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*
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520; and
University of Utrecht, Department of Cell Biology, Medical School ACU, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimers assemble in the endoplasmic reticulum
(ER)4 with a trimer of the
nonpolymorphic invariant chain to form a nonameric complex that is
transported through the Golgi apparatus to the trans-Golgi
network (TGN). In the trans-Golgi network, signals in the
invariant chain cytosolic tail target the complex to organelles in the
endocytic pathway. There invariant chain is degraded, and class II
molecules are loaded with peptides derived from Ags that have been
internalized and partially degraded. Following peptide loading, class
II is transported to the cell surface by an undefined route (for
review, see 1 . The endocytic system of human B cells has recently been described in detail (2). Briefly, six endocytic subcompartments can be distinguished on the basis of morphologic criteria and the presence of marker proteins: two types of early endosomes (EEs), three types of late endosomes (LEs)/lysosomes, and an intermediate type kinetically positioned between EEs and LEs. The majority of intracellular class II molecules are located in the compartments downstream of EEs, collectively called MIICs (for MHC class II-enriched compartments) (2, 3). Typical MIICs are characterized by the presence of internal vesicles and membrane sheets. The multivesicular MIICs probably represent the classical multivesicular bodies or late endosomes in B cells, while MIICs with internal membrane sheets correlate with lysosomes. No exclusive class II-containing organelle has been found, indicating that class II molecules localize to conventional endocytic compartments (2). Loss of the lumenal domain of the invariant chain correlates with the transition from multivesicular to multilamellar MIICs and suggests that invariant chain is degraded in this group of organelles (4). Subcellular fractionation and pulse-chase analysis have shown that peptide loading occurs in compartments with characteristics of MIICs and, following loading, class II molecules move from these organelles to the cell surface.
Two other class II-like molecules encoded in the MHC, HLA-DM, and HLA-DO have been localized to MIICs. Experiments in mutant B cell lines and knockout mice have demonstrated that DM is required for efficient Ag presentation by many class II alleles (5, 6, 7, 8). In vitro studies have shown that DM facilitates the removal of a fragment of the invariant chain (class II-associated invariant chain peptide (CLIP)) from the peptide binding groove and stabilizes empty class II molecules awaiting antigenic peptide (9, 10, 11, 12, 13, 14). A direct low affinity interaction between DM and class II in MIICs has been demonstrated by coprecipitation analysis (10, 15). HLA-DO molecules also bind to DM. However, this interaction is strong and is initiated in the ER, after which the DM/DO complex is transported to the MIIC (16). HLA-DO inhibits the ability of DM to remove CLIP and facilitate peptide loading (17, 18, 19).
To identify new molecules involved in class II processing and presentation, we raised mAbs to immunoisolated MIICs. We report here that one molecule identified by this approach was CD82, a member of the tetraspan (also tetraspanin, transmembrane 4) superfamily of proteins that has previously been found on the surface of B cells, activated T cells, macrophages, and granulocytes (20, 21, 22). The tetraspan family consists of at least 18 members with a proposed structure of four transmembrane segments and two extracellular loops of unequal size (reviewed in Refs. 23 and 24). These proteins form large complexes on the cell surface composed of several different tetraspan molecules bound to many different cell surface proteins. CD82 itself associates with the tetraspan proteins CD81, CD63, CD53, CD9, and CD37 as well as CD21, CD19, MHC class I, MHC class II, CD4, and integrins (25, 26, 27, 28, 29, 30). To date, no function has been unambiguously assigned to any of the tetraspan proteins.
In this study we demonstrate that CD82 is highly enriched in MIICs of human B lymphoblastoid cell lines. In these compartments, CD82 and CD63, another tetraspan protein previously localized to MIICs, specifically associate with class II, DM, and DO. The results suggest that the late stages of class II maturation may occur in large multimolecular complexes in MIICs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human B lymphoblastoid lines Swei (31), Pala (32), RN (33), and 6H5.DM (34) and the TxB hybrid T2.DM (32) have been previously described. The following Abs used for immunofluorescence microscopy, immunoprecipitation, and immunoblotting have been previously described: L243 (35), R.DRAB (36), HB10 (37), XD5.A11 (38), R.DMB-C (32), and MaP.DMB/C (32). The polyclonal anti-cathepsin D Ab was purchased from Dako (Carpinteria, CA). Abs for immunoelectron microscopy were as follows: HLA-DM (15), HLA-DR (39), human invariant chain C-terminus (40), Lamp-1 (41), CD63 (42), and CD82 (this report). The anti-CD63 mAb H5C6 developed by Dr. J. Thomas August was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), and the Department of Biologic Sciences, University of Iowa (Iowa City, IA).
Subcellular fractionation and immunoisolation of MIICs
For each fractionation, 4 x 107 Pala cells were pelleted, washed once in homogenization buffer (HB; 250 mM sucrose, 10 mM HEPES, and 1 mM EDTA, pH 7.4), and resuspended in 4.75 ml of HB. Cells were disrupted with two to four passes through a ballbearing homogenizer using a 0.0013 inch gap. Cell breakage was monitored by phase contrast microscopy. Nuclei were pelleted by two successive centrifugation steps at 4°C for 5 min at 1500 x g in a microfuge. Two milliliters of postnuclear supernatant was mixed with 9 ml of Percoll in HB to give a final Percoll concentration of 27% in Beckman 344059 ultracentrifuge tubes (Palo Alto, CA). The mixture was underlayed with a 0.5-ml isotonic Nycodenz (27.6%) cushion buffered with 10 mM HEPES to pH 7.4. The gradients were centrifuged in Sorvall TH-641 rotor (Randolph, MA) for 1 h at 17,500 rpm (41,000 x gave) at 4°C. MIICs were collected in 0.5 ml from the interface with the cushion by side puncture. For further purification, 1 ml of collected MIICs was mixed with Percoll, and the separation was repeated. MIICs purified on sequential gradients was used as a starting material for immunoisolation. The MIIC fraction was first precleared of any residual ER and plasma membrane contaminants with 100 µl of Biomag magnetic protein A that was prebound with both a rabbit polyclonal Ab that recognizes the C-terminal 19 amino acids of calnexin (43) and w6/32, which recognizes assembled MHC class I molecules. Binding to Biomag protein A (Perseptive Diagnostics, Cambridge, MA) and subsequent washing of unbound Ab were performed in PBS with 1% IgG-free BSA. Preclearing was performed by rotating for 1 h at 4°C and subsequent removal of the beads on a magnet. Immunoisolation was performed by rotating the precleared MIIC fraction with 100 µl of Biomag protein A bound to MaP.DMB/c, an mAb that recognizes the C-terminus of HLA-DMB, for 3 h at 4°C. Beads were then washed three times for 10 min each time by rotating with HB.
Immunoelectron microscopy
For immunogold labeling, cells were allowed to internalize 5-nm gold particles coated with BSA for 10 min. Cells were then fixed in 2% paraformaldehyde in 0.1 M sodium phosphate buffer at pH 7.4 for 2 h and stored in 1% paraformaldehyde in the same buffer until cryosectioning. Cryosectioning and immunolabeling were performed essentially as described previously (44), except that the cryosections were embedded in a mixture of sucrose and methylcellulose to better visualize membranes (45). Ultrathin cryosections were indirectly single or double immunolabeled with 10- and 15-nm protein A/gold particles (44).
For visualization of immunoisolated MIICs, organelles bound to Biomag protein A were pelleted and fixed for 1 h in a buffer containing 1.5% glutaraldehyde, 146 mM sucrose, and 100 mM sodium cacodylate. Cells were then embedded in Epon, sectioned, and counterstained with 3% uranyl acetate and Reynolds lead citrate solutions.
PCR cloning of CD82 and transfection of HeLa cells
The cDNA for CD82 was cloned from a Raji cell cDNA library (32) using Pfu polymerase (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Primers used for amplification were 5'-GGTACCGCCACCATGGGCTCAGCCTGTATCAAAG-3' and 5'-ATCGATTCAGTACTTGGGGACCTTGC-3'. The PCR product was cloned into the TA cloning vector pCR 2.1 (Invitrogen, Carlsbad, CA) and sequenced using an ABI DNA sequencer (Applied Biosystems, Foster City, CA). For expression in HeLa cells the CD82 cDNA was subcloned into the mammalian expression vector pMCFR-PAC (32). HeLa cells were transfected by electroporation in a 0.4-cm gap cuvette using a Bio-Rad gene pulser set at 0.23 kV and 960 µF. Transfectants were selected in medium containing 0.75 µg/ml puromycin and were screened for CD82 expression by immunofluorescence microscopy.
Immunofluorescence microscopy
For staining nonadherent cells, coverslips (12 mm in diameter) were treated with a 1% solution of alcian blue that had been heated to the boiling point. Excess dye was subsequently washed away with water. Cells were washed once in serum-free Iscove’s modified Dulbecco’s medium (SFM) and plated on coverslips in a 24-well plate at a density of 3 x 104 cells/well in 500 µl of SFM. Cells were allowed to adhere for 20 to 30 min. Spreading of the cells was monitored by phase contrast microscopy. Cells were then fixed for 15 min in a freshly prepared solution of 3.7% formaldehyde in SFM. After washing away the fixative with SFM, cells were permeabilized and blocked for 15 min in either PBS or SFM containing 10% bovine serum, 0.05% saponin, 10 mM glycine, and 0.05% sodium azide (permeabilization buffer (PB)). Samples were labeled for 30 min in a moist chamber at room temperature by placing coverslips cell side down on parafilm in a 20-µl drop of Ab diluted in PB. Cells were washed in the 24-well plate three times for 5 min each time in 500 µl of PB after incubation in primary Ab or secondary Abs conjugated to FITC or Texas red. After the washes, cells were washed twice in 1 ml water and mounted on glass coverslips in Mowiol 4–88 (Calbiochem-Novabiochem Corp., San Diego, CA). Cells were viewed with a Zeiss axiophot 2 fluorescence microscope (Rockleigh, NJ).
For internalization of anti-CD82 Abs (IgG1) or control mouse IgG, Fab were prepared on an immobilized ficin column (Pierce, Rockford, IL) according to the manufacturer’s directions. Fab were further purified by high performance size exclusion chromatography. In uptake experiments, cells at a concentration of 1 x 106/ml were incubated in IMDM containing 5% bovine serum with 10 µg/ml Fabs for 90 min, then washed in SFM and plated on coverslips for processing as described above.
Analysis of radiolabeled CD82-associated components
Pala cells (1.5 x 108) were labeled with [35S]methionine/cysteine (ICN, Costa Mesa, CA; 2.5 mCi) for 4.5 h and chased in the presence of cold methionine/cysteine for 1.5 h. The cells were fractionated on Percoll gradients, and the dense (MIIC) and light (plasma membrane/ER) fractions were collected. Membranes were solubilized by the addition of 10% (w/v) CHAPS in 0.15 M NaCl/0.01 M Tris, pH 7.4, and PMSF (0.5 mM final concentration) and iodoacetamide (5 mM final concentration) were added. Each extract was applied to an affinity column (1 ml) of 2 mg MaP.CD82 mAb conjugated to Bio-Gel A15 m agarose with a similar precolumn of normal mouse IgG, as previously described for similar affinity purifications (9). After washing with 0.6% CHAPS in 0.15 M NaCl/0.01 M Tris, pH 7.4, CD82-associated proteins were eluted with 1% sodium deoxycholate in 0.15 M NaCl/0.01 M Tris, pH 8.2. Control total glycoproteins were isolated from material that failed to bind to the MaP.CD82 column by the addition of concanavalin A-Sepharose beads (50 µl/0.75 ml of extract). CD82-associated proteins were ethanol precipitated and dissolved in sample buffer for two-dimensional gels, and concanavalin A-bound proteins were eluted in the same buffer. Two-dimensional gels used a nonequilibrium pH gradient as the first dimension and SDS-PAGE as the second (46). Gels were dried and exposed as previously described (32).
Coimmunoprecipitation
For coprecipitation analysis of MIIC fractions, 4 x 107 Pala cells were fractionated on a Percoll gradient, and the MIIC fraction of the gradient was collected in 600 µl by side puncture. Each immunoprecipitation used one-third of the fraction and was mixed with 400 µl of 2% CHAPS in a pH 7.4 buffer containing 130 mM NaCl, 25 mM HEPES, 1 mM PMSF, and 2% BSA. The organelles were lysed at 4°C for 15 min, and then specific Abs covalently coupled to Bio-Rad A15 M beads or a combination of protein G Sepharose and Ab were added to the lysate. Immunoprecipitation was conducted by rotating at 4°C for 1.5 h. Lysates were then washed three times in pH 7.4 buffer containing 2% CHAPS, 130 mM NaCl, and 25 mM HEPES. After the final wash, nonreducing sample buffer was added, and the samples were heated to 95°C for 1 min. When Abs used for both immunoprecipitation and immunoblotting were from the same species or when immunoprecipitating Abs were bound to protein G-Sepharose rather than covalently coupled, coprecipitating material was first eluted from the beads with 15 µl of 0.1% Triton X-100 and 0.05% SDS in 10 mM Tris/300 mM NaCl, pH 8.0. Samples were then analyzed by 10% SDS page followed by transfer to an Immobilon (Millipore, Bedford, MA) membrane for immunoblotting. Immunoblots were visualized with Pierce chemiluminescence reagents.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Partial purification of MIICs for mAb production was performed on
Percoll density gradients (Fig. 1
A). Fractionation of the
human B lymphoblastoid cell line Pala showed MHC class I, which is
predominantly in the plasma membrane, and calnexin, a marker for the
endoplasmic reticulum, to be in the low density fractions of the
gradient. MHC class II molecules (DR,ß) were predominantly located
in the same fractions but were also present in a small peak in the high
density portion of the gradient (fraction 15). This small peak
cofractionated with the major peak of DM and was thus taken to contain
MIIC. MIIC were defined throughout this study by the presence of DM,
which has previously been shown by us and others to be almost
exclusively localized to these compartments at steady state in human B
lymphoblastoid cells (47, 48). DM in the lighter portions of the
gradient may have been present in lower density MIIC or ER. These
lighter fractions were not characterized further.
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B). Identification of CD82 as an MIIC component
BALB/C mice were initially s.c. injected with an MIIC
preparation from 4 x 107 Pala cells still
associated with the magnetic matrix. They were boosted i.v. before
fusion with MIIC that had been eluted from the magnetic substrate with
the peptide used to generate mAb MaP.DMB/c. mAbs were produced by
standard methods, and hybridoma supernatants were screened by
immunofluorescence microscopy using Pala cells. Four Abs that gave an
MIIC-like staining in Pala cells but not in nonlymphoid cells (HeLa)
were chosen for further analysis. Two of these Abs (MaP.DM1 and
MaP.DM2) recognized DM, one recognized Ig 
-chain (MaP.IgK), and one
(MaP.CD82) recognized a heavily glycosylated protein of 40 to 70 kDa
(Fig. 1C). This protein was purified on an affinity column
made from the mAb. Sequencing of a tryptic fragment of the protein
identified it as CD82, a member of the tetraspan family of integral
membrane proteins that has three extensively sialated
N-linked glycans. The lower 35-kDa band was shown to be a
core glycosylated precursor of CD82 by pulse-chase analysis and
enzymatic digestion of the carbohydrate (data not shown). Western
blotting of Percoll gradient fractions showed a peak of CD82 protein in
the most dense portion of the gradient, coinciding with the peak of DM
(Fig. 1A).
Double-label immunofluorescence studies in Pala cells confirmed that
CD82 was highly enriched in MIICs (Fig. 2). CD82 was present in perinuclear
vesicles that contained DM, class II, and another member of the
tetraspan family, CD63, which has previously been characterized as a
marker of MIICs and lysosomes (42). Similar patterns of intracellular
CD82 staining were seen in other human B lymphoblastoid lines,
including Swei, LOO-1, and Raji. The cDNA for CD82 was cloned by PCR
from a Raji cDNA library and expressed in HeLa cells to determine
whether any B cell-specific factors were responsible for the cellular
distribution of CD82. Similar to B lymphoblastoid lines, CD82 expressed
in HeLa is present on the plasma membrane and in intracellular vesicles
that colocalize with the lysosomal hydrolase cathepsin D (Fig. 2, J–L). Apparently, no B cell-specific factors are required
for its localization.
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and
4A). Interestingly, the
majority of CD82 was found on the internal membranes of the MIICs
(Figs. 3 and 4A), similar to the distribution of the
lysosomal tetraspan protein CD63 (not illustrated, see 49 . On the
other hand, HLA-DM (Fig. 4A) and the lysosomal membrane
protein Lamp-1 (Fig. 4B) predominantly localized to the
limiting membrane of the MIICs. Multilaminar MIICs showed a higher CD82
labeling in the internal membranes than multivesicular MIICs (Figs. 3 and 4).
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shows an irregularly shaped type of MIIC, termed early MIIC to
indicate that it represents the most likely entrance site for
biosynthetic class II/invariant chain complexes into the endocytic
pathway (2, 50). Typical MIICs are globular, while the shape of early
MIICs reflect their formation from the tubulovesicular EEs. After
endocytosis for 10 min, 5-nm BSA-gold particles reached the early MIICs
but not the later MIIC types (Fig. 3), (2). Early MIICs often showed
vesicles in a process of budding into the MIIC to form the internal
vesicles (Fig. 3). Early MIICs showed abundant labeling for the
C-terminus of the invariant chain and colabeling for HLA-DM (2) and
CD82.
To determine whether CD82 can be internalized from the cell surface to
the MIIC, purified anti-CD82 Fab were added to Pala cells in
culture. The MaP.CD82 Fabs were rapidly internalized to DM-positive
compartments (Fig. 2, M–O), whereas Fab prepared from
normal mouse IgG showed no detectable uptake (not shown).
Colocalization of Fab with DM after 1 h of internalization and 30
min of chase in the absence of Ab is shown in Figure 2. Fabs were
detectable in MIIC compartments as early as 30 min after addition (data
not shown).
CD82 specifically associates with proteins in the MIIC
A characteristic of tetraspan proteins is the formation of large
multimolecular complexes (23, 24). We therefore determined whether CD82
associates with resident and itinerant molecules in the MIIC (Fig. 5). Pala cells were labeled with
[35S]methionine/cysteine for 4.5 h and were chased
for an additional 1.5 h. The cells were fractionated on Percoll
gradients, and the most and least dense fractions that contained either
MIIC or plasma membrane/ER, respectively, were collected. Fractions
were solubilized in 1% CHAPS, and the lysate was run over an MaP.CD82
affinity column. In parallel, total glycoproteins were affinity
purified using Lens culinaris lectin. Proteins bound to the
CD82 column were eluted with the more stringent detergent deoxycholate,
which removes all proteins with the exception of CD82 (data not shown),
and those bound to the lectin beads were eluted with high pH. Eluted
material was analyzed by two-dimensional nonequilibrium pH
gradient/SDS-gel electrophoresis and fluorography. The total
glycoprotein precipitation from each fraction contained a large number
of proteins. In contrast, very few labeled proteins eluted from the
CD82 column. In the plasma membrane/ER fractions, spots identifiable as
MHC class I heavy chain, ß2m, and MHC class II - and
ß-chains were prominent, in agreement with previous reports. The MIIC
fractions showed only class II and ß and another unidentified
spot. Thus, CD82 appeared to quite specifically interact with class II
in the MIIC.
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A). For these experiments the
MIIC-enriched fractions from Percoll gradients (fraction 15) were lysed
in 2% CHAPS (similar results were obtained with 1% Brij 58) and
immunoprecipitated with agarose beads covalently coupled to MaP.CD82.
Control beads were coupled to an isotype-matched Ab, the
anti--chain Ab, which was found in our immunofluorescence screen
and recognizes free Ig light chain in the MIIC (data not shown). In
agreement with the labeling results, MHC class II was found to
coprecipitate with CD82 (Fig. 6A, lanes 1
and 2). Immunoblotting also showed DM and CD63 to be
associated with CD82 (Fig. 6A, lanes 3–6). Beads
bound to an anti-CD63 mAb, H5C6, also specifically coprecipitated
MHC class II and DM as well as CD82 (Fig. 6B). Similar
results were obtained for both CD82 and CD63 coprecipitation using
highly purified, immunoisolated MIIC as starting material, thus
confirming the subcellular location of the interactions (not shown).
Importantly, Lamp 1, a very abundant protein in the MIIC that was
easily detected in MIIC lysates, did not coprecipitate with either CD82
or CD63 (Fig. 6, A and B, lanes 7
and 8).
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C, lane 5) and not
in the mixed samples. Thus, the interaction of DM with CD82 exists in
cells before lysis.
The fraction of total DM and class II bound by CD82 in the cell is
difficult to determine by coprecipitation because it is not clear how
many interactions are broken even by mild detergents. Since previous
studies have provided a clear function for the direct interaction of
class II and DM, we compared the amount of DM associated with class II
with that associated with CD82 (Fig. 7,
lanes 1–4). In Pala cells, the amount of class
II-associated DM was somewhat greater than that bound to CD82. In Swei
cells, however, the amount of coprecipitating DM was nearly equal.
These results show that the amount of DM associated with CD82 is
similar to that found in complexes of known functional significance.
Whether DM could bind to CD82 in the absence of class II and DO was
determined by coprecipitation from T2.DM cells (Fig. 7, lanes
5 and 6). These cells have a chromosomal deletion in
the MHC but have been transfected with cDNA encoding the DM and ß
subunits. DM was specifically coprecipitated with CD82 from these cells
in the absence of the other proteins.
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). Pala cells were lysed in 2% CHAPS,
and the lysates were precleared with either a mixture of anti-class
II mAbs or a control Ab. Preclearing was repeated until DM
coprecipitation with class II was greatly diminished (Fig. 8, lane 6 vs lane 8), then the lysates were
immunoprecipitated with either anti-CD82 (lanes 1
and 2) or anti-CD63 (lanes 3 and4) beads. All coprecipitating material was immunoblotted for DM,
the vast majority of which was located in MIIC at steady state. The
results show that removing class II-associated DM substantially
depleted the pool of DM bound to CD82 and CD63. This demonstrates that
complexes of the tetraspan proteins, class II, and DM exist in
MIICs and also supports the fractionation data (Fig. 6, A
and B, lanes 1 and 2) showing that
class II in MIICs, localized here by association with DM, can interact
with CD82 and CD63.
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).
As with DM, DO was found to coprecipitate with beads coupled to mAbs
recognizing CD82, CD63, and class II, but not a control Ab
(lanes 1–4). Preclearing DM-associated DO from cell
lysates substantially diminished the amount of DO bound to the
tetraspans and class II (lanes 5–10). This shows
that much of the DO in tetraspan complexes is DM associated and agrees
with our observation that little if any DO exists free of DM in MIICs
(L. K. Denzin and P. Cresswell, unpublished observations). The
association between DO and class II is particularly interesting with
regard to a possible mechanism for DO inhibition of DM. Our results
would seem to rule out the possibility that DO acts by preventing DM
binding to class II. A likely explanation for the discrepancy between
our results and those in a previous report (51), which did not show an
interaction between DO and class II, is the use in the present study of
the detergent CHAPS for coprecipitation rather than Triton X-100.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible, cysteine
protease cathepsin S, which is highly expressed in APCs and is capable
of degrading invariant chain (52). In this study we raised mAbs to
highly purified MIICs to identify additional proteins involved in class
II-mediated Ag presentation. CD82, a member of the tetraspan family of
integral membrane proteins, was found by this approach to be highly
enriched in MIICs and bound to class II, HLA-DM, and HLA-DO proteins.
These findings identify CD82 as another specialized component of MIICs
that is potentially important in the later stages of class II
maturation. Taken together with the tendency of tetraspan proteins to form large multimolecular structures, our findings of CD82 and CD63 association with class II, DM, and DO in MIICs strongly suggest that the late stages of class II maturation occur in the context of tetraspan complexes. Additional tetraspan proteins may also be present in these organelles. CD37, a tetraspan protein found in mature B cells, is highly enriched in intracellular vesicles that are probably MIICs (53) (C. Hammond, H. Geuze, and P. Cresswell, unpublished observations). Our results also suggest MIICs may contain tetraspan protein complexes of several different compositions. Preclearing experiments demonstrate that class II-associated DM binds both CD63 and CD82. Coprecipitation of DM with CD82 from T2 cells shows that neither class II nor DO is needed for association, which leaves open the possibility that free DM and DM/DO complexes may bind tetraspan proteins in the MIICs of normal B cells. The existence of CD82-CD63-class II complexes at the cell surface, where there is essentially no DM or DO, has been shown by several groups (25, 29, 30). Since a minority of the class II molecules in MIICs are thought to be bound to DM (54), it is likely that CD82 and CD63 bind free class II in these compartments. Given that much of the class II coprecipitated with CD82 from purified MIICs is loaded with peptide, as assessed by stability in SDS, and CD82 does not associate with class II-invariant chain complexes (C. Hammond and P. Cresswell, unpublished observations), CD82 seems to associate with class II molecules nearing the end of their stay in the MIIC.
The domains of CD82 responsible for the interactions described here are unknown. Preventing carbohydrate processing does not affect the interaction of CD82 with either class II (C. Hammond and P. Cresswell, unpublished observations) or class I (27), thus demonstrating that the molecule’s highly modified N-linked glycans are not necessary for these associations. Studies using chimeras between the tetraspan molecule CD9 and CD82 suggest that both the large C-terminal extracellular loop of CD9 and the most C-terminal transmembrane domain may be involved in the interaction between CD9 and ß1 integrin (29). For CD82 association, the extracellular and cytosolic regions of CD4 have been implicated, again pointing to the extracellular domain of the tetraspan being important for binding (26).
The pathway by which CD82 arrives in the MIIC is unknown, but the rapid
internalization of anti-CD82 Fab suggests that it may reach the
compartment after internalization from the cell surface. The C-terminal
cytosolic tail of CD82 contains a recently identified clathrin
activating protein-2 binding motif (YSKV) that may serve as an
internalization signal (55). Expression of CD82 in HeLa cells, which
have undetectable levels of endogenous CD82 protein, results in its
localization to late endocytic vesicles (Fig. 2, M–O). This
result and the observation of a similar distribution in .174 cells,
which have deleted the class II region of the MHC, suggest that other
components of the class II processing machinery are not necessary for
localization of CD82 to the MIIC. Given the rapid internalization of
CD82 and its long half-life (>4 h; our unpublished
observations), it is possible that CD82 may recycle between the MIIC
and the plasma membrane. Further investigation should address whether
CD82 travels with class II from the MIIC, as the two molecules are
known to associate on the cell surface. Recent evidence showing that
activating protein-2 and clathrin bind to lysosomes under certain
experimental conditions (56) opens the possibility that the YSKV motif
on CD82 may act in the MIIC as well, perhaps facilitating the exit of
class II molecules.
Additional possibilities for CD82 function in the MIIC are suggested by
the known properties of the tetraspans. CD82 and other tetraspans form
large protein networks or membrane microdomains that may limit
diffusion of included molecules and facilitate interactions between
molecules by keeping them in close proximity (57). In MIICs, class II
and DM are obvious candidates for such a facilitated interaction.
Specificity for protein association with different tetraspan networks
would presumably come from their particular composition. Work with the
tetraspan CD9, which associates with the heparin binding epidermal
growth factor-like receptor has shown that CD9 can enhance ability of
the heparin binding epidermal growth factor-like receptor to bind
diptheria toxin and stimulate the growth of neighboring cells by
juxtacrine signaling through the epidermal growth factor receptor
(57, 58, 59). These studies suggest that tetraspans can facilitate protein
interactions outside the plane of the membrane. The differential
distribution of CD82 and DM within the MIIC (Fig. 4A) may
hint at such an interaction in this case also.
CD82 may also be involved in the recruitment of ancillary molecules to
the MIIC. Kinase, GTPase, and phosphatase activities have been found to
associate with tetraspan proteins (3, 54, 56), although in many cases
these interactions are probably indirect. This scenario is made more
attractive by the results of Pierce and co-workers, who have found that
signaling through the B cell receptor not only increases the efficiency
of class II-mediated Ag presentation in some cases, but also causes an
increase in phosphoproteins and GTP binding proteins in the MIIC
fractions of Percoll gradients (60). A final possibility for tetraspan
function in the MIIC is suggested by the presence of CD63 on regulated
secretory vesicles, including Weibel-Palade bodies, cytolytic granules
of cytotoxic T cells (3), platelet granules, and basophilic granules
(61). Platelet -granules, which are connected to late endocytic
compartments, contain the tetraspan proteins CD9, CD63, and CD151 (H.
Heijnen and H. Geuze, unpublished observation). Perhaps CD82, CD63, and
other tetraspans have a role in regulated secretion and are somehow
involved in the recently reported transport and fusion of MIICs with
the plasma membrane (33, 62). During the process, proteins present in
the MIIC limiting membrane are inserted into the plasma membrane,
whereas the MIIC internal vesicles are released as exosomes (33).
Exosomes express peptide-loaded class II molecules and are enriched in
tetraspan proteins (H. Geuze, data not shown), in accordance with the
abundant labeling of CD82 on the internal membranes of MIICs shown
here.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Immunology, Duke University School of Medicine, 106 Jones Building, Research Drive, Durham, NC 27710.
3 Address correspondence and reprint requests to Dr. Peter Cresswell, Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address:
4 Abbreviations used in this paper: ER, endoplasmic reticulum: MIIC, major histocompatibility complex class II-enriched compartment; CLIP, class II-associated invariant chain peptide; HB, homogenization buffer; SFM, serum-free medium; PB, permeabilization buffer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate.
Received for publication March 20, 1998. Accepted for publication May 28, 1998.
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ß dimers and facilitates peptide loading. Cell 82:155.[Medline]
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ß: class II-associated invariant chain peptide complexes to mature peptide-bound class II ß dimers in a normal B-cell line. J. Immunol. 156:2196.[Abstract]
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