From the Division of Gastroenterology, Department of
Medicine, Brigham and Women's Hospital, Harvard Medical School,
Boston, Massachusetts 02115, and the ¶ Division of
Hematology/Oncology, Department of Medicine, Beth Israel-Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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CD1d is a major histocompatibility complex class
I-like molecule that exhibits a distinct antigen processing pathway
that functions in the presentation of hydrophobic antigens to T cells. CD1d has been previously shown to be expressed on the cell surface of
human intestinal epithelial cell lines in vivo and a
transfected cell line in vitro independently of
First identified on cortical thymocytes, the CD1 gene family
encodes a group of nonpolymorphic proteins, which have several features
in common with both the major histocompatibility complex (MHC)1 class I and class II
molecules (1). CD1 is a system of proteins encoded by five genes,
CD1A-E, which, although homologous to classic MHC molecules, are
likely distinct in their structure and function. The human CD1 gene
family falls into two groups based on sequence homology: CD1A-C and
CD1D. In mice, two CD1D homologues, MCD1.1 and MCD1.2, have been
identified (2, 3). There is increasing recognition that the CD1
molecules may serve a unique role in antigen presentation distinct from
both MHC class I and class II by presenting nonpeptide antigens to a
discrete subset of T cells. CD1b and CD1c, for example, have been shown
to mediate CD4 In rodents, a subset of CD4+ T cells exist that express the
natural killer cell (NK) marker 1.1, called NK1.1+
CD4+ T cells, and carry an invariant T cell receptor- Previous studies have shown that CD1d is also expressed on intestinal
epithelial cells of human (21, 22), mouse (23), and rat (24, 25).
However, in this location, the major form of CD1d identifiable on the
cell surface of human intestinal epithelial cells has been defined as a
37-kDa, nonglycosylated, Given these previous observations on the biochemical characterization
of CD1d in intestinal epithelium and the recognition that T cell clones
can be identified that appear to be capable of recognizing
Transfected Cells--
The Antibodies--
A series of monoclonal and polyclonal antibodies
were generated against CD1d-glutathione S-transferase (GST)
fusion protein. CD1d-GST fusion proteins containing the
Monoclonal Ab (mAb) against the pooled CD1d-GST fusion proteins was
generated by fusing hyperimmune BALB/c spleen cells to murine myeloma
(NS-1) cells. Wells were screened by enzyme-linked immunosorbent assay
against the pooled fusion proteins, and positives were subsequently
screened against GST to eliminate GST-reactive clones. One Ab from this
screen, D5 (IgG2b isotype) reacts in immunoblots to an epitope in the
CD1d
CD1d-Ig fusion protein was produced by generating a BamHI
site in the 3' end of the CD1d Flow Cytometry--
Analyses were carried out using
approximately 1 × 106 cells in 50-100 µl of PBS
containing 0.05% sodium azide and 1% fetal calf serum. The primary
Abs were each used at 10-20 µg/ml for 20-30 min at 4 °C. The
secondary Abs were fluorescein isothiocyanate-conjugated anti-mouse or
anti-rabbit F(ab')2 fragments (DAKO, Carpinteria, CA). Flow
cytometry analyses were performed using a Becton Dickinson immunocytometry system (Franklin Lakes, NJ).
Radiolabeling, Immunoprecipitation, Western Blotting, and
SDS-Polyacrylamide Gel Electrophoresis--
Using 1-2 × 107 cells, surface proteins were radiolabeled with 1-2 mCi
of 125I using the lactoperoxidase-catalyzed method, as
described previously (26). After washing, labeled cells were lysed in
immunoprecipitation buffer (0.15 M NaCl, 1 mM
EDTA, 50 mM Tris, pH 7.8, 10 mM iodoacetamide) containing protease inhibitors (phenylmethylsulfonyl fluoride (0.17 mg/ml), leupeptin (2 µg/ml), aprotinin (2 µg/ml), chymostatin (2 µg/ml), pepstatin (2 µg/ml), antipain (2 µg/ml), and diisopropryl fluorophosphate (1 µl/ml)) and 1% Nonidet-P40 as detergent, as described previously (26). Lysis was performed at 4 °C for 30 min
and sequentially centrifuged at 14,000 × g and
100,000 × g for 15 and 30 min, respectively.
Immunoprecipitations were performed using Abs coupled to protein G
(IgG1 mAb) or protein A-Sepharose beads (Pierce). To minimize background from eluted IgG, the Ab in some cases was covalently coupled
to the beads using dimethylpimelimidate. Lysates were cleared by 2-3
incubations with nonimmune serum bound to protein A and/or G-Sepharose
beads. Lysates were then incubated with specific antibodies coupled to
Sepharose beads, washed 10 times with immunoprecipitation buffer,
resuspended in Laemmeli buffer in the absence or presence of reducing
agents and analyzed by autoradiography after resolution of proteins by
SDS-PAGE under reducing or nonreducing conditions.
Nonlabeled CD1d was detected by Western blotting as described
previously using the affinity purified rabbit anti-CD1d antibody, followed by an anti-rabbit horseradish peroxidase conjugate and ECL.
Pulse-Chase Metabolic Labeling--
Semi-confluent 1-2 × 107 FO-1 CD1d and FO-1 CD1d/ CD1d Is Expressed on the Cell Surface in the Absence of
Structure of CD1d Expressed by FO-1 Cells in the Absence of
The double transfected, CD1d plus
The size of the CD1d heavy chain expressed by the
FO-1d/ CD1d Structure in Cells Expressing Endogenous
Immunoblotting with an anti-
Interestingly, the BBM.1 mAb in these experiments did not
co-precipitate CD1d (Fig. 5, bottom), a result which
indicates that CD1d Glycosylation Pattern in the Absence of
Examination of CD1d Biosynthetic Pathway by Pulse-Chase Metabolic
Labeling--
To confirm these observations on cell surface expression
of CD1d in the absence and presence of
The examination of CD1d biosynthesis in the FO-1 CD1d/ Several studies have demonstrated expression of human CD1d and its
murine homologue in association with Although CD1d expressed by the The importance of In summary, using cell lines that expressed either endogenous (C1R and
CHO) or exogenous (FO-1) 2-microglobulin (
2m). To define the
relationship between CD1d and
2m and characterize the
biochemical structure of CD1d in the absence of
2m, we
have used a newly generated series of CD1d transfectants and
CD1d-specific antibodies. These studies show that in the absence of
2m, CD1d is expressed on the cell surface as a 45-kDa
glycoprotein that is sensitive to endoglycosidase-H and is reduced to
37-kDa after N-glycanase digestion. In contrast, in the presence of
2m, CD1d is expressed on the cell surface as a 48-kDa
endoglycosidase-H-resistant glycoprotein. Pulse-chase metabolic
labeling studies demonstrate that acquisition of endoglycosidase-H
resistance of CD1d is observed in the presence of
2m but
not in the absence of
2m even after a 24-h chase period.
Thus, CD1d is able to be transported to the cell surface independently
of
2m; however, in the absence of
2m, the
glycosylation pattern of CD1d is altered and consistent with an
immature glycoprotein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CD8
double negative T cell
recognition of bacterial lipid and glycolipid antigens (4-6). Similar
functions are predicted for the CD1d subtype based on the recent
crystallographic structural characterization of mouse CD1d (7) and the
recent identification of glycophosphatidylinositol anchors (8) and
glycosylceramides (9) as antigens involved in CD1d presentation. In
addition, unlike MHC class I, which presents peptide antigens in
transporter in antigen processing proteindependent manner, CD1d
has been shown by several investigators to be transporter in antigen
processing-independent with respect to CD1d surface expression and
antigen presentation (10-12).
chain in co-association with a limited range of T cell receptor-
chain variable regions (13). These T cells directly recognize
2-microglobulin (
2m)-associated mouse CD1
in the absence of an exogenous antigen (14). CD1d mediated activation
of these V
14+ NK1.1+ T cells by
glycosylceramides has also been demonstrated (9). Highly enriched in
the liver, these mouse CD1d reactive NK1.1+
CD4+ T cells have a high likelihood of playing a central
role in immunoregulation of cytokine responses through production of
interleukin-4 and/or interferon
and in anti-tumor immunity
(14-17). Recently, double negative T cells with similar use of a
highly homologous T cell receptor-
chain and recognition of CD1d has
been identified in humans (18-20). These studies suggest, therefore,
that CD1d in association with
2m represents the cognate
ligand for a distinct subset of T cells.
2m-independent molecule (26).
Although, it has been recently shown that intestinal epithelial cells
also express a
2m-associated, fully glycosylated form of
CD1d (27), the fact that a
2m-unassociated form of CD1d
could be identified on the cell surface of a native cell type raises
the possibility that post-translational modifications of CD1d, and the
interactions between CD1d and
2m are likely to be key
factors in determining and/or modifying the function of CD1d as a
ligand for T cells. For example, in addition to NK1.1+ T
cells, which appear to recognize
2m-associated CD1d, T
cell clones have been identified that may be capable of recognizing CD1d on the cell surface of
2m-deficient antigen
presenting cells (28, 29).
2m-independent forms of CD1d, we have investigated the
relationship between cell surface expression of CD1d and
2m. To examine this interaction, we have established a
transfected model system using stably transfected cell lines that
express CD1d and/or
2m. FO-1 is a CD1d and
2m-negative cell line derived from human melanoma cells,
which we have stably transfected with either CD1d alone or CD1d plus
2m. These studies clearly show that CD1d is capable of
being expressed on the cell surface independently of
2m.
In addition, we have shown that, although the absence of
2m does not prevent CD1d glycosylation, the
glycosylation pattern is altered such that CD1d is expressed on the
cell surface with an immature glycosylation pattern.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2m-deficient human
melanoma cell line, FO-1 (30) (kindly provided by Dr. Soldano Ferrone,
New York Medical College, Valhalla, NY), and CD1d transfectant of this
cell line have been described previously (26). FO-1 clones expressing
CD1d and
2m were generated similarly by co-transfection
of the CD1d cDNA in the pSR
-neo vector (31) with the human
2m gene in pEMBL9 (kindly provided by Dr. Hidde Ploegh,
Harvard Medical School, Boston, MA) (32). C1R, a human HLA A- and
B-deficient lymphoblastoid cell line (33) was transfected with CD1d
cDNA in the pSR
-neo vector by electroporation as described
previously (18). Chinese hamster ovary (CHO) cells expressing CD1d were
generated by calcium phosphate transfection method as described
previously (18).
1 domain
alone, the
1 and
2 domains, or the
1-
3 domains were
constructed using convenient restriction sites in the CD1d cDNA
(34) or sites were introduced by polymerase chain reaction. These
proteins were expressed as GST fusion proteins and purified on
glutathione agarose beads (Sigma) (35). The purified proteins were
eluted sequentially with glutathione and SDS, and the pooled proteins
were used to immunize rabbits and mice. Rabbit antiserum was affinity
purified by multiple passes through a GST column followed by absorption to a column of agarose beads (Affi-Gel, Bio-Rad) covalently conjugated with a mixture of the three CD1d-GST fusion proteins. Bound Abs were
acid eluted by a pH step gradient from pH 4.2 to 2.6, followed by
alkaline elution from pH 9.5 to 11.5, and pooled as described previously (36).
1 domain,2 and it
immunoprecipitates native CD1d solubilized in ionic or nonionic
detergents.3
3 domain by polymerase chain
reaction. This BamHI site was then used to fuse the CD1d
leader sequence through the
3 domain to the Fc portion of murine
IgG2b, using an Ig Fc expression vector (kindly provided by Dr. Terry
Strom, Beth Israel Deaconess Medical Center, Boston) (37). The fusion protein was secreted as a
2m-associated disulfide-linked
dimer when expressed in hamster (CHO), murine (NSO), or human (C1R) cells.2 Mice were immunized with the fusion protein
produced in murine NSO cells, and hybridomas were subsequently screened
by enzyme-linked immunosorbent assay using the protein. The anti-CD1d
mAbs raised against the CD1d-Ig fusion protein, 55.1, 42.1, and 51.1, were of the IgG1 isotype as described previously (18). BBM.1 is a mouse
anti-human
2m mAb of the IgG1 isotype (38).
2m was detected by Western blotting of proteins resolved under nonreducing conditions with the BBM.1 mAb. N-linked
carbohydrates were removed by digestion with N-glycanase
(New England Biolabs, Beverly, MA), as described previously (26).
Endoglycosidase-H (New England Biolabs) digestion was carried out by
denaturing glycoproteins in 0.5% SDS, 1%
-mercaptoethanol, boiling
at 100 °C for 5 min, and incubating with 1 µl of endoglycosidase-H
overnight at 37 °C in reaction buffer as defined by the manufacturer
containing 50 mM sodium citrate, pH 5.5.
2m-transfected
cells grown in 60- × 15-mm tissue culture dish (Becton Dickinson; Falcon 3002) were used for metabolic labelings. After washing cells
three times with Hanks' balanced salt solution containing divalent
cations, Dulbecco's modified Eagle's medium without cysteine, and
methionine supplemented with 0.15% dialyzed fetal calf serum and
penicillin/streptomycin was added to the cells and incubated at
37 °C for 30 min. Tran35S-labeled cysteine/methionine
(ICN, Costa Mesa, CA) 0.75-1 mCi was added for 10 min at 37 °C to
label the cells. After discarding the radioactive medium, cold RPMI
supplemented with 10% dialyzed fetal calf serum was added, and cells
were lysed at 0, 2, 4, and 24 h of the chase period with 1%
Nonidet P-40 lysis buffer containing protease inhibitors.
Immunoprecipitations of the lysates with the D5 and the 51.1.3 mAb were
performed followed by the N-glycanase and endoglycosidase-H
digestion protocols as mentioned above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2m--
Cell surface expression of CD1d by transfected,
2m-deficient FO-1 human melanoma cells has been shown
previously using flow cytometry with a cross-reactive rat anti-mouse
CD1d mAb (26). Expression of human CD1d by these cells was confirmed by
flow cytometry using a polyclonal affinity purified rabbit anti-human CD1d antibody and two murine anti-human CD1d mAbs, 42.1 and 51.1 (Fig.
1; wild type versus CD1d). As
expected, there was no binding by the anti-
2m mAb,
BBM.1, and wild type FO-1 cells did not react with any of the
CD1d-specific antibodies. FO-1 cells co-transfected with CD1d and human
2m were also examined (Fig. 1;
CD1d/
2m). These cells stained with each of the anti-CD1d
antibodies and with the BBM.1 mAb. It should be noted that staining of
each transfected line with the murine mAbs suggested two populations of
cells, although this was not apparent with the rabbit antibody.
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Fig. 1.
Analysis of FO-1 transfectants by flow
cytometry. FO-1 wild type (WT), CD1d-transfected FO-1,
and CD1d plus 2m-transfected
(D/B2) FO-1 cells were examined by flow cytometry
using the indicated primary antibodies. NRS, normal rabbit
serum; GST-d, affinity purified rabbit anti-CD1d;
NMS, normal mouse serum; 42.1 and
51.1, mouse anti-CD1d mAbs; BBM.1, mouse
anti-
2m mAb.
2m--
A previous report showed that CD1d expressed on
the surface of intestinal epithelial cells lacked
2m and
N-linked carbohydrates (26). To assess the structure of CD1d
expressed by FO-1 cells in the absence of
2m, a series
of immunoprecipitations were carried out. FO-1 wild type and
CD1d-transfected cells were surface iodinated and immunoprecipitated
with the affinity purified rabbit anti-CD1d antibody (Fig.
2A) or the mouse anti-human
CD1d mAb, D5 (Fig. 2B). Both antibodies immunoprecipitated
heavy chains of approximately 45 kDa from the CD1d-transfected cells
but not the wild type cells (Fig. 2, A and B,
lanes
). There were no bands detected at 37 kDa,
indicating that most or all of the CD1d expressed by these cells was
glycosylated. To confirm that the CD1d transfected FO-1 cells were
expressing glycosylated CD1d, the CD1d immunoprecipitates were treated
with N-glycanase to remove N-linked
carbohydrates. Subsequent analysis of these proteins showed that they
were reduced to about 37 kDa, consistent with the CD1d polypeptide
backbone (Fig. 2, A and B, lanes
+). These results demonstrated that CD1d expressed by FO-1
cells in the absence of
2m contained N-linked
carbohydrate, in contrast to the CD1d protein expressed by intestinal
epithelial cells (26).
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Fig. 2.
Immunoprecipitation of cell surface CD1d from
CD1d-transfected FO-1 cells. Wild type (FO-1) or
transfected (FO-1d) cells were radiolabeled with
125I and lysates were immunoprecipitated with either GST-d,
a rabbit anti-CD1d mAb, and NRS (normal rabbit serum) as a control
(A) or D5, an anti-CD1d mAb, and NMS (normal mouse serum) as
a control (B). The immunoprecipitates were analyzed by
SDS-PAGE on 12% gels under reducing conditions. (+) and ( ) indicate
either treatment or lack of treatment with N-glycanase. The
molecular weight markers in kilodaltons are shown on the
left.
2m FO-1 cells were
examined similarly by immunoprecipitation. The rabbit polyclonal
antibody (Fig. 3A)
immunoprecipitated from these cells a heavy chain of about 48 kDa
(lane
, thick arrow), which was associated with a 12-kDa protein (lane
, open arrow),
consistent with
2m. The D5 mAb (Fig.
3B) immunoprecipitated the 48-kDa CD1d heavy chain (lane
, thick arrow). Note the absence of the
12-kDa band in the D5 immunoprecipitate of the FO-1d/
2m
transfectant (Fig. 3B, lane
, open
arrow). The lack of
2m in the D5 mAb
immunoprecipitates was similarly observed in cells that expressed
endogenous
2m and appeared to reflect disruption of
2m binding by this mAb (see below).
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Fig. 3.
Altered glycosylation pattern of CD1d on cell
surface of CD1d-transfected FO-1 cells. FO-1d cells and FO-1 cells
transfected with CD1d plus 2m
(FO-1d/
2m) were radiolabeled with
125I, and lysates were immunoprecipitated with either GST-d
(rabbit anti-CD1d Ab) and NRS (normal rabbit serum) as a control
(A) or D5, an anti-CD1d mAb, and NMS (normal mouse serum) as
a control (B). (+) and (
) indicate either treatment or
lack of treatment with N-glycanase. Molecular weight markers
in kilodaltons are shown on the left. Thick
arrow, 48-kDa glycosylated CD1d; thin arrow, 45-kDa
glycosylated CD1d; arrowhead, 37-kDa deglycosylated CD1d;
open arrow, 12-kDa
2m.
2m transfectant appeared to be about 3-kDa larger
than the FO-1d transfectant expressing only CD1d. This difference could
be clearly appreciated when either rabbit polyclonal anti-CD1d Ab (Fig.
3A) or D5 mAb (Fig. 3B)
immunoprecipitates were prepared from both cell types and analyzed on
the same gel (Fig. 3, A and B, respectively, lanes
, thin arrow). In contrast,
the CD1d bands immunoprecipitated from both cell types of transfectants
migrated similarly at 37 kDa after N-glycanase digestion
(Fig. 3, A and B, lanes +,
arrowhead), indicating that differences in
N-linked carbohydrate accounted for the smaller apparent
size of CD1d expressed in the absence of
2m.
2m--
Additional CD1d-transfected cell lines that
expressed endogenous
2m were analyzed similarly by
iodination and immunoprecipitation. Both C1R cells, a human B cell
line, and CHO cells yielded CD1d heavy chains of approximately 48 kDa
that migrated identically to the CD1d heavy chain identified from the
FO-1 CD1d/
2m double transfectant (Fig.
4). The rabbit polyclonal antibody
immunoprecipitates from these cells again demonstrated an associated
12-kDa protein consistent with
2m. This 12-kDa protein
was not detected in the D5 precipitates, again indicating that D5 may
disrupt CD1d-
2m binding.
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Fig. 4.
Immunoprecipitation of cell surface CD1d from
C1R and CHO cells transfected with CD1d. Immunoprecipitates from
lysates prepared from surface iodinated CD1d-transfected CHO cells
(CHO-d), wild-type CHO cells (CHO), and CD1d
transfected C1R cells (C1R-d) are shown. Lanes 1,
5, and 11, normal mouse serum; lanes
2, 6, and 12, D5 anti-CD1d mAb; lanes
3, 7, and 9, normal rabbit serum;
lanes 4, 8, and 10, rabbit anti-CD1d
Ab. Samples were analyzed by SDS-PAGE on a 13% gel under reducing
conditions. Molecular weight markers in kilodaltons are shown in the
middle of the figure.
2m mAb, BBM.1, was used to
confirm the identity of the 12-kDa protein as
2m.
CD1d-transfected C1R cells were immunoprecipitated with a series of
CD1d mAbs and immunoblotted with the
2m-specific mAb,
BBM.1. Immunoblotting confirmed a CD1d association with
2m when lysates were precipitated with the 42.1, 51.1, or 55.3 anti-CD1d mAbs (Fig. 5,
top), but not with the D5 mAb. The precipitation of CD1d by
each of the anti-CD1d mAbs was confirmed by subsequent immunoblotting
with the rabbit anti-CD1d antibody (Fig. 5, bottom).
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Fig. 5.
Western blotting of CD1d-transfected C1R
cells. Unlabeled lysates of CD1d transfected C1R cells were
immunoprecipitated with various Abs and resolved by SDS-PAGE in 15%
gels under reducing conditions and Western blotted with either mouse
anti-human 2m mAb, BBM.1 (anti-beta 2 blot),
or rabbit anti-CD1d antibody (anti-CD1d blot). Lane
1, D5 anti-CD1d mAb; lane 2, BBM.1 anti-
2 m mAb;
lane 3, 55.1 anti-CD1d mAb; lane 4, 51.1 anti-CD1d mAb; lane 5, 42.1 anti-CD1d mAb. The location of
the 14- and 46-kDa markers are shown on the left.
2m interacts differently with CD1d
versus HLA class I proteins, which are co-precipitated by
this mAb (data not shown). However, we have observed BBM.1
co-precipitation of CD1d when cells were iodinated (data not shown),
indicating that the association may be stabilized by oxidation. This is
consistent with a previous report showing BBM.1 precipitation of CD1d
from iodinated tumor cells (39).
2m--
The previous studies described above showed
that CD1d is glycosylated differently in the absence of
2m. One possible explanation is that in the absence of
2m, the CD1d on the cell surface represented an immature
form of CD1d that was not processed to maturity after initial
modification by N-glycosyltransferase. This was raised as a
possibility because preliminary partial N-glycanase
experiments suggested that all four potential sites for
N-linked carbohydrate side chains were modified, yet the
protein migrated faster in SDS-PAGE (data not shown). To test for this
possibility, the sensitivity of CD1d to endoglycosidase-H (Endo-H) on
the cell surface of the FO-1d single transfectant in the absence of
2m was evaluated. Radiolabeled cell surface proteins of
the FO-1d transfectant were immunoprecipitated with either the D5 or
51.1 anti-CD1d mAbs, and the immunoprecipitates subjected to either
digestion with N-glycanase (+N) to remove all
N-linked modifications or endoglycosidase-H (+H)
to remove immature N-linked carbohydrate side-chain
modifications that had not been further modified beyond the initial
glycosyltransferase transfer. As can be seen in Fig.
6, the CD1d protein on the cell surface
of the FO-1d single transfectant migrated as a 45-kDa endoglycosidase-H-sensitive protein (arrowhead), whereas the
CD1d protein on the cell surface of the FO-1d/
2m double
transfectant migrated as a 48-kDa endoglycosidase-H-resistant protein
(arrow). This indicates that, in the absence of
2m, transport of an immature CD1d glycoprotein had
occurred to the cell surface.
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Fig. 6.
Characterization of endoglycosidase-H
sensitivity of cell surface CD1d in either the presence or absence
of 2m. FO-1 cells transfected
either CD1d alone (FO-1d) or CD1d plus
2m
(FO-1d/
2m) were radiolabeled with
125I, and the lysates were immunoprecipitated with the D5
and 51.1.3 mAbs with normal mouse serum serving as a control
(lanes marked C). The immunoprecipitates were
subjected to digestion with either N-glycanase
(lanes marked +N) or endoglycosidase-H
(lanes marked +H) and resolved by SDS-PAGE on
13% gels under reducing conditions. The untreated (lanes
marked
) immunoprecipitates were resolved similarly. Autoradiography
of the immunoprecipitates is shown. The open arrow indicates
12-kDa
2m. The molecular weight (MW) markers
in kilodaltons are indicated on the left.
2m, metabolic
labeling studies were performed. Semi-confluent FO-1 CD1d and FO-1
CD1d/
2m cells were labeled with 35S for 10 min and subsequently chased for 0, 2, 4, and 24 h. The pulse-chase
studies of the FO-1 cells transfected with CD1d alone are shown in Fig.
7 after immunoprecipitation with the D5
and 51.1.3 mAbs. Throughout the 24-h chase, CD1d in the
2m negative FO-1 cells remained completely sensitive to
Endo-H, consistent with the predicted molecular weight of the
polypeptide backbone, resolving as 37 kDa after this type of digestion
(open arrow; lanes +H). At 0 h,
the glycosylated 45-kDa CD1d (open arrowhead; lanes
N) was observed to be sensitive to Endo-H
(lanes +H) and N-glycanase
(lanes +N). At 2, 4, and 24 h, the 45-kDa
form of CD1d (open arrowhead) chased into a 48-kDa form of
CD1d (closed arrowhead). Although the nature of the
carbohydrate side-chain modification of this 48 kDa form is presently
unknown, these post-translational changes exhibited a complete
sensitivity to Endo-H. Interestingly, because only the 45-kDa
glycoprotein was identifiable on the cell surface (see Fig. 6) it is
likely that this 48-kDa glycoprotein was retained intracellularly. Of
additional note, the D5 mAb, which was raised against a GST-CD1d fusion
protein, but not the 51.1.3 mAb, which was raised against a
2m-associated Fc-fusion protein of CD1d, readily
detected the
2m-independent form of CD1d. Although the
51.1.3 mAb was able to immunoprecipitate the 48-kDa form of CD1d in the
absence of
2m, it did so very late in the chase and with
significantly less avidity than the D5 mAb.
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Fig. 7.
Pulse-chase metabolic labeling of CD1d in the
FO-1 CD1d cell line. FO-1 CD1d cells were radiolabeled with 1 mCi
of Tran35S-labeled cysteine and methionine for 10 min in
Dulbecco's modified Eagle's medium in the absence of cysteine and
methionine and chased for 0, 2, 4, and 24 h. Cells were lysed in
1% Nonidet P-40, and immunoprecipitations were performed using the D5
and 51.1.3 mAbs with normal mouse serum serving as a negative control
(lanes marked C). The immunoprecipitates
untreated with glycanases ( N), or treated with either
N-glycanase (lanes marked +N) or
endoglycosidase-H (lanes marked +H) digestions,
and the proteins were resolved by SDS-PAGE on 13% gels under reducing
conditions. Closed arrowhead, 48-kDa form of CD1d;
open arrowhead, 45-kDa form of CD1d; and open
arrow, deglycosylated CD1d. The molecular weight (MW)
markers in kilodaltons are shown on the left.
2m
double transfectants is shown in Fig. 8.
In this cell line, at 0 h of the chase, a 45-kDa band
(open arrowhead) was identifiable that was
completely sensitive to Endo-H digestion (open
arrow) but not recognized by the 51.1.3 mAb. In contrast to
the single transfectant (see Fig. 7), this 45-kDa band, rapidly chased
into a 48-kDa band (closed arrowhead) that was
recognized by the 51.1.3 mAb and was resistant to digestion with
Endo-H. A slight reduction of the molecular weight of the 51.1.3 immunoprecipitable material was observed, which we speculate is related
to either contamination of the endoglycosidase-H enzyme with
neuraminidase or incomplete maturation of one of the four carbohydrate
side-chain modifications on CD1d. This observation needs clarification
in future studies. By 4 h, the majority of CD1d had reached
maturity based on Endo-H sensitivity, which was complete at 24 h
in contrast to the single transfectant. The 51.1.3 mAb also notably
preserved the interaction of CD1d with
2m
(closed arrow), which, taken together with the other observations described above, suggest that the 51.1.3 mAb recognizes a conformational epitope associated with CD1d maturity. These metabolic-labeling studies of CD1d in the FO-1 CD1d and FO-1
CD1d/
2m transfectants thus confirm the cell surface
radiolabeling studies.
View larger version (43K):
[in a new window]
Fig. 8.
Pulse-chase metabolic labeling of CD1d in the
FO-1 CD1d/ 2m cell line. FO-1
CD1d/
2m cells were radiolabeled with 1 mCi of
Tran35S-labeled cysteine and methionine for 10 min in
Dulbecco's modified Eagle's medium in the absence of cysteine and
methionine and chased for 0, 2, 4, and 24 h. Immunoprecipitations
and analyses were performed as described in the legend of Fig. 7. In
addition, the closed arrow indicates the location of a
12-kDa band consistent with
2m. The molecular weight
(MW) markers in kilodaltons are shown on the
left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2m (22) and some have suggested that
2m may be required for murine CD1d
expression (40). In contrast, we reported previously that endogenous
CD1d could be detected on the cell surface of human intestinal
epithelial cells in the absence of
2m and that
transfected human and mouse CD1d could be expressed at the cell surface
in a
2m-deficient cell line as defined by cell surface
staining with a cross-reactive rat anti-mouse CD1d mAb (26). However,
this initial report could not be corroborated by a second group working
with mouse CD1d (40), perhaps because of the use of a transient
transfection system. Using the original human CD1d transfectant of FO-1
plus a variety of other newly generated transfectants and newly
generated CD1d-specific antibodies, we now provide definitive data that prove the ability of CD1d to be displayed on the cell surface in the
absence of
2m. Cell surface expression of transfected human CD1d by the
2m deficient FO-1 human melanoma cell
line was confirmed by flow cytometry with several CD1d-specific
antibodies. Moreover, confirmation of this staining data was provided
by immunoprecipitation and autoradiography of cell surface labeled
proteins. The immunoprecipitations also showed that CD1d expressed by
2m-deficient FO-1 cells contained N-linked
carbohydrate. This is in contrast to the
non-
2m-associated form of CD1d expressed by intestinal
epithelial cells, which does not carry N-linked carbohydrate
(26). These results confirm that CD1d expression is not strictly
2m-dependent and is distinct from human
CD1a, -b, and -c (41). In addition, these studies also indicate that
the absence of
2m binding is probably not the primary
event directing the expression of CD1d without N-linked carbohydrate in intestinal epithelial cells as previously reported (26,
27).
2m-deficient FO-1 cells
was glycosylated, it was not identical to CD1d expressed by FO-1 cells transfected with both CD1d and
2m. CD1d expressed by
this double transfectant was
2m associated and migrated
at about 48 kDa, similar to CD1d in other cells, which express
2m endogenously such as C1R and CHO. In contrast, cell
surface CD1d expressed by the
2m deficient FO-1 cells
migrated at about 45 kDa. This difference in mobility was due to
N-linked carbohydrate as CD1d from both the
2m-deficient and
2m-transfected FO-1
cells migrated to 37 kDa after N-glycanase digestion.
However, interestingly, the N-linked carbohydrate associated
with CD1d on the cell surface of the
2m-negative
transfectant exhibited complete sensitivity to Endo-H. This indicates
that all of the CD1d expressed on the cell surface in the absence of
2m was immature and thus not subjected to further
processing beyond that which occurred in the endoplasmic reticulum.
Whether this altered form of CD1d is able to present processed antigen
or form multimers with similarly processed CD1d or other molecules is
not known. However, preliminary data suggest that the abnormally
glycosylated CD1d expressed by the
2m-deficient FO-1
cells may be biologically relevant. Co-cultivation of the FO-1d cell
line, but not the untransfected FO-1 cell line, leads to the
phosphorylation of the T cell receptor-associated kinase p59fyn.4 In addition,
a recent study of CD1d expression on
2m
/
mice splenic B cells indicate that CD1 is expressed at low levels independently of
2m and this form of CD1 is capable of
inducing T cell proliferation, which can be blocked by a rat anti-mouse CD1d mAb, 3C11 (42). These observations by others provide strong support to our previous report of
2m independence of
CD1d expression on human intestinal epithelial cell surface (26).
2m association with CD1d with respect
to the CD1d biosynthetic pathway has not been adequately explored. As
demonstrated by surface labeling and pulse-chase metabolic labeling of
CD1d, the association of
2m with CD1d appears to have an
important role in regulating the extent of CD1d glycosylation and the
maturity of the attached carbohydrate side chains. It is possible that
in the presence of
2m, CD1d may be associating with
chaperoning protein(s) in the endoplasmic reticulum that recognize both
CD1d and
2m as a heterodimer and which function to delay
the biosynthetic process until CD1d becomes fully glycosylated and
reaches its maturity. Given the differences in the glycosylation pattern and Endo-H sensitivity in the presence and the absence of
2m in FO-1 CD1d cells, it is likely that CD1d
synthesized in these two cell lines may have different chaperoning
proteins involved in its assembly/biosynthesis.
2m, human CD1d was displayed on
the cell surface as a
2m-associated heavy chain that was
modified with N-linked carbohydrates that were processed to
maturity. In contrast, in the absence of
2m, although
CD1d transport to the cell surface was not significantly disrupted,
CD1d associated N-linked carbohydrate modifications remained
immature. These data provide another example of how CD1d biosynthesis
is distinct from classical MHC class I (43) and suggest that
2m may regulate the expression of structurally distinct
forms of CD1d that may deliver unique signals to CD1d reactive
lymphocytes as may occur in intestinal neoplasia wherein
2m expression is often disrupted (44).
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ACKNOWLEDGEMENTS |
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We are thankful for expert technical support from John Polischuk.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Grants AI 09682-02 and K08 DK02549-01.
Supported by National Institutes of Health Grants DK44319,
DK53056, and DK51362 and a grant from the Crohn's and Colitis
Foundation of America. To whom correspondence should be addressed: Div.
of Gastroenterology, Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-6917; Fax:
617-264-5185; E-mail: rblumberg{at}rics.bwh.harvard.edu.
** Supported by National Institutes of Health Grant AI33911 and AI42955.
2 S. P. Balk, unpublished observation.
3 S. P. Balk and R. S. Blumberg, unpublished observation.
4 N. A. Campbell, H. S. Kim, K. N. Evans, R. S. Blumberg, L. Mayer, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
MHC, major
histocompatibility complex;
2m,
2-microglobulin;
NK, natural killer;
GST, glutathione
S-transferase;
Ab, antibody;
mAb, monoclonal antibody;
CHO, Chinese hamster ovary;
PAGE, polyacrylamide gel electrophoresis;
Endo-H, endoglycosidase-H.
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REFERENCES |
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