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The Journal of Immunology, 1998, 160: 5797-5806.
Copyright © 1998 by The American Association of Immunologists

Intracellular Formation and Cell Surface Expression of a Complex of an Intact Lysosomal Protein and MHC Class II Molecules1

Balasubramanian Arunachalam*, Mary Pan{dagger} and Peter Cresswell2,{dagger}

* Department of Surgery and Section of Immunobiology, Yale University School of Medicine, and {dagger} Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The generation of invariant chain-free MHC class II molecules and their association with endocytically generated peptides are thought to occur in specialized lysosome-like compartments called MIICs (MHC class II compartments). A number of in vitro studies have shown that large denatured proteins can bind to class II molecules, and that class II association can protect the bound segment of protein from proteolytic degradation. In this work, we present what we believe is the first example of an intact endogenous protein (IP30) binding in an allele-dependent fashion to class II molecules in vivo. IP30 is an IFN-{gamma}-inducible 35-kDa glycoprotein that localizes in MIICs. In this study, we show that intact IP30 binds to certain HLA-DR alleles via an N-terminal prosequence. The association takes place in the endocytic pathway following removal of invariant chain from class II molecules and before their cell surface expression. We also show that DR-IP30 complexes are SDS stable. The potential precursor-product relationship between DR-IP30 complexes and the DR-peptide complex is discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II molecules are expressed on the cell surface as heterodimers of {alpha} and ß subunits. Immediately after synthesis, the {alpha}- and ß-chains associate with trimers of the nonpolymorphic invariant chain forming a nonameric complex (reviewed in 1 . The invariant chain promotes the efficient assembly of {alpha}ß dimers and prevents premature loading of class II molecules with peptides or intact proteins (2). Following transit through the Golgi apparatus, the complex is driven into the endosomal-lysosomal pathway by targeting signals in the cytoplasmic domain of the invariant chain (3). Following proteolytic degradation of the invariant chain, its residual fragments (class II-associated invariant chain peptides, CLIP3 (4)) in the peptide-binding groove of class II molecules are replaced with lysosomally generated peptides by the action of HLA-DM (5, 6, 7).

Proteins entering the endocytic pathway encounter an increasingly hydrolytic environment imposed by a progressive decrease in pH and an increase in protease concentrations. Ultimately, most of them are degraded in lysosomes to small peptides and free amino acids. In APCs, peptides of up to 20 or more amino acids with appropriate sequence motifs are rescued from complete degradation by binding to class II molecules (8, 9, 10). CLIP release and peptide binding are believed to occur in specialized lysosome-like compartments containing HLA-DM called MIICs (MHC class II compartments) (11, 12, 13, 14). Characteristics of the compartment, e.g., relatively reduced proteolytic activity or specialized protective mechanisms (15), may be responsible for the generation of relatively long peptides associated with class II molecules.

Davidson et al. (16) showed using Ag-specific B cell lines that class II molecules bind large fragments of radiolabeled tetanus toxoid following Ig-mediated endocytosis. This suggests that binding of such large fragments to class II molecules may be followed by proteolytic trimming in the MIIC, resulting in the generation of class II-peptide complexes that are then transported to the cell surface. An extreme hypothetical variation of such a mechanism is that unfolded, but otherwise intact, proteins may bind to class II molecules before their degradation. A number of in vitro studies have shown that large denatured proteins can bind to class II molecules (reviewed in Refs. 17 and 18). This is not surprising from a structural point of view, because the ends of the class II peptide-binding groove, unlike those of MHC class I molecules, are open (19). Studies from various laboratories suggest that the reduction of disulfide bonds (17, 18, 20, 21, 22) and unfolding (23, 24, 25, 26, 27) are crucial steps in the binding of most proteins to class II. Sercarz and others have shown that class II association can protect a bound segment of a protein from proteolytic degradation (28, 29, 30, 31), leading to the determinant capture model that suggests that class II binding may indeed precede proteolysis.

In this study, we present what we believe is the first example of an intact endogenous protein that binds in an allele-dependent fashion to class II molecules in vivo. The protein is an IFN-{gamma}-inducible glycoprotein, originally defined by Luster et al. (32) and called IP30, which is expressed constitutively in B-lymphoblastoid cell lines (B-LCL). IP30 is synthesized as a 224-amino-acid 35-kDa precursor, derivatized by mannose-6-phosphate addition, and cleaved in the endosomal/lysosomal pathway, giving rise to a mature 30-kDa form after removal of both N- and C-terminal peptides (Arunachalam and Cresswell, manuscript in preparation). Peptides derived from the N-terminal prosequence of IP30 region were isolated previously from HLA-DR molecules (33). In this study, we show that intact IP30 binds to certain DR alleles in vivo and that the association takes place in the endocytic pathway following removal of invariant chain from the class II molecules and before their cell surface expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Abs

The EBV-transformed cell lines Pala (DR3, DRw52), Swei (DR11, DRw52), Raji (DR3, DR6, DRw52), WT-20 (DR3, DRw52), A2m (DR4), and 8.1.6 have all been previously described (33, 34, 35). T1.DR3 (DR3, DR7) and T2.DR3 (DR3) are previously described T x B hybrid cell lines (36).

Antisera were raised in rabbits against synthetic peptides derived from N-terminal (SPLQALDFFGNGPPVNC) (residues 1–16), internal (CVLDELDMELAFLT) (residues 106–119), and C-terminal (CPSSTSSLRSVCFK) (residues 211–224) sequences of IP30, and are referred as R.IP30N, R.IP30i, and R.IP30C, respectively. Note that the C-terminal sequence is different from that proposed by Luster et al. (32). The complete correct sequence will be published elsewhere (Arunachalam and Cresswell, manuscript in preparation). The peptides were coupled to keyhole limpet hemocyanin (Calbiochem-Behring, La Jolla, CA) via the sulfhydryl group of the natural or underlined added cysteine residue (37) for immunization. Mouse mAbs used were DA6.147 (anti-HLA-DR{alpha}-chain (38)), L243 (anti-HLA-DR{alpha}ß dimer (39)), XD5.A11 (anti-class II ß-chain (39)), HB10A (anti-HLA-DRß-chain (40)), and PIN.1 (anti-invariant chain N-terminal (41)). The rabbit antisera R.DRAB1 (anti-HLA-DR{alpha}ß dimer (42)), R.DMB-C (anti-HLA-DMß-chain C-terminal peptide (43)), have been described. A rabbit antiserum to the mannose-6-phosphate/insulin-like growth factor II receptor (44) was a kind gift from Dr. Stuart Kornfeld. Rabbit anti-cathepsin D serum was from Dako (Carpenteria, CA). A rabbit antiserum specific for the chicken hepatic lectin (CHL; 45) was obtained from Dr. K. Drickamer. Horseradish peroxidase (HRP)-coupled goat anti-rabbit Ig and anti-mouse Ig, FITC-coupled goat anti-rabbit Ig, and Texas Red (TR)-coupled donkey anti-mouse Ig were purchased from Jackson ImmunoResearch (West Grove, PA).

Generation of mouse mAb specific for IP30

IP30 was partially purified from extracts of different B-LCLs using Con A affinity chromatography, followed by ion-exchange chromatography and gel filtration, and used to immunize mice. Cells were extracted for 1 h on ice in 150 mM NaCl, 10 mM Tris, pH 7.4 (TS), containing 2% C12E9, 0.5 mM PMSF, 0.1 mM tosyl lysyl chloromethyl ketone, and 5 mM iodoacetamide. The glycoproteins from the postnuclear extract were purified by passing through a Con A-Sepharose 4B (Sigma, St. Louis, MO) column and eluted in TS containing 0.1% C12E9, 5% {alpha}-methyl D-mannoside (Sigma). The Con A-purified glycoproteins were applied to a DEAE-Sephacel column, and most of bound IP30 (detected by Western blot) was eluted with 0.2 M NaCl in 10 mM phosphate buffer, pH 7.4. This was applied to a Sephacryl S-300 column, and fractions containing IP30 were pooled. At this stage, IP30 was the major protein based on SDS-PAGE and silver staining. Mice were immunized with the partially purified material. Spleen cells from an immunized mouse were fused with Ag.8 myeloma cells, and the culture supernatants were screened for a vesicular staining pattern by indirect immunofluorescence. Potential clones were further analyzed by immunoprecipitation for their specificity. One of the clones (MAP.IP30) was found to secrete an IP30-specific IgG1 Ab and was used in this study. MAP.IP30 is a conformation-specific Ab that precipitates the mature form of IP30 more efficiently than the proform (Arunachalam and Cresswell, manuscript in preparation).

Metabolic radiolabeling and immunoprecipitations

Cells were metabolically labeled, chased, and extracted for immunoprecipitation, as previously described (46). In brief, Pala cells (106–107) were deprived of methionine and cysteine by incubation for 1 h in L-methionine- and L-cysteine-free DMEM (Life Technologies, Grand Island, NY) containing 3% dialyzed FCS, and then pulsed with L-[35S]methionine and L-[35S]cysteine (Amersham Life Science, Cleveland, OH) (0.5–1 mCi) for 1 h at 37°C in the presence of 5 µg/ml brefeldin A (BfA; Epicentre Technology, Madison, WI). For chasing, the cells were washed after labeling and cultured in medium containing 10% FCS and 15-fold excess of nonradioactive L-methionine and L-cysteine for different periods of time. Cells were extracted in 150 mM NaCl, 10 mM Tris, pH 7.4 (TS), containing 1% Triton X-100, 0.5 mM PMSF, 0.1 mM tosyl lysyl chloromethyl ketone, and 5 mM iodoacetamide. Postnuclear supernatants were precleared overnight with normal rabbit serum and either protein A- or G-Sepharose (Pharmacia, Piscataway, NJ), and then precipitated with specific Ab and protein A- or G-Sepharose. Pellets were washed three times with TS/0.1% Triton X-100 and either analyzed by SDS-PAGE or stored at -20°C.

Immunoprecipitation of cell surface vs intracellular DR-IP30 complex and secreted vs intracellular IP30

Cells were labeled and chased for different periods of time, as described above. At each time point, culture supernatant was collected for immunoprecipitation of secreted IP30. To precipitate cell surface DR-IP30 complexes, the cells were washed once with cold, serum-free medium containing 0.1% BSA (SFA) and incubated with the DR-specific mAb L243 in cold SFA for 1 h at 4°C. Cells were then washed twice with cold SFA to remove unbound Abs and detergent extracted as above. Ab-bound complexes were precipitated from the extract using protein A-Sepharose beads. Extracts precleared of cell surface complexes were then incubated with the mAb HB10A and protein A-Sepharose beads for 1 h at 4°C to precipitate intracellular DR-IP30 complexes. The precleared extracts were then used to isolate the residual IP30. R.IP30i recognizes both pro- and mature forms of IP30, but only after reduction and denaturation. Hence, glycoproteins from the cell extracts were isolated using Con A-Sepharose beads (Pharmacia). These were stripped of associated glycoprotein by boiling in 1% SDS under reducing conditions and IP30 isolated following alkylation with iodoacetamide by immunoprecipitation using affinity-purified R.IP30i-Abs and protein A-Sepharose similar to previously described methods (47). Similarly, L243 and HB10A precipitates were SDS stripped, and the released IP30 was reprecipitated with R.IP30N.

Electrophoresis

SDS-PAGE was performed as described (48). 14C-labeled m.w. markers (Amersham, Arlington Heights, IL) were used. Gels were fixed, equilibrated in 150 mM sodium salicylate, dried, and exposed to Kodak Biomax MR film at -70°C. Intensity of specific bands was quantitated with a Bio-Rad (Richmond, CA) GS-250 molecular imager after subtracting a background obtained by integrating a blank area of the appropriate size on each gel.

Western blotting

Samples were separated by SDS-PAGE under reducing or nonreducing conditions. Proteins from the gels were electrophoretically transferred to Immobilon P membranes (Millipore, Bedford, MA). Membranes were incubated in PBS containing 1% BSA and 0.3% Tween-20 for 1 h at room temperature or overnight at 4°C to block nonspecific binding to the membrane. The blots were then incubated for 1 to 3 h at room temperature with specific primary Abs, followed by secondary Abs (HRP-conjugated antispecies Ig) diluted in the blocking solution. The blots were washed extensively after each incubation using PBS containing 0.3% Tween-20. The blots were incubated with SuperSignal CL-HRP substrate working solution (Pierce, Rockford, IL) and exposed to Kodak Biomax MR film to visualize the specific bands. Prestained m.w. markers (Life Technologies, Gaithersburg, MD) were used. For analysis of the SDS stability of DR-IP30 complexes, gels after electrophoresis were incubated in 8 M urea containing 50 mM DTT for 30 min at 80°C. Treated gels were washed three times with water before electrophoretic transfer.

Immunofluorescence

Indirect immunofluorescence was performed as previously described (46, 49). In brief, 8.1.6 cells at a concentration of 3 x 104 cells/well were incubated for 30 min at 37°C on Alcian blue (Sigma)-treated coverslips in 24-well tissue culture dishes. Cells were fixed with 3.7% formaldehyde in Iscove’s modified Dulbecco’s medium (IMDM) containing 10 mM HEPES, pH 7.4, for 20 min at room temperature. Fixing was quenched and cells were permeabilized with 0.05% Saponin in IMDM containing 5% calf serum, 10 mM glycine, and 10 mM HEPES, pH 7.4. Cells were incubated with primary Abs (specific to different proteins) as culture supernatant, diluted ascites, or serum, followed by secondary Abs (TR/FITC-conjugated antispecies Ig) on ice in the presence of 0.05% saponin. Coverslips were washed extensively after each incubation and mounted onto slides using mounting solution (10% Mowiol 4-88 (Calbiochem, San Diego, CA), 2.5% 1,4-diasabicyclo[2,2,2]octane (DABCO), 25% glycerol in 0.1 M Tris, pH 8.5). Samples were examined using a fluorescent microscope (Zeiss) under x157.5 magnification.

Flow-cytometric analysis

A total of 0.5 to 1 x 106 cells was washed once with ice-cold PBS containing 1% BSA (PBA) and incubated with primary Abs for 30 min on ice. Cells were washed three times with cold PBA and incubated with secondary Abs (1/50 dilution of goat anti-rabbit IgG conjugated to FITC) on ice. Cells were washed three times with cold PBA and finally once with cold PBS, and suspended in 200 µl of PBS containing 3% formaldehyde. Samples were analyzed on a Becton Dickinson FACScan (Mount View, CA).

SDS-stability analysis

HLA-DR was purified from Pala and A2m cell detergent extracts using L243 affinity column, as previously described (34). Purified DR was mixed with reducing SDS-PAGE sample buffer and either boiled or incubated at room temperature for 5 min. Samples were separated on 12% SDS-PAGE, followed by Western blotting, as described above. IP30 on the membranes was detected using either R.IP30N or R.IP30i sera and DR{alpha}ß-chains using DA6.147 and XD5.A11 Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intracellular colocalization of IP30 with HLA-DR, -DM, and lysosomal markers

In the original description of IP30, we were struck by the observation that the molecule was localized to intracellular vesicles, possibly lysosomes. This, combined with the fact that the molecule was IFN-{gamma} inducible, suggested a possible involvement with MHC class II Ag processing. To more precisely determine the intracellular distribution of IP30, we examined human B-LCL by indirect immunofluorescence. The cells were stained for IP30, HLA-DR, HLA-DM, and the late endosomal and lysosomal markers mannose-6-phosphate receptor and cathepsin D. The results are shown in Figure 1Go. IP30 was clearly localized to intracellular vesicles containing DR, DM, mannose-6-phosphate receptor, and cathepsin D. The colocalization with intracellular DR and DM suggests that the IP30-containing vesicles are MIICs. This has been confirmed recently by immunoelectron microscopy (H. Geuze, personal communication).



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  		FIGURE 1. Intracellular localization of IP30 by indirect immunofluorescence. 8.1.6 cells were permeabilized and stained with MAP.IP30 for IP30 (A, D, G, and J), rabbit anti-mannose-6-phosphate receptor (B), rabbit anti-cathepsin D (E), R.DRAB for HLA-DR {alpha}ß dimer (H), and R.DMB-C for HLA-DM (K). Colocalization is shown for IP30 and mannose-6-phosphate receptor in C, IP30 and cathepsin D in F, IP30 and DR in I, and IP30 and DM in L. FITC-conjugated goat anti-rabbit Ig and TR-conjugated donkey anti-mouse Ig were used as secondary Abs. Control experiments showed no background immunofluorescence with single-stained cells when using the inappropriate filter. Cells were pictured under x157.5 magnification.

 
Association of IP30 with HLA-DR molecules

The colocalization of IP30 with intracellular HLA-DR and HLA-DM was consistent with a potential role for IP30 in Ag processing. We therefore looked for possible physical associations between IP30 and components of the class II complex. Detergent extracts of a number of cell lines were immunoprecipitated with mAbs specific for invariant chain (PIN.1) or DR{alpha}-chain (DA6.147), and the precipitates were separated by SDS-PAGE and electrophoretically transferred to membranes. The membranes were then probed with a rabbit Ab to an internal peptide of IP30 (R.IP30i). The anti-DR{alpha} mAb DA6.147 coprecipitated IP30 (Fig. 2Go). No IP30 was coprecipitated by the invariant chain-specific Ab, indicating that IP30 is not associated with DR-invariant chain complexes. In other experiments, Abs specific for HLA-DM or MHC class I molecules did not coprecipitate IP30 (data not shown). Interestingly, IP30 association with HLA-DR molecules was detected only in cells expressing the DRw52 supertypic allele, namely Pala (DR3), Swei (DR11), Raji (DR3, DR6), and WT-20 (DR3). No association was seen in T1 cells (DR7) transfected with a genomic clone encoding the DR3ß-chain or T2 cells transfected with genomic clones encoding DR{alpha}- and DR3ß-chains. Additional cell lines tested, including DR1 and DR2 homozygous lines, showed no association. Thus, among the alleles analyzed, IP30 only appears to associate detectably with DRw52. In separate experiments, extracts from metabolically labeled DRw52-negative cells were incubated with unlabeled extracts from DRw52-positive cells. Anti-class II Abs did not coprecipitate labeled IP30 from the mixture, which argues against potential postlysis association (data not shown). All of the cell lines shown in Figure 2Go expressed similar amounts of IP30, and reprobing of immunoblots with DA6.147 indicated that for each cell line, comparable amounts of class II molecules were precipitated (data not shown). These data suggested that the class II-IP30 association was class II restricted and might be mediated by the peptide-binding groove.



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  		FIGURE 2. Association of IP30 with HLA-DR in different cell types. Extracts from 2.5 x 106 cells of different types (indicated on the top of the figure) were precipitated with anti-invariant chain (PIN.1, A) or anti-DR{alpha}-chain (DA6.147, B) Ab. Precipitates were subjected to SDS-PAGE (12%) under reducing conditions, transferred to a membrane, and probed with purified R.IP30i, followed by goat anti-rabbit Ig conjugated to HRP. The position of IP30 is indicated on the right, and those of molecular mass markers (kDa) on the left. The bands seen below 30 kDa represent the light chain of precipitating Ab that cross-reacts with the HRP-conjugated secondary Ab.

 
Stability of DR-IP30 complexes in SDS

The possibility that the IP30 molecule might associate with a subset of DR molecules by the peptide-binding groove is consistent with the finding of Chicz et al. (33) that two peptides derived from the N-terminal region of IP30 could be isolated from DR3/DRw52 molecules purified from the B-LCL WT-20. Many class II-peptide complexes are stable in SDS unless heated, suggesting that the DR-IP30 complex might also be stable in SDS if the association was via the binding groove. To assess this, DR molecules were affinity purified from detergent extracts of unlabeled Pala (DR3, DRw52) and A2m (DR4) using an L243 column. After incubation in SDS-containing sample buffer under reducing conditions and with or without boiling, proteins were separated by SDS-PAGE. Before transfer to membranes, the gels were incubated in 8 M urea containing 50 mM DTT for 30 min at 80°C. This treatment was found to dramatically improve the subsequent detection of unheated samples of IP30 by R.IP30i. Blots were probed with either a mixture of DA6.147 and XD5.A11 (Fig. 3GoA), R.IP30N (Fig. 3GoB), or R.IP30i (Fig. 3GoC). SDS-stable DR{alpha}ß dimers were detected both in A2m and Pala under nonboiling conditions, and upon heating to 100°C they separated into free {alpha}- and ß-chains, as expected (panel A). IP30 was detected only in purified DR from Pala cells (panels B and C). More free IP30 was detected both with R.IP30N and R.IP30i when the samples were first heated to 100°C (last lane in panels B and C). In the nonboiled samples, both R.IP30N and R.IP30i detected a band with an approximate m.w. of 90 to 100 kDa only in the Pala-derived DR preparation. This is the expected size for an {alpha}ß.IP30 complex, and the result suggests that the complex is SDS stable. There are R.IP30N- and R.IP30i-reactive bands below the dominant high m.w. band in the nonboiled sample and below the IP30 band in the boiled sample (panels B and C). This may correspond to C-terminally proteolyzed IP30. We have been unable to confirm this by probing with R.IP30C, which is unfortunately a less sensitive reagent in Western blots.



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  		FIGURE 3. SDS stability of the DR-IP30 complex. Affinity-purified DR from Pala cells (last two lanes in A, B, and C) or A2m cells (first two lanes in A, B, and C) was mixed with reducing SDS-PAGE sample buffer, and incubated at room temperature (NB) or boiled (B) for 5 min. The samples were separated on SDS-PAGE (12%), transferred to membrane, and probed with DA6.147 (DR{alpha}) and XD5.A11 (class II ß) Abs (A) or R.IP30N (B) or R.IP30i (C) serum, followed by goat anti-mouse/rabbit Ig coupled to HRP. The positions of molecular mass markers (kDa) are indicated on the left.

 
DR-IP30 complexes are expressed on the cell surface

The two IP30-derived peptides isolated from DR3/DRw52 by Chicz et al. (33) correspond to residues 1–20 and 1–22 of the protein following signal sequence cleavage. The R.IP30N antiserum was raised to residues 1–16. Thus, it seemed possible that this antiserum might react with the peptide, presumably DRw52 associated (Fig. 2Go), at the cell surface. To test this, Pala (DR3, DRw52), Raji (DR3, DR6, putatively DRw52), and A2m cells (DR4) were stained with R.IP30N and analyzed by flow cytometry (Fig. 4Go). Pala and Raji cells were both positive, and the reactivity of the Ab was inhibited by the immunizing peptide. Swei cells (DR11, DRw52) were also positive with R.IP30N (data not shown). The A2m cell line was negative, as were a number of other DRw52-negative B cell lines (data not shown).



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  		FIGURE 4. Detection of IP30 on the cell surface by flow cytometry. The cells indicated were stained with the indicated Abs, followed by goat anti-rabbit Ig conjugated to FITC. The dotted line represents the negative control Ab, and the solid line represents the specific Ab, as indicated on the top of the histogram. In panels in which R.DRAB Ab was used, an irrelevant rabbit serum specific for CHL was used as negative control. In the panels in which R.IP30N or R.IP30C was used, antiserum containing the peptide against which it was raised (10 µM) was used as negative control, and serum containing an irrelevant peptide was used to stain for cell surface IP30. Data are plotted as fluorescence intensity vs cell number.

 
The observed staining pattern could reflect surface expression of DRw52-peptide complexes and/or DRw52-IP30 complexes. The intact protein should react with R.IP30C, raised to the C-terminal sequence of IP30. Figure 4Go shows that Pala and Raji cells are also positive with this Ab, and again the reactivity is inhibited by the immunizing peptide (residues 211–224; Arunachalam and Cresswell, manuscript in preparation). The staining intensity with the C-terminal-specific Ab is lower than that of the N-terminal-specific Ab. This may be because the N-terminal-specific Ab is also recognizing DRw52-peptide complexes, although inherent differences in efficiency of the two antisera may be responsible.

Alternative explanations for the reactivity of the IP30 C-terminal-specific Ab with the cell surface could be that the C-terminal peptide is independently expressed or that the intact molecule is present, but not class II associated. To approach this question, we isolated surface HLA-DR molecules by prebinding the anti-DR mAb L243 to intact Pala cells that had been radiolabeled and chased for 6 h to ensure cell surface expression of labeled class II molecules. The cells were washed to remove free Ab, and detergent solubilized. The DR-Ab complexes were isolated using protein A-Sepharose, and dissociated by boiling in SDS under reducing conditions. After alkylation with iodoacetamide, the supernatants were diluted with Triton X-100 and reprecipitated with HB10A, R.IP30N, R.IP30i, or R.IP30C. The results are shown in Figure 5Go. Lanes 1 to 4show that while DA6.147 (lane 1) (which reacts with the cytoplasmic domain of DR{alpha} and therefore fails to bind at the cell surface) and an irrelevant rabbit antiserum (anti-CHL, lane 2) failed to bind surface DR molecules, L243 (lane 4) effectively captured them, as assessed by reprecipitation of DRß subunits with HB10A. While the irrelevant rabbit anti-CHL Ab (lane 3) failed to react with any protein released from the surface class II molecules, all three IP30-specific Abs specifically immunoprecipitated released IP30 (lanes 5–7). Thus, complexes of DR with intact IP30 protein are clearly present at the cell surface.



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  		FIGURE 5. Presence of DR-IP30 complexes on the cell surface. Pala cells were metabolically radiolabeled for 1 h in the presence of BfA (5 µg/ml), washed, and chased for 6 h at 37°C. The cells were then incubated at 4°C for 1 h with anti-DR Ab (L243, lanes 3–7), Ab specific to cytoplasmic segment of DR{alpha}-chain (DA6.147, lane 1), or Ab specific to CHL (lane 2). Cells were detergent extracted after removal of unbound Abs, and class II-Ab complexes were precipitated using protein A-Sepharose. Proteins were eluted from the beads in SDS under reducing conditions, diluted with 1% Triton X-100 containing 10 mM iodoacetamide, and reprecipitated with the anti-DRß Ab HB10A (lanes 1, 2, and 4), control anti-CHL Ab (lane 3), R.IP30N (lane 5), R.IP30i (lane 6), or R.IP30C (lane 7). The positions of molecular mass markers (kDa) are indicated on the left.

 
DR-IP30 association occurs in a chloroquine-sensitive compartment

The observation that IP30 is detectable in MIICs suggested that binding to DR molecules might occur in this compartment before expression of the complex on the cell surface. If this were true, the level of association could potentially be affected by reagents that affect peptide loading. Alternatively, binding could occur in the ER, before invariant chain association. To test these ideas, we examined the effects on the class II-IP30 interaction of various drugs that perturb intracellular trafficking and processing. Pala cells were incubated with BfA, monensin, chloroquine, or leupeptin for 13 h at 37°C, and extracted with detergent, and HLA-DR molecules were immunoprecipitated. Preliminary experiments showed that all of these agents induced the accumulation of the proform of IP30 (data not shown), similar to results for many lysosomal enzymes (reviewed in 50 . Associated IP30 was detected by SDS-PAGE, followed by Western blotting. The only agent that substantially affected the amount of DR-associated IP30 was chloroquine, which induced a significant increase (Fig. 6Go). BfA caused a reproducible, but slight, decrease. The most straightforward explanation of these results is that the class II-IP30 association occurs in an acidic compartment, putatively the MIIC. Neither BfA nor monensin has a significant effect because both prevent newly synthesized {alpha}ß-invariant chain complexes, IP30, and other lysosomal proteins from getting to the MIIC, arresting transport in the ER and medial Golgi, respectively. Chloroquine, by neutralizing the MIIC, presumably causes the accumulation of intact IP30 by inhibiting the proteolysis of the N- and C-terminal prosequences, favoring the association of intact IP30 with liberated class II molecules. Leupeptin might have been expected to have a similar effect, except that it profoundly affects invariant chain degradation, causing the accumulation in MIICs of class II complexes with the LIP (leupeptin-induced protein) fragment (51, 52). LIP consists of approximately the N-terminal two-thirds of the invariant chain, and includes the CLIP region (53). Thus, class II-LIP complexes are not capable of binding peptides. Chloroquine also can inhibit invariant chain degradation (54), but this effect is incomplete at the concentration used (data not shown). In fact, the processing and presentation of certain epitopes are unaffected by chloroquine under similar conditions (55, 56).



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  		FIGURE 6. Effect of different drugs on DR-IP30 complex formation. Pala cells were incubated at 37°C for 13 h either alone or with BfA (5 µg/ml), monensin (10 µM), chloroquine (30 µM), or leupeptin (0.5 mM). Extracts from 2.5 x 106 viable cells were precipitated with anti-DR{alpha}-chain (DA6.147, lane 2) or anti-invariant chain (PIN.1, lane 1) Abs coupled to beads. Precipitates were subjected to SDS-PAGE (12%) under reducing conditions, transferred to a membrane, and probed with purified R.IP30i, followed by goat anti-rabbit Ig conjugated to HRP. The positions of molecular mass markers (kDa) are indicated on the left.

 
Kinetics of DR-IP30 complex formation

The enhanced association of IP30 with DR molecules induced by chloroquine treatment (Fig. 6Go) is consistent with it occurring in an acidic compartment. In addition, the SDS-stable nature of DR-IP30 complexes suggests that they have probably been exposed to endosomal environment (27). Together with the observation that IP30 is localized in MIICs, the data suggest that IP30 association is likely to occur intracellularly, before cell surface expression of the complex. To test this, we wished to determine the kinetics of DR-IP30 association, and to compare it with that of cell surface expression of the complex. This experiment was combined with an analysis of IP30 processing to the mature form, as well as secretion of the molecule.

Pala cells were labeled for 1 h with [35S]methionine and [35S]cysteine in the presence of BfA to accumulate sufficient labeled IP30 in the ER for analysis. BfA was removed by washing, and the cells were resuspended in chase medium at 37°C containing unlabeled methionine and cysteine. At various times, supernatants were collected and secreted IP30 immunoprecipitated with R.IP30N. Surface DR molecules were isolated by prebinding the L243 mAb before solubilization, as described above. After isolation of the surface complexes with protein A-Sepharose, intracellular DR molecules were immunoprecipitated using the DRß-specific mAb HB10A and protein A-Sepharose. Finally, free intracellular IP30 together with the mature 30-kDa form was isolated from the extract by binding the total cellular glycoproteins to Con A-Sepharose, stripping by heating to 100°C in SDS under reducing conditions, and reprecipitating with R.IP30i after alkylating with iodoacetamide and diluting the samples with Triton X-100. IP30 was similarly released from L243 and HB10A precipitates and reprecipitated with R.IP30N.

Figure 7Go shows the kinetics of IP30 secretion and intracellular processing to the mature 30-kDa form. IP30 secretion begins at 1.5 h and reaches a maximum at 6 h. The accumulation of the intracellular mature form begins at approximately the same time as secretion, but does not increase significantly after 3 h. The 30-kDa form does not react with the N-terminal- and C-terminal-specific antisera (data not shown). The fraction that is secreted is difficult to assess from this experiment, because of the many manipulations involved in generating the data describing the intracellular material. However, other experiments involving more direct comparisons suggest that approximately 25% is secreted, which is consistent with the fraction of starting material that remains intracellular in the mature form in the lower right-hand panel of the figure.



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  		FIGURE 7. Kinetics of IP30 transport, secretion, and processing. Pala cells were metabolically labeled for 1 h in the presence of BfA (5 µg/ml), washed, and chased for different periods of time. Culture supernatants and detergent extracts at each chase point were used to isolate the secreted and intracellular IP30, respectively. Secreted IP30 was precipitated with R.IP30N serum. For intracellular IP30, total glycoproteins from extracts from which all DR-IP30 complexes had been removed (see legend to Fig. 8Go) were bound to Con A-Sepharose. The glycoproteins were eluted in SDS under reducing conditions and reprecipitated with purified R.IP30i. Samples were analyzed on SDS-PAGE (12%) under reducing conditions. The positions of molecular mass markers (kDa) are indicated on the left. The intensity of specific bands was quantitated and is plotted as relative intensity (PD, pixel density) vs time (h).

 
The kinetics of cell surface expression and intracellular generation of DR-IP30 complexes is shown in Figure 8Go. Intracellular complexes are close to maximal at 1.5 h of chase, and after 3 h they begin to decline until a plateau level is reached at 8 h. Cell surface DR-IP30 complexes reach a peak at 3 h, when they also decrease, with the rate of decline also decreasing at 8 h. Thus, at the time when IP30 degradation begins to be seen (1.5 h, Fig. 7Go), DR-IP30 association is close to maximal. This suggests that the IP30 interaction with DR molecules may occur before it is exposed to the proteolytic machinery normally involved in its maturation. Accumulation of DR-IP30 complexes on the cell surface lags behind their intracellular detection, consistent with the proposed model that binding occurs in an acidic intracellular vesicle and is followed by transport of at least some of these complexes to the cell surface. The remaining fraction presumably undergoes intracellular proteolytic degradation, perhaps generating DR-IP30 peptide complexes that are also expressed on the cell surface. The accumulation of steady state levels of DR-IP30 on the cell surface is clear from Figures 4Go and 5Go. This presumably reflects a balance between the decline visible in Figure 8Go and new synthesis. Similar kinetics of IP30 secretion and intracellular processing and DR-IP30 complex generation was observed when cells were chased after pulse labeling in the absence of BfA (data not shown).



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  		FIGURE 8. Kinetics of appearance of the DR-IP30 complex. Pala cells labeled and chased as in Figure 7Go were incubated with the mAb L243 for 1 h at 4°C and detergent extracted after removal of unbound Ab, and the surface-specific complexes were isolated using protein A-Sepharose beads. Intracellular DR from extracts depleted of cell surface DR was then precipitated with the mAb HB10A, followed by protein A-Sepharose. Proteins were eluted from the beads under reducing condition, reprecipitated with R.IP30N serum, and analyzed on SDS-PAGE (12%) under reducing conditions. The positions of molecular mass markers (kDa) are indicated on the left. Intensity of specific bands was quantitated and is plotted as relative intensity (PD, pixel density) vs time (h).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our interest in the IP30 molecule began with the observation that it localizes to MIICs (Fig 1Go). The presence of an IFN-{gamma}-inducible protein in MIICs has potential implications for Ag processing. While searching for a function for IP30, we determined that it associates with DR molecules (Fig. 2Go) and that the complex is expressed on the cell surface (Figs. 4Go, 5Go, and 8Go). Based on analysis of a number of cell lines, all of which express IP30 at similar levels, we believe that it associates with the product of the DRw52 allele (Fig. 2Go), and that the binding is therefore likely to occur via the peptide-binding groove. Evidence in favor of this is that the association is SDS stable (Fig. 3Go), as are many class II-peptide interactions (57). Further evidence is that N-terminally derived peptides from IP30 have previously been isolated from DR molecules purified from the DR3/DRw52-positive cell line, WT20 (33), one of the cell lines in which we have shown the DR-IP30 interaction (Fig. 2Go). Although exogenous intact proteins have been shown to bind to class II molecules (17, 18, 58, 59), this is the first example of which we are aware of class II association with a defined intact endogenous protein other than the invariant chain. The fact that DR-IP30 peptide and DR-IP30 protein complexes are present in the same cell line raises a number of questions concerning regulation of the class II-binding process.

Following synthesis in the ER, IP30 is transported through the Golgi apparatus and a fraction is secreted. The majority, however, remains intracellular, presumably being transported to MIICs, where proteolytic processing takes place (Fig. 7Go). The near simultaneous appearance of intracellular processed IP30 and unprocessed IP30 in the supernatant argues for an intracellular segregation of the two pools. We have evidence that IP30 is modified by addition of mannose-6-phosphate residues to one or more of its N-linked glycans (Arunachalam and Cresswell, manuscript in preparation), which argues that this segregation probably occurs at the Trans Golgi Network (TGN), in which lysosomal enzymes so derivatized are captured by the mannose-6-phosphate receptor for delivery to late endosomes or prelysosomes (reviewed in 60 . Missorted lysosomal enzymes are also often secreted. Processing to remove the DR-binding N-terminal peptide, as well as the C-terminal peptide, of IP30 would be expected to begin when the molecule enters proteolytically active lysosomal compartments, or MIICs. Similarly, invariant chain proteolysis and release of class II {alpha}ß dimers are thought to occur in MIICs following transport of {alpha}ß-invariant chain complexes from the TGN (reviewed in Refs. 1, 61, and 62). Current models suggest that this results in the generation of {alpha}ßCLIP complexes, and that CLIP release induced by HLA-DM molecules reveals the peptide-binding groove (reviewed in 63 . Thus, a plausible scenario is that peptide-free DR{alpha}ß dimers are being generated in proteolytically active, DM-positive organelles in which IP30 processing is simultaneously occurring.

Simultaneous processing of IP30 and DR-invariant chain complexes in MIICs would appear to present newly generated DRw52 {alpha}ß dimers with a choice of binding N-terminal IP30-derived peptides, among other peptides, or intact IP30 protein. The DR-IP30 interaction is initiated intracellularly, consistent with its occurring in MIICs (Fig. 8Go). Chloroquine treatment enhances the association of IP30 with DR molecules (Fig. 6Go), which could be explained if it reduces N-terminal processing of IP30 by raising the pH of MIICs. Chloroquine would appear to have a greater inhibitory effect on IP30 processing than on invariant chain degradation, because invariant chain-associated class II molecules do not bind IP30 (Fig. 2Go), and yet IP30-class II association is increased. Following their association, DR-IP30 complexes are transported from MIICs to the plasma membrane with a lag time of 1 to 2 h (Fig. 8Go). Anderson et al. (64) observed that a small fraction of invariant chain-free I-Ak molecules is also associated with a protein on the cell surface. This protein had an apparent Mr of 55 kDa, but remains uncharacterized.

An alternative idea to the "choice" hypothesis presented above is that DR-IP30 protein complexes, once formed, are processed to DR-IP30 peptide complexes by proteolysis at the C-terminal end of the N-terminal propeptide of IP30. This would be a natural example of determinant capture (31). It is clear that the number of intracellular and cell surface DR-IP30 complexes declines between 4 and 8 h (Fig. 8Go), which could reflect such processing. It is also clear that the number of DR-IP30 complexes is almost maximal at a time (1.5 h) when processed IP30 is considerably submaximal (Fig. 8Go). This could reflect initial DR-IP30 complex formation and subsequent rapid cleavage to generate DR-IP30 peptide complexes. The maintenance of an approximately steady state level of intracellular DR-IP30 protein complexes between 1.5 and 4 h (Fig. 8Go) may reflect a balance between new complex formation and degradation of preformed complexes into DR molecules associated with IP30 peptide. However, we have been unable to date to show a clear precursor-product relationship between DR-IP30 protein complexes and DR-IP30 peptide complexes. The decline between 4 and 8 h may in fact represent loss of the protein-protein complex independent of the generation of the DR-peptide complex. Binding of IP30 by its N-terminal prosequence could conceivably inhibit the cleavage at position 31, which normally occurs during maturation of IP30 (Arunachalam and Cresswell, manuscript in preparation).

The function of IP30 is unknown, although the combination of MIIC/lysosomal localization and IFN-{gamma} inducibility is intriguing and suggests a possible role in Ag processing. It is unclear whether the formation and surface expression of a DRw52-IP30 complex have any functional significance, or whether they represent an opportunistic interaction of a class II molecule with a region of a protein extended and available because its normal function is to be cleaved and removed. If the DR-IP30 complex represents a processing intermediate, then it may provide an avenue to the identification of additional molecules involved in Ag processing. These questions await further analysis.


   Acknowledgments
 
We thank Ms. Nancy Dometios for preparation of this manuscript, Dr. Craig Hammond for technical advice, and Mr. Will Stephen for daily support. We also thank Dr. Roman M. Chicz for providing WT-20 cells, Dr. Stuart Kornfeld for providing rabbit anti-mannose-6-phosphate receptor serum, Dr. Betsy Mellins for providing the 8.1.6 cell line, and Dr. Jeffrey Ravetch for providing complementary DNA clones.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI23081 and Howard Hughes Medical Research Institute. Back

2 Address correspondence and reprint requests to Dr. Peter Cresswell, Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510. E-mail address: Back

3 Abbreviations used in this paper: CLIP, class II-associated invariant chain peptide; B-LCL, B-lymphoblastoid cell line; BfA, brefeldin A; CHL, chicken hepatic lectin; ER, endoplasmic reticulum; HRP, horseradish peroxidase; LIP, leupeptin-induced protein; MIIC, major histocompatibility complex class II compartment; TR, Texas Red. Back

Received for publication August 28, 1997. Accepted for publication February 13, 1998.


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