Identification of a sulfate-bearing molecule associated with HLA class ...

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 1534-1538, March 1984 Immunology

Identification of a sulfate-bearing molecule associated with HLA class II antigens (major histocompatibility complex/Ia antigens/proteoglycan)

ANDREA J. SANT*, SUSAN E. CULLEN*,

AND

BENJAMIN D. SCHWARTZtt

Departments of *Microbiology and Immunology and of tMedicine and tThe Howard Hughes Medical Institute Laboratories, Washington University School of Medicine, St. Louis, MO 63110

Communicated by David M. Kipnis, November 16, 1983

(Fred Hutchison Cancer Research Center, Seattle); the homozygous DR4 cell line 3164 (HLA-A2, A24; B27, B-; DR4, DR4) was obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). Human tonsil was obtained from a 14-year-old male who had undergone elective tonsillectomy. The subject was not HLA-typed. A single cell suspension was prepared in phosphate-buffered saline (P1/NaCl) containing 1% dialyzed fetal calf serum (hereafter referred to as dialyzed serum) and the cells were washed twice in Pi/NaCl and dialyzed serum prior to labeling. Antibodies. The monoclonal anti-Ia antibodies used in this study, the Ia molecule detected by them on Swei or 3164 (or both), and their sources are as follows: SG171 (DR) and SG465 (DS), both from J. Silver and S. Goyert (Michigan State University, East Lansing, MI) (8, 9); 227 (DR), from the American Type Culture Collection (Rockville, MD); K14 (DR) and K19 (a non-DR, non-DS, non-SB Ta molecule unique to B cells), both developed and characterized by M. Nahm and M. Shipp (Washington University School of Medicine, St. Louis, MO); and B4A9 (DR), produced in our own laboratory. SG171 and SG465 may detect additional Ia molecules on the other cells used in this study. RbO3, a rabbit anti-DS xenoserum was generously supplied by J. Silver and S. Goyert (8), and rabbit anti-p2-microglobulin (f32m) was purchased from Accurate Scientific (Westbury, NY). Biosynthetic Labeling, Solubilization, and Immunoprecipitation. Prior to biosynthetic labeling, cells were washed three times in P1/NaCl containing 1% dialyzed serum, then preincubated in precursor-deficient medium for 45 min at 37°C, pelleted by centrifugation and resuspended in medium containing the radioactive precursor. For [3H]leucine labeling, the medium used was RPMI 1640 (GIBCO) lacking leucine and supplemented with glutamine, penicillin, streptomycin, Hepes, and 10% dialyzed serum. [3H]Leucine (Amersham; 130 Ci/mmol; 1 Ci = 37 GBq) was added at a concentration of 250 ,Ci/ml. For labeling with [35S]sulfate, the medium used was Earle's balanced salt solution, with MgCl2 substituted for MgSO4, supplemented with amino acids and vitamins from an RPMI 1640 Selectamine kit (GIBCO), glutamine, Hepes, 10% dialyzed serum, and [35S]sulfate (Amersham; 1000-1500 Ci/mmol) at a concentration of 250-300 ,Ci/ml. Lymphoblastoid lines and human tonsil cells were labeled at a density of 2 x 106 and 1 x 107 cells/ml, respectively, for 5 hr at 37°C in a humidified atmosphere containing 7% CO2 in air. After labeling, cell pellets were solubilized with Nonidet P-40 (Particle Data Laboratories, Elmhurst, IL) as described (10), except that the lectin purification step was omitted. Aliquots of the lysate were immunoprecipitated with various antibodies and antigenantibody complexes were isolated with Staphylococcus aureus Cowan strain I (11). NaDodSO4/PAGE and Two-Dimensional Gel Electrophoresis. NaDodSO4/PAGE analysis of immunoprecipitated ma-

The human Ia antigens (DR, DS, and SB), deABSTRACT termined by genes contained within the HLA complex on chromosome 6, are glycoprotein heterodimers consisting of a Mr w34,000 a chain and a Mr =28,000 (3 chain. As a result of studies exploring the possibility that a or j3 (or both) might be sulfated, a unique component of the oligomeric Ia antigen complex was discovered. When anti-Ia immunoprecipitates from Nonidet P-40 lysates of [35S]sulfate-labeled lymphoid cells were analyzed by NaDodSO4/PAGE, a molecule of considerable size heterogeneity (Mr 40,000-70,000) was observed. This component was present in both anti-DR and anti-DS immunoprecipitates prepared from both human tonsil cells and lymphoblastoid B-cell lines but was not observed in control precipitates or in association with immunoglobulin or class I HLA molecules. Preliminary biochemical studies indicate that this Mr 40,000-70,000 molecule is polyanionic, disperse in molecular weight, and sensitive to protease digestion. The sulfatebearing moiety of this component was resistant to Pronase but sensitive to chondroitinase ABC, indicating that this molecule belongs to the chondroitin sulfate class of proteoglycans.

The human class II or Ia molecules, determined by genes of the HLA complex, have been shown to be involved in the interactions of immunocompetent cells. Efforts to understand how these molecules mediate their function have been directed to an elucidation of their structural features. Early studies (1, 2) demonstrated that the human Ia molecules, like their counterparts in other species, consisted of a relatively nonpolymorphic a-chain glycoprotein of Mr -34,000 and a polymorphic P-chain glycoprotein of Mr -29,000, which formed a noncovalently associated heterodimer. In addition, several nonpolymorphic proteins and glycoproteins, including basic invariant chain (homologous to the murine Ij) and acidic invariant chain, have been found to be associated with the a-/ heterodimer during different stages of processing and maturation (3-6). Although it is unclear exactly what the role of the latter nonpolymorphic chains may be, the concept is emerging of an "oligomeric" Ta antigen complex. Recently, in studies originally designed to examine the basis of murine Ia a- and -chain charge heterogeneity, we discovered a component unique to the Ia oligomeric complex (7). This component has biochemical characteristics suggesting that it is a proteoglycan. To determine if this component was unique to murine Ia oligomers or, like other murine Ia components, would have homologues in other species, we examined human Ta oligomers for the presence of a similar component. Our results indicate that such a proteoglycanlike component is indeed found associated with human DR, DS, and other Ia a-B heterodimers.

MATERIALS AND METHODS Cells. The homozygous DR5 cell line Swei (HLA-A29, A29, B40, B40; DR5; DR5) was obtained from John Hansen The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations:

VO,

1534

void

f32m,

832-microglobulin; IEF, isoelectric focusing;

volume; Vi, included volume.

Immunology: Sant et aL terial was performed under reducing conditions exactly as described (11), by using a modified Laemmli-Maizel discontinuous PAGE system (12, 13) consisting of a 4% acrylamide stacking gel and a 10% acrylamide running gel. Two-dimensional separations (two-dimensional PAGE) were performed as described by O'Farrell (14) with modifications (15). The first dimension [isoelectric focusing (IEF)] was performed in 5 x 130 mm cylindrical gels by using a pH 3.5-10 gradient and the second dimension (NaDodSO4/PAGE) was performed by using an 11% polyacrylamide running gel. Protease Treatments. Staphylococcal V8 protease (S. aureus V8, lot 13) was purchased from Miles. A stock solution of enzyme (1 mg/ml) was prepared in NaDodSO4 elution buffer and added to NaDodSO4 eluates of immunoprecipitates at a final concentration of 125 ug/ml. Enzyme-treated and untreated control samples were incubated at 370C for 2 hr and then boiled for 2 min to inactivate enzyme. For Pronase digestion, eluates of S. aureus immunoprecipitates were precipitated with 95% ethanol (16) and then resolubilized in 0.5 ml of 10 mM Tris.HCl/1 mM CaCl2, pH 8. Predigested Pronase (Streptomyces griseus protease, lot 202867, Calbiochem-Behring) was added to a final concentration of 5 mg/ml. Samples were incubated in a toluene atmosphere at 50°C for 12 hr, a second aliquot of Pronase was added, and digestion was allowed to continue for an additional 12 hr. After digestion, samples were boiled for 2 min to inactivate enzyme. Anion-Exchange Chromatography. [3H]Leucine- and 35 o4labeled immunoprecipitates were solubilized for 18 hr at 25°C in 0.5 ml of column starting buffer (8 M urea/0.15 M NaCl/0.05 M sodium acetate/0.5% Triton X-100, pH 6) (17). Insoluble material was pelleted by centrifugation and the supernatant was applied to a 75 x 200 mm DEAE-Sephacel column (Pharmacia), equilibrated in the same buffer. The column was washed with 5.0 ml of the starting buffer, after which bound material was eluted by using a linear salt gradient (0.15-1.4 M NaCl). Fractions of 0.7 ml were collected and aliquots of each fraction were assayed for radioactivity and conductivity. Bio-Gel P-10 Chromatography. Pronase-treated samples (0.5 ml) were applied to a Bio-Gel P-10 (Bio-Rad) column (200-400 mesh, 40 x 1 cm) equilibrated in 0.1 M ammonium bicarbonate. The column was eluted with the same buffer at a flow rate of 5 ml/hr. Fractions of 0.9 ml were collected and the radioactivity in an aliquot from each fraction was determined. Void (V0) and included (V1) volumes were determined for each column run by monitoring hemoglobin and mannose, which were added to each sample prior to application to the column. Bio-Gel P-10 has an exclusion limit of Mr 20,000 for globular proteins. Alkaline Borohydride Treatment. An aliquot of the 35 o4labeled fraction peak obtained by Bio-Gel P-10 chromatography of Pronase-digested material was lyophilized to dryness, resuspended in 1.0 ml of hydrolysis buffer (0.05 M NaOH/50 ,uM N-acetylglucosamine/1 M NaBH4), and incubated at 45°C for 16 hr (18). Acetic acid was then added dropwise to neutralize the sample. The neutralized sample was dried, resuspended in 0.1 M ammonium bicarbonate, reconstituted with hemoglobin and mannose, and rechromatographed on the Bio-Gel P-10 column. Chondroitinase Digestion. Immunoprecipitates were subjected to chondroitinase ABC digestion as described (7), except that digestion was performed directly on S. aureus pellets and the incubation time with enzyme was 2 hr. Mockand enzyme-treated samples were centrifuged at 2000 x g for 10 min. The supernatants were concentrated and analyzed by descending paper chromatography with a solvent system of butanol/acetic acid/1 M NH40H (2:3:1). Pellets were analyzed by NaDodSO4/PAGE.

Proc. NatL. Acad Sci USA 81 (1984)

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RESULTS In initial experiments, cultured lymphoblastoid B-cell lines were examined for expression of an Ia-associated sulfated product. Cells were biosynthetically labeled either with [3H]leucine or with [35S]sulfate. Aliquots of Nonidet P-40 lysates of labeled cells were immunoprecipitated with antibodies reactive with several different B-cell alloantigens. Fig. 1A shows NaDodSO4 gel profiles of immunoprecipitated material from the [3H]leucine-labeled DR homozygous cell lines Swei (DR5) and 3164 (DR4). HLA class I antigens were readily detected in both Swei and 3164 preparations (lanes 1 and 6, respectively). HLA class II a and p chains from Swei, cells were detected by monoclonal anti-DR antibodies SG171 and 227 (lanes 2 and 5) and from 3164 by SG171 (lane 7). Weak affinity of SG465 (lanes 3 and 8) and 227 on 3164 (lane 10) yielded insufficient material to be visualized on the gel. When NaDodSO4 gels of immunoprecipitates made from the 35S04-labeled cells were examined (Fig. 1B), several observations were made. First, a diffusely migrating sulfatebearing molecule was detected in several anti-Ia immunoprecipitates. From Swei, it was detected in the SG171 and 227 immunoprecipitates (lanes 2 and 5, respectively), and from 3164, it was present in the SG171 immunoprecipitate (lane 7). This product is heterogeneous in apparent molecular weight, spanning the Mr 40,000-70,000 region of the gel. The second observation made was that the Mr 40,000-70,000 molecule is

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FIG. 1. Identification of a Mr 40,000-70,000 sulfated molecule associated with human HLA class II antigens. Lymphoblastoid Bcell lines Swei (lanes 1-5) and 3164 (lanes 6-10) were biosynthetically labeled with either ['H]leucine (A) or [35S]sulfate (B). Detergent extracts of labeled cells were immunoprecipitated with rabbit antihuman ,82m (lanes 1 and 6), SG171 (lanes 2 and 7), SG465 (lanes 3 and 8), control ascites fluid (lanes 4 and 9), or 227 (lanes 5 and 10). Immunoprecipitated material was analyzed by NaDodSO4/PAGE (1o acrylamide). The positions of "4C-labeled molecular weight markers (Mr X 10-3) run in an adjacent lane are indicated.

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Immunology: Sant et aL

Proc. NatL Acad. Sci. USA 81 (1984)

not found in control (lanes 4 and 9) or anti-class I (lanes 1 and 6) immunoprecipitates. Anti-class I immunoprecipitates from both cell lines did contain sulfate-labeled material, but this material was of high apparent molecular weight and was clearly distinct from the Mr 40,000-70,000 molecule. To establish that the class II-associated, sulfate-bearing product is expressed in normal tissues as well as in continuous cell lines, human tonsil cells were examined for expression of the Mr 40,000-70,000 molecule. Fig. 2 shows the results of NaDodSO4/PAGE analysis of immunoprecipitates prepared from detergent lysates of the [3H]leucine- (Fig. 2A) and 35S04-labeled (Fig. 2B) tonsil cells. In Fig. 2A, IgG and IgM are readily detected (lanes 1-3); as are HLA class I molecules (lane 6). Labeled class II glycoproteins are detectable in immunoprecipitates prepared with the anti-DS heteroantiserum RbO3 (lane 7) and the anti-DR monoclonal antibodies K14, SG171, and B4A9 (lanes 8, 10, and 11, respectively). Fig. 2B shows that in the control (lane 1) and anti-immunoglobulin precipitates (lanes 2 and 3) there was no detectable labeled product. Anti-032m (lane 6) precipitated a high molecular weight species similar to that seen with the lymphoblastoid cell lines. The Mr 40,000-70,000 molecule was detected in the anti-class II precipitates made with the RbO3, K14, SG171, and B4A9 reagents (lanes 7, 8, 10, and 11, respectively). It is noteworthy that the relative amount of the Mr 40,000-70,000 molecules precipitated is directly correlated

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with the relative amount of class II molecules precipitated (Fig. 2, SG171 > K14 > RbO3 > B4A9). A similar sulfatelabeled component was detected in Ia immunoprecipitates prepared from the peripheral mononuclear cells of two normal individuals (data not shown). Our next series of experiments was designed to gain preliminary information on the structural features of this molecule. The Mr 40,000-70,000 product was not detected in NaDodSO4 gels of [3H]leucine-labeled immunoprecipitates (Figs. lA and 2A). To determine if the failure to label with leucine was due to a lack of protein in the molecule, we tested its sensitivity to protease digestion. K14-immunoprecipitated material from 35SO4-labeled Swei cells was subjected to degradation by S. aureus V8 protease, which cleaves at aspartyl and glutamyl residues (19). After digestion, mocktreated and enzyme-digested material was analyzed by NaDodSO4/PAGE (Fig. 3). Comparison of the untreated sample (lane 4) with the enzyme-treated sample (lane 8) indicates that all of the Mr 40,000-70,000 molecule was sensitive to V8 protease and thus contains polypeptide components. The failure to label with [3H]leucine may be due to a relatively long turnover time or to low representation of leucine in the protein moiety of the molecule. We next wished to determine the degree of charge heterogeneity in the Mr 40,000-70,000 molecule. Therefore, K14 immunoprecipitates derived from aliquots of the 35S04-labeled and [3H]leucine-labeled Swei preparations were analyzed by two-dimensional (IEF/NaDodSO4/PAGE) electrophoresis (Fig. 4). This analysis (Fig. 4A) shows that the class II-associated sulfated product is extremely heterogeneous in charge as well as in molecular weight, focusing in the pH 4.5-6.5 region of the IEF gel. The polydispersity of the sulfated molecule in charge and molecular weight and its poor incorporation of leucine under short-term labeling conditions, despite the fact that it contains protein, suggested that this molecule might be a proteoglycan (16). Proteoglycans are polyanionic molecules with a high average charge-to-mass ratio due to their high content of negatively charged sugars (glucuronic or iduronic acid) and sulfate groups (20), and consequently they can be distinguished from proteins and glycoproteins on the basis of net charge. To investigate whether the Mr 40,000-70,000 product belonged to this class of macromolecules, K14-im-

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FIG. 2. NaDodSO4/PAGE analysis of [3H]leucine- and [35S]sulfate-labeled human tonsil cell immunoprecipitates. Human tonsil cells were biosynthetically labeled with [3H]leucine (A) or [35S]sulfate (B), and detergent extracts were precipitated with normal rabbit serum (lane 1) or rabbit anti-human Ig (lane 2). Extracts that had been precleared with S. aureus were immunoprecipitated with rabbit anti-human Ig (lane 3), control ascites fluid (lane 4), normal rabbit serum (lane 5), rabbit anti-human 82m (lane 6), RbO3 (lane 7), K14 (lane 8), K19 (lane 9), SG171 (lane 10), or B4A9 (lane 11). Molecular weights are indicated as Mr X 10-3.

FIG. 3. Treatment of the HLA class II-associated sulfated component with staphylococcal V8 protease. NaDodSO4/PAGE profile of mock-digested (lanes 1-4) or V8 protease-digested (lanes 5-8) samples. [14C]Bovine serum albumin (lanes 1 and 5) and [14C]IgG (lanes 2 and 6) were used as positive controls for digestion. The 15S04-labeled Mr 40,000-70,000 component precipitated from Swei by K14 (lane 4) is reduced to a low molecular weight species after treatment with V8 protease (lane 8). Lanes 3 and 7 are control immunoprecipitates prepared from 15S04-labeled Swei. Molecular weights are indicated as Mr X 10-3.

Proc. NatL. Acad ScL USA 81 (1984)

Immunology: Sant et a'L A.

B.

a

FIG. 4. Two-dimensional gel electrophoresis of K14 immunoprecipitates prepared from lysates of "SO4-labeled (A) or [3H]leucinelabeled (B) Swei cells. The 35SO4-labeled component (A, bracket) migrates as a series of spots heterogeneous in size and charge. In B, the [3H]leucine-labeled a, ,3, basic invariant (Ii) and acidic invariant (braces) chains are shown for comparison.

munoprecipitated material from labeled Swei cells was fractionated on the anion-exchange resin DEAE-Sephacel in a dissociating solvent under conditions in which proteoglycans, but not proteins or glycoproteins, would bind (17). Fig. 5 shows the results of this experiment. When a [3H]leucinelabeled K14 immunoprecipitate was fractionated on DEAESephacel (Fig. 5A), >95% of the recovered radioactivity failed to bind to the DEAE-Sephacel. This is the expected result because the majority of the [3H]leucine is incorporated into proteins and glycoproteins. Conventional Ia a and f3 chains were found in this fraction (data not shown). In contrast, when the 35S04-labeled immunoprecipitated material was fractionated on DEAE-Sephacel (Fig. 5B), =80% of the recovered 35 04 was bound by the resin and could be eluted with 0.3-0.4 M NaCl. These results support our hypothesis that the Mr 40,000-70,000 molecule is a proteoglycan. Finally, we wished to investigate the nature of the moiety to which the sulfate is linked in this molecule. Sulfate has been identified on macromolecules in several different forms. In proteins, it can be linked directly to an amino acid residue, most commonly tyrosine (21). In glycoproteins, it can be linked to an amino acid or can be attached to either 0or N-linked carbohydrate groups (22-25). In proteoglycans, it is linked to the large repeating disaccharide units that are 0-linked to the core protein (20). To determine the type of moiety to which 35 04 groups were linked in the class IIassociated Mr 40,000-70,000 molecule, K14-immunoprecipitated material from 35S04-labeled Swei cells was exhaustively digested with Pronase and then fractionated on a sizing column of Bio-Gel P-10. (Fig. 6A). If the sulfate were attached to an amino acid or to a carbohydrate group of a conventional glycoprotein, the radioactivity would be expected to elute in the V, of the column. If the sulfate were attached to a large Pronase-resistant glycan, the radioactivity would elute in the V0. As can be seen from the radioactivity profile of the Bio-Gel P-10 column (Fig. 6A, closed circles), all of the Pronase-treated, sulfated material eluted in the V0, indicating that the S04 residues in this molecule are, in fact, bound to a large, Pronase-resistant structure. This result is consistent with the idea that the Mr 40,000-70,000 molecule has a proteoglycan type of structure. However, the result is

1537

also consistent with the possibility that this molecule could be a conventional glycoprotein bearing a Pronase-resistant structure (17, 26) consisting of closely linked serine or threonine residues to which small sulfated O-linked oligosaccharide groups are attached. To evaluate this latter possibility, the Pronase-digested P-10 pool was subjected to mild alkaline hydrolysis to release 0-linked oligosaccharides. When the O-linked oligosaccharides released by the alkali treatment were rechromatographed on the Bio-Gel P-10 column (Fig. 6A, open circles), 92% of the 35SQ4 remained excluded by Bio-Gel P-10, indicating that almost all of the 35 04 residues are attached to large macromolecules, typical of proteoglycans. To test directly whether this molecule was indeed a proteoglycan, its sensitivity to enzymes specific for carbohydrate structures found on this class of macromolecules was investigated. A K14 immunoprecipitate derived from 35S04labeled Swei cells was mock-digested or digested with chondroitinase ABC. The material remaining on the pellet was analyzed by NaDodSO4/PAGE (Fig. 6B), whereas the material released into the supernatant was analyzed by paper chromatography (Fig. 6C). Chondroitinase ABC treatment removed the vast majority of the 35S04 label from the Mr 40,000-70,000 molecule (Fig. 6B, compare lane 2 to lane 1). Analysis of the 35S04-labeled material released by chondroitinase ABC (Fig. 6C, closed circles) shows the label migrating with the standard 4-sulfated N-acetylgalactosamineuronic acid. Thus, the Mr 40,000-70,000 molecule belongs to the chondroitin sulfate class of proteoglycans.

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FIG. 5. DEAE-Sephacel column chromatography of K14-immunoprecipitated material from [3H]leucine-labeled (A) or [35S]sulfate-labeled (B) Swei cells.

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Proc. Natl. Acad. Sci. USA 81 (1984)

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DISCUSSION In the studies reported here, we have identified a subcomponent in the oligomeric HLA class II antigen complex. This product was identified by examination of anti-class II immunoprecipitates prepared from lymphoid cells biosynthetically labeled with [35S]sulfate. On NaDodSO4/polyacrylamide gels, the sulfated product migrates as a diffuse band, ranging in apparent Mr from 40,000-70,000. This molecule could be immunoprecipitated by using antibodies specific for both DR and DS class II glycoproteins but was not seen in control precipitates or in association with other lymphocyte antigens such as immunoglobulin or MHC class I alloantigens. The Mr 40,000-70,000 molecule was found to be expressed on all Ia-positive cells examined thus far, including lymphoblastoid B-cell lines, human tonsil cells, and peripheral blood mononuclear cells. Preliminary biochemical studies indicate this molecule to be extremely heterogeneous, both in apparent molecular weight and in isoelectric point, to be highly negatively charged, to contain protein, and to bear the SO4 residues on a large Pronase-resistant moiety that is susceptible to chondroitinase ABC digestion. All of these features are consistent with those expected of the chondroitin sulfate class of proteoglycans. The studies using the lymphoblastoid B-cell lines indicate that the proteoglycan-like component can be synthesized by B cells in the absence of other cells, whereas the studies using tonsils and peripheral blood cells indicate that it is a normally synthesized component. The finding that this component could be immunoprecipitated by several different antihuman Ia monoclonal antibodies and antisera, each of which detects separate Ia species, underscores the intimate association of this component with the and invariant chains in the Ia oligomeric complex. The uniqueness of the Mr 40,000-70,000 molecule to the Ia complex suggests a possible role of this component in the function of that complex. It is as yet unclear what that role may be. Possibilities include stabilization of a particular conformation of the Ia glycoproteins, a direct role in intercellular recognition and communication, and involvement in maturation and processing of Ia glycoproteins. Preliminary evidence suggests that the sulfated component is associated with Ia glycoproteins present on the cell surface, but it is not yet known at what point in the biosynthesis of these molecules the association begins. Further studies are necessary to elucidate any possible role this component may have in the maturation or function of Ia. a,

/3,

The authors are grateful to Ms. Peggy Finan and Ms. Judy Craig for excellent secretarial assistance. This work was supported in part by U.S. Public Health Service Grants CA-20500, CA-33529, AI15353, and AI-18925.

FIG. 6. (A) Bio-Gel P-10 column chromatography of Pronase-digested (-*e) and alkaline borohydride-treated (o----o) K14 immunoprecipitate prepared from 35SO4-labeled Swei cells. (B) NaDodSO4/PAGE analysis of 35S04-labeled material remaining on a K14 immunoprecipitate after mock digestion (lane 1) or chondroitinase ABC digestion (lane 2). Molecular weights are indicated as Mr x 10'3. (C) Paper chromatographic analysis of 35S04-labeled material released from K14 immunoprecipitates by mock digestion (o-o) or by chondroitinase ABC digestion (.-). The mock-digested released material remains at the origin. Arrows indicate the positions of the standards, from left 30 to right, 6-sulfated, 4-sulfated, and nonsulfated N-acetylgalactosamine-uronic acids.

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