and IA-mCDd1 cell
lines were labeled with [3H]inositol. CD1d1 from both cell lines
were affinity purified from postnuclear fractions of detergent lysates, and the amount of radioactivity in each eluted fraction was
monitored by scintillation counting. The data revealed that the
[3H]inositol label was associated with CD1d1 and not with H2Kb
(Fig. 7D, a and d). Interestingly, about the same amount of
[3H]inositol-labeled compound was associated with CD1d1
expressed by GPI-positive K562-mCD1d1 and GPI-deficient
IA-mCD1d1 cells (Fig. 7D, b and c). Thus, CD1d1 assembles with
an inositol-containing lipid even in the absence of GPI.
To conclusively prove that the CD1d1-associated [3H]inositol is
indeed incorporated into PI, the associated ligand(s) was extracted,
separated by TLC, and detected by phosphorimaging. The data
revealed that one of the three detectable lipids extracted from
CD1d1 is indeed PI, because the Rf of this lipid is similar to the
mammalian [3H]PI standard (Fig. 7D) and because this spot extracted from mCD1d1 is sensitive to PI-PLC (Fig. 4A). Thus,
CD1d1 assembles with PI in GPI-deficient cells.
The association of CD1d1 with PI in the IA-mCD1d1 cell line
could be due to its assembly with human ␤2m. Therefore, mouse
cell lines S49a and Bw5147e deficient in Pig A and Pig E (complementation group E mutant that is unable to mannosylate glucosaminylated PI), respectively (22), were labeled with [3H]inositol, and their endogenous CD1d1 and H2 class I molecules were
affinity purified. Scintillation counting of the eluted fractions revealed that [3H]inositol-derived radioactivity was associated with
affinity purified CD1d1 only but not with control class I molecules
(data not shown). Thus, the association of CD1d1 with PI in IAmCD1d is not due to assembly with human ␤2m. Instead, the data
suggest that CD1d1 use lipids indigenous to the ER to assemble
stable CD1d1 in vivo.
Discussion
In summary, the data presented herein affirm that CD1d1, a member of the CD1 family of lipid binding proteins, selectively associates with PI and GPI in vivo. Additional data revealed that the
association of CD1d1 with the phospholipids occurs during the
assembly of CD1 in the ER. These findings are consistent with
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Therefore, the assembly and traffic of wild-type CD1d1 was studied in human GPI-positive K562 and the derived GPI-deficient cell
line IA. IA lacks functional Pig A and, hence, they are deficient in
the first glycosylated product of PI (23). Upon CD1d1 cDNA
transfer and selection, both wild-type (K562-mCD1d1) and the Pig
A mutant (IA-mCD1d1) cells express similar levels of CD1d1 (Fig.
7A). This expression pattern is consistent with the ability of
V␣14J␣15 NKT cells and derived hybridomas to recognize
CD1d1 expressed by Pig A-deficient mouse cell lines (Fig. 7B and
Ref. 20).
An explanation for normal expression of CD1d1 in GPI-positive
and GPI-deficient cells could be that a few CD1 molecules assemble in the absence of GPI and, hence, traffic to the plasma membrane. Because CD1d1 has a long half-life (⬎1 day; data not
shown), they can accumulate at the cell surface over time. Thus the
rate of intracellular traffic of newly synthesized CD1d1 was determined by pulse-chase analysis. The data revealed that CD1d1 in
both GPI-positive and GPI-negative cells egress from the ER with
similar kinetics (Fig. 7C). The t1/2 of ER residence is ⬃2 h (Fig.
7C). Thus, CD1d1 assembles normally in GPI-deficient cell lines.
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ASSEMBLING A CD1 MOLECULE IN VIVO
the discontinuous electron density observed within the ligand binding site in the mouse CD1d1 crystal structure. Note that this structure was determined without the addition of exogenous lipids during protein purification (7). Together the data suggest that lipids
indigenous to the ER may play a chaperone-like role in the assembly of CD1d1 in vivo akin to the function of invariant chain in
the assembly of MHC class II molecules in the ER.
Selective binding of lipids to CD1d1 in vivo
Among the numerous [3H]2-mannose-, [3H]inositol-, and [3H]mevalonic acid-labeled lipids, only PI and GPI assemble with CD1d1.
The label in [3H]2-mannose does not transfer into another sugar
but into fucose (31). Therefore, if a fucosylated lipid(s) is indeed
synthesized by the cell, it is not associated with CD1d1 because
such lipids are resistant to PI-PLC. Among the mannosylated lipids
synthesized in the ER are GPI and DPM-glycan. The latter are
precursors of N-linked glycans attached to glycoproteins (36).
They were not associated with CD1d1 in the [3H]2-mannose and
the [3H]mevalonic acid labeling experiments. Thus CD1d1 selects
GPI over DPM-glycans.
Two [3H]inositol-labeled lipids specifically associate with
CD1d1, one of which is PI-PLC-sensitive PI. The character of the
second, slow-migrating, [3H]inositol-labeled compound is unknown because no inositol-containing lipid other than PI is known
to exist in mammalian cells. That notwithstanding, inositol-containing ceramides are synthesized by yeast (41) in lieu of sphingomyelin (42). Although not described in mammalian cells, inositolceramide or a similar compound may be synthesized by certain
mammalian cells (e.g., tumor cells such as in NS0 plasmacytoma
and K562 erythroleukemia lines) used in the present study. Alternatively, because inositol can be converted to glucose, this novel
compound associated with CD1d1 could be a glucose-containing
lipid other than a PI-glycan. Additionally, this novel compound is
resistant to both PI-PLC and PLA2. Thus, further biochemical
studies are required to solve the structure of the novel CD1d1associated compound.
Mammalian cells synthesize nonacylated and acylated inositolcontaining GPI anchors for proteins (43), and protein-free GPI
anchors although usually contain acylated inositol (44) can contain
nonacylated inositol. The GPI extracted from CD1d1 is sensitive to
PI-PLC. Therefore, the GPI associated with CD1d1 does not contain an acylated inositol. Furthermore, the PI associated with
CD1d1 is resistant to PLA2 (45). PLA2 is sensitive to the stereochemistry of the asymmetric C-atom 2 of glycerophosphatides
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FIGURE 7. Normal CD1d1 assembly and intracellular traffic in GPI-deficient cells. A, Surface expression of CD1d1 in GPI-positive K562-mCD1d1 and
GPI-negative IA-mCD1d1 cells was determined by immunofluorescent staining of cells with biotinylated-1B1 (CD1d1-specific) mAb. Staining was
detected by flow cytometry. Data are representative of two similar experiments. B, K562-mCD1d1, IA-mCD1d1, mCD1d1 (positive control), or sCD1d1-er
(negative control) was individually cocultured with two V␣14J␣15-positive NKT cell hybridomas, N38-2C12 and N37-1A12. Stimulation of the hybridomas was measured by quantitating the secreted IL-2 in the coculture supernatant. Data are representative of two similar experiments. C, K562-mCD1d1
and IA-mCD1d1 cells were pulse labeled with [35S]cysteine/[35S]methionine and chased in the presence of excess cold cysteine and methionine for the
indicated periods of time. The postnuclear fractions of detergent solubilized cells were immune precipitated with CD1d1-specific hAs. The immune
complexes were digested with endo H, separated by 15% SDS-PAGE, and detected by autoradiography. r, Endo H resistant; s, endo H sensitive. Data are
representative of three similar experiments. D, CD1d1-positive cell lines, mCD1d1, GPI-positive K562-mCD1d1, and GPI-negative IA-mCD1d1, as well
as Kb-high were labeled with [3H]inositol. CD1d1 and H2Kb were affinity purified from the labeled cells; radioactivity within each of the 10 eluted fractions
was monitored by scintillation counting. E, [3H]Inositol-labeled lipids were extracted from affinity-purified CD1d1 expressed by GPI-negative IA-mCD1d1
(lane 1), GPI-positive K562-mCD1d1 (lane 2), and mCD1d1 (lane 4), as well as control class I H2Kb (lane 3). 3H-labeled lipids were separated by TLC
along with 0.025 ␮Ci of [3H]PI and [3H]DPM standards as in Fig. 2B and detected by phosphorimaging.
The Journal of Immunology
(33). Therefore, one plausible reason for the resistance of the
CD1d1-associated ligand to PLA2 might be the stereochemistry of
PI. Whereas the glycerol in cellular PI has its asymmetric C-atom
2 in L-configuration (45), that in the CD1d1 ligand might be in the
D-configuration. Alternatively, C-atom 2 in CD1d1-associated PI
may be modified by a PLA2-resistant ether linkage to an alkyl
group as opposed to the PLA2-sensitive ester linkage. The CD1d1associated PI is less likely to contain an alkyl group because its
mass would be distinct from the observed mass of CD1d1-associated PI reported previously (19).
Thus, CD1d1 exhibits selectivity in the type of natural ligands it
binds: selectivity is observed in the type of lipids that bind (e.g.,
CD1d1 selects PI and GPI over DPM-glycans and numerous other
cellular lipids). CD1d1-associated PI is resistant to PLA2, and
hence the C-atom 2 has a distinct configuration. Finally, CD1d1
associates with GPI distinct from the common mammalian GPI
(i.e., it does not contain an acylated inositol common to
mammalian GPI).
The association of PI and GPI with CD1d1 raised interest in the
biological significance of this finding. A thorough search for the
function of CD1d1-GPI complex in vivo revealed that it is inert to
the immune system. Thus, NKT cells do not recognize the CD1d1GPI complex (Ref. 20 and data not shown). However, the possibility remains that the CD1d1-PI can be recognized by some NKT
cells. Consistent with this possibility, one study reported the recognition of PI in the context of CD1d1 by an autoreactive
V␣14J␣15-positive NKT cell hybridoma (18). Thus, this finding
supports the biological relevance of the CD1d1-associated PI described herein. That notwithstanding, the prevalence of PI-reactive
NKT cells in vivo and their physiological role remain undefined.
The data presented herein support the idea that the lipids associated with CD1d1 play a chaperone-like role in the assembly of
CD1d1. Being a type I integral membrane protein, akin to the
classical Ag-presenting molecules for which much is known regarding assembly in vivo, the assembly of CD1d begins as it is
cotranslationally inserted into the ER. Soon thereafter it binds calnexin, a step inferred from the latter’s association with a group I
CD1 synthesized in the absence of ␤2m (46) as well as with MHC
class I molecules (47– 49). Calnexin probably prevents aggregation of unassembled CD1 H chains. The monomeric H chain then
assembles with ␤2m, forming a heterodimer that is receptive to
lipids of the ER and/or the downstream vesicular compartment(s).
The ability to extract PI from both wild-type CD1d1 and derived
mutants that do not negotiate the recycling compartment (19) suggests that the association of the cellular ligand with CD1 occurred
in the secretory pathway. The association of lipids with CD1d1
during biosynthesis probably satisfies two structural requirements:
1) protecting the deep, nonpolar hydrophobic Ag binding groove
from collapse, and 2) occupying that groove with a ligand that
might be easily exchanged for an Ag in a late secretory vesicle.
Thus, PI and GPI might play a chaperone-like role (Fig. 8) akin to
the function of MHC class II-associated invariant chain in class II
assembly in vivo (50).
CD1d1 expression in PIG-A mutants is normal. Additionally,
V␣14J␣15-positive and -negative NKT cells recognize CD1d1 expressed by the mutant as effectively as they recognize those expressed by wild-type cells. Thus, GPI, albeit a major natural ligand
of CD1d1, is neither essential for the assembly of CD1d in vivo
nor the natural Ag of NKT cells (20). Therefore, in the absence of
GPI, PI or other lipids of the ER may assist CD1d1 assembly in
vivo. After assembly of CD1d in the ER, it negotiates the secretory
FIGURE 8. Predicted pathway for the assembly and intracellular traffic
of CD1d1 molecules. Because CD1d1 is a type 1 integral membrane protein, its folding and assembly are predicted to occur in the rough ER. The
folding of the CD1d1 H chain (␣) is assisted by calnexin. The L chain ␤2m
(␤) presumably associates with partially folded H chain-calnexin complex.
This H chain-␤2m-calnexin complex forms a structure receptive to ER
resident lipids and/or glycolipids (cPL). In this model, the lipid ligand is PI
or GPI. Upon stable association, the CD1d1-glycolipid complexes dissociate from calnexin, egress from the ER, and negotiate the secretory pathway to the plasma membrane. Because CD1d1 has the YQDI internalization sequence within its cytosolic tail, it is rapidly internalized and recycled
back to the plasma membrane. During its time in the endosome/lysosome
compartment, CD1d1 exchanges cPL for another, presumably cellular, glycolipid whence it comes to the plasma membrane and is presented to
V␣14J␣15-positive NKT cells (from Ref. 1).
pathway and arrives at the MHC class II-enriched vesicles either
directly from the trans-Golgi or via the plasma membrane. Targeting to the class II-enriched vesicles depends on the Yxx␾ internalization sequence found at the cytosolic tail of CD1d (39, 51).
Here, CD1d meets the naturally processed Ag(s) whence they arrive at the plasma membrane for presentation to NKT cells (Fig. 8)
(39, 51). Because human CD1 show structural (11, 52–56) and
functional (57–59) similarities to CD1d1, we predict that the above
assembly and intracellular traffic mechanism may be a common
feature of this family of lipid Ag-presenting molecules.
To support our model that PI and PI-glycans act as chaperones,
an in vitro competition assay was performed. sCD1d1, when plate
bound, does not activate NKT cell hybridomas. The addition
␣GalCer but not PI to plate-bound sCD1d1 specifically activates
V␣14J␣15 NKT cell hybridomas. This stimulatory activity of
␣GalCer can be inhibited by titrating amounts of PI (A. K. Stanic,
L. Van Kaer, and S. Joyce, unpublished data). Considering that PI
and ␣GalCer have similar affinities for CD1d1 (19, 60), the reverse
can also occur. Thus, PI and PI-glycans assembled with CD1d1 in
the ER can be exchanged for another lipid.
Two recent reports indicate that CD1 molecules can be assembled in vitro by oxidative refolding chromatography (61, 62). Albeit an inefficient process, these reports suggest that CD1 can assemble in the ER of mammalian cells without cellular lipids; i.e.,
CD1 assemble as empty molecules in the ER. However, it is possible that the lipids contained within the H and L chain inclusion
bodies could have been included in the in vitro assembly process,
thus achieving a low level of folding of CD1. A similar argument
can be levied against our data as well, in that lipids resulting from
cell death could have contributed to the elution of radio-labeled
lipids from CD1d1. However, this co-elution is less likely because,
as discussed above, CD1d1 did show selectivity in associating with
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Model for CD1 assembly, intracellular traffic, and Ag
presentation
731
732
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgments
We thank M. H. Hunsinger, A. J. Joyce, and J. Weaver for technical help;
M. E. Embers for generating sCD1d1-Kbtail and sCD1d1-er cell lines; and
A. Bendelac and K. Hayakawa for NKT cell hybridomas. We are grateful
to Drs. J. R. Bennink, L. Van Kaer, and J. W. Yewdell for continued
discussions and encouragement during the course of this project as well as
for the critical evaluation of the data and comments on this manuscript. We
also thank Dr. C. Aiken for critical reading of this manuscript, as well as
members of the M. Boothby, W. Khan, and G. Oltz laboratories at Vanderbilt University for helpful discussions. Excellent secretarial help from
A. L. Karns is gratefully acknowledged.
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assembly of CD1 family of proteins. Finally, and most importantly, the development of this method sets the stage for the isolation and characterization of the elusive natural Ag(s) of CD1drestricted NKT cells.
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