N-Linked Oligosaccharides of Murine Major Histocompatibility Complex Class I1 Molecule ROLE IN ANTIGENIC PEPTIDE BINDING, T CELL RECOGNITION, AND CLONAL

To evaluate the functional role of the N-linked oli- gosaccharides of major histocompatibility complex (MHC) class I1 molecules, affinity-purified murine IA" class I1 molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IA" molecules were characterized by 12% SDS-polyacryl-amide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the a10 heterodimer was confirmed by lectin-link Western blot analysis. Like the native IA" molecules, the deglycosylated IA" mole- cules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IA".MBP(89-101) and deglycosylated IA"-MBP(89-101) were compared at 4 and at 37 "C. Both complexes were equally stable at 4 "C; however, at 37 "C the deglycosylated IA". MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IA"-MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that rec- ognize and respond to IA".MBP(89-101). Finally, the complexes of deglycosylated IA"-MBP(89-101) lysate method. For T cell binding experiments, radiolabeled IA" or deglycosylated IA" was incubated with unlabeled peptides under identical conditions and purified. Specific activities of purified radiolabeled complexes were 6.7 X IO6 and 6.83 X IO6 cpm/ pg for IA".MBP(89-101) and deglycosylated IA". MBP(89-101), re-spectively. Induction of in Vitro T Cell Nonresponsiueness-Residual antigen- presenting cells were removed by subjecting cloned HS-1 T cells (10 days following antigen pulsing) to two rounds of 19% metrizamide density-gradient centrifugation, followed by two washes in RPMI 1640 medium. HS-1 cells (1 X lo6) were cultured with either 5 or 20 pg of preformed IA".MBP(89-101) or deglycosylated IA".MBP (89- 101) complexes for 26 h at 37 "C. A 10-fold molar excess of MBP(89-101) peptide, IA" alone, deglycosylated IAs alone, and IAn.MBP(l- 14) or deglycosylated IA".MBP (1-14) complexes were used as con-trols. The cells were washed four times and 8 X IO4 T cells in triplicate were cultured with 5 X lo5 freshly irradiated SJL/J spleen cells in the presence of 0,5, 10, or 20 pg/ml of MBP(89-101) peptide. During the last 8 h of a 72-h incubation, 1 pCi (1 Ci = 37 GBs) of [3H] thymidine was added, and the degree of proliferation was measured by incorporated radioactivity.


N-Linked Oligosaccharides of Murine Major Histocompatibility Complex Class I1 Molecule
To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class I1 molecules, affinity-purified murine IA" class I1 molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IA" molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three Nlinked sugar components from the a10 heterodimer was confirmed by lectin-link Western blot analysis. Like the native IA" molecules, the deglycosylated IA" molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IA".MBP(89-101) and deglycosylated IA"-MBP(89-101) were compared at 4 and at 37 "C. Both complexes were equally stable at 4 "C; however, at 37 "C the deglycosylated IA". MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IA"-MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IA".MBP(89-101). Finally, the complexes of deglycosylated IA"-MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IA".MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class I1 molecules may not be essential for either antigenic peptide binding or T cell recognition.
In addition results obtained here provide evidence that the carbohydrate moities of MHC class I1 molecules may not be involved in induction of T cell clonal anergy.
Murine MHC class I1 molecules (or Ia antigens) are cell surface glycoproteins which in conjunction with specific an- It has been reported that the a/@ chains expressed by various cell types or different cell lines show heterogeneity in the oligosaccharide patterns (5-6). The heterogeneity of carbohydrate components has been shown earlier to have some association with the differences in the capacity of antigen-presenting cells (APC) to activate T lymphocytes in an antigen-specific manner (7)(8)(9).
In contrast, Krieger et al. (10) reported that neuraminidasetreated IAd molecules did not show any alteration in their ability to present ovalbumin peptide to T hybridoma cells using a planar membrane system of antigen presentation. Several reports have been published indicating that the carbohydrate moieties of MHC class I antigens are not essential for their recognition by T lymphocytes (11-15). Although a number of studies have evaluated the role of N-linked oligosaccharides in the expression and function of MHC class I, relatively little is known about the role of N-linked sugar entities in MHC class I1 function. A study in this regard involved site-directed mutagenesis to selectively modify the N-linked acceptor sites of a and p chains, showing that the N-linked oligosaccharides may affect the secondary structure of class I1 molecules, and deletion of these oligosaccharides may have either a negative or positive effect on antigen presentation to T hybridomas (16). There is no evidence available that correlates the role of N-linked carbohydrate components of MHC class I1 and antigen-specific T cell tolerance or nonresponsiveness, a phenomenon referred to as clonal anergy (17). In this study, affinity-purified murine IA" molecules were deglycosylated with N-glycanase, and the deglycosylated IA" was used to examine the role of N-linked oligosaccharide components in (i) antigenic peptide binding, (ii) stability of IA". MBP peptide complexes, (iii) binding of deglycosylated IA" . MBP complexes to TCR on cloned T cells, and (iv) in vitro induction of T cell nonresponsiveness.

MATERIALS AND METHODS
Purification of Murine IA"-IA" was purified from an Nonidet P-40 extract of membranes prepared from SJL/J mouse spleen cells using an affinity support prepared by coupling monoclonal antibody, 10-2.16 (specific for both IAk and IA"), with Sepharose 4B beads by the standard cyanogen bromide coupling method as described earlier (18,19). Briefly, a high-speed (100,000 X g ) membrane fraction was detergent-extracted in a buffer containing 10 mM Tris-HC1, pH 8.3, 0.5% Nonidet P-40, 0.1 M NaCl, 5 mM EDTA, 0.02% sodium azide, and 1 mM PMSF, and the lysate was recycled over the pre-equilibrated antibody column at 4 "C for 16 h. The column was washed with 10 bed volumes of deoxycholate buffer containing 10 mM Tris-HC1, pH 8.3, 0.5% deoxycholate, 0.1 M NaCI, 5 mM EDTA, 0.02% sodium azide, and 1 mM PMSF followed by 5 bed volumes of PBS containing 1% n-octyl-P-D-glucopyranoside (OG) detergent. Finally, IA" was eluted by 20 mM phosphate buffer, pH 11, containing 0.1 M NaCl, 1% OG, 0.02% sodium azide, and 1 mM PMSF. Each fraction was immediately neutralized with acetic acid to a final concentration of 12 mM, and the pooled IA" preparation was concentrated using an Amicon Centriprep-10 concentrator. Affinity-purified IA" was characterized by 12% one-dimensional SDS-polyacrylamide gel electrophoresis under native conditions.
Lectin-link Western Blot Analysis-Lectin-link Western blot kit was purchased from Genzyme Inc. Cambridge, MA, and the manufacturer's recommended protocol was followed with some modifications. Proteins (1 pg) were electrophoretically transferred onto a nitrocellulose membrane at 250 mA constant current for 3 h at 25 "C using Bio-Rad's miniblot apparatus in standard Tris/glycine/methanol buffer, pH 8.8 (1 liter of transfer buffer contains 2.4 g of Tris base, 11.52 g of glycine, and 200 ml of methanol). The nitrocellulose membrane containing protein bands was blocked with carbohydratefree blocking reagent and was incubated with the biotinylated lectin at 25 "C for 1 h. The blot was washed with washing buffer and incubated with avidin-alkaline phosphatase for another 1 h at 25 "C. Following incubation, the blot was washed extensively with washing buffer and developed with the staining substrate. Among five different lectins tested for blot analysis, Datura stramonium agglutinin (DSA) worked best for our application.
Synthesis of Peptide Analogs-The bovine MBP peptide analogs with the sequence MBP(89-101)YS9 with the sequence Ac-YFKNIVTPRTPPP-NH2; MBP(1-14)A4 with the sequence Ac-ASQARPSQRHGSKY-NH2 and ovalbumin OVA(323-340)Y340 with the sequence Ac-ISQAVHAAHAEINEAGRY-NH2 were synthesized by standard solid-phase methodology using side chain-protected Fmoc amino acids on an automated peptide synthesizer (Applied Biosystems 431 model). A tyrosine residue was added in both MBP(89-101) and OVA(323-340) peptides either to the N-or Cterminal end to enable radiolabeling with Iz5I. Deprotected crude peptides were purified by reverse-phase high performance liquid chromatography, and the homogeneity and the identity of the purified peptides were confirmed by mass spectroscopic analysis.
Radiolabeling of Peptide and IA" Molecules-Radiolabeling of peptides was achieved by the standard chloramine-T labeling procedure (20). Typically, 2.5 mg of peptide in 500-pl volume was incubated with 2 mCi of Nal2'1 in 0.1 M sodium phosphate buffer, pH 7.7. The labeled peptides were separated from free lZ5I by G-10 Sephadex gel filtration chromatography, and the specific activities calculated ranged from 0.5 to 1.0 X lo6 cpm/pg. Labeling of IA" and deglycosylated IA" with was achieved using Pierce Chemical Co. IODO-BEADS and the manufacturers recommended procedure. 100 pCi of N a Y in 0.1 M sodium phosphate buffer, pH 7.5, was added to 200 pg of Ia molecules. Pre-loaded IODO-BEADS were prepared by washing with 50 mM phosphate buffer, pH 7.5 (1 ml of buffer for two beads) and dried on Whatman paper. Two beads were added to the reaction mixture and left at room temperature for 15 min. Labeled IA" or deglycosylated IA" was separated from the beads, and free '''1 was removed by extensive dialysis against PBS containing 0.1% OG at 4 "C. Specific activities of IA" and deglycosylated IA" ranged from 6.5 t o 6.8 X lo6 cpm/pg.
Peptide Binding Assay-The binding of radiolabeled peptides was measured by the silica gel TLC plate assay method as described earlier (18, 19). Briefly, Ia molecules at a concentration of 400-500 pg/ml were incubated with 30-fold molar excess of radiolabeled peptides at 37 "C for 48 h. In competition experiments, 300-fold molar excess of unlabeled peptide over Ia was also added in the same reaction mixture prior to the incubation. The unbound peptide was removed by dialysis against PBS containing 0.1% OG, and 1-pl samples were applied in triplicate at the origin of silica gel TLC plate. Ascending TLC was performed using plastic-supported silica gel plates (DC-Plastikfolien Kieselgel60 F254) obtained from EM Sciences Inc. The solvent system consists of 5% ammonium acetate in 50% aqueous methanol. The solvent front was allowed to run 5 cm from the origin in a standard TLC chamber. Plates were subjected to autoradiography, and the distribution of radioactivity was estimated by excising and y counting strips at RF (relative frequency)= 0-0.2 and RF = 0.2-1.0. In parallel experiments, an equivalent amount of radiolabeled peptides incubated and dialyzed in the absence of Ia were also spotted at the origin of the TLC plate in triplicates, and the radioactivity values were subtracted in calculating the percent peptide occupancy of Ia molecules. The percent of IA" or deglycosylated IA" with bound labeled peptides was calculated from the specific activities of the respective peptides.
Preparation of Class II-Peptide Complexes-Two types of complexes were prepared; unlabeled complexes of either IA" or deglycosylated IA" with MBP(89-101) or MBP(1-14) were prepared and purified as described earlier (21) with some modifications and were used for all in vitro T cell functional assays. Briefly, affinity-purified IA" or deglycosylated IA" molecules at 400-500 yg/ml were incubated at 37 "C for 48 h with 30-fold molar excess of unlabeled peptides in a buffer containing 10 mM Tris-HC1, pH 8.3, 0.02% sodium azide, 1 mM PMSF, and 1% OG. The unbound free peptide was removed by extensive dialysis of the complex against serum-free RPMI medium, and the final complex preparation was tested for endotoxin using limulus amebocyte lysate method. For T cell binding experiments, radiolabeled IA" or deglycosylated IA" was incubated with unlabeled peptides under identical conditions and purified. Specific activities of purified radiolabeled complexes were 6.7 X IO6 and 6.83 X IO6 cpm/ pg for IA".MBP(89-101) and deglycosylated IA". MBP(89-101), respectively.
Induction of in Vitro T Cell Nonresponsiueness-Residual antigenpresenting cells were removed by subjecting cloned HS-1 T cells (10 days following antigen pulsing) to two rounds of 19% metrizamide density-gradient centrifugation, followed by two washes in RPMI thymidine was added, and the degree of proliferation was measured by incorporated radioactivity.

RESULTS
In order to evaluate the potential role of N-linked oligosaccharides of MHC class I1 molecules in various in vitro studies, affinity-purified IA" from SJL/J mice was subjected to Nglycanase treatment. IA" eluted from a 10-2.16 monoclonal antibody-coupled affinity column was concentrated to 400-500 pg/ml and denatured in the presence of SDS and mercaptoethanol. An attempt to remove N-linked sugar residues of IA" under fully native conditions (ix. in the absence of SDS and ME) was totally unsuccessful even with a 20 times increased concentration of N-glycanase. Enzymatic digestion was carried out for 16 h at 37 "C. The deglycosylated IA" did not react with 10-2.16 monoclonal antibody when tested in a dot blot assay using peroxidase-conjugated anti-mouse IgG (data not shown). Therefore the recovery of deglycosylated IA" from the reaction mixture was achieved by ethanol precipitation at -20 "C followed by centrifugal vacuum concentration. Concentrated deglycosylated IA" was reconstituted by detergent buffer (20 mM phosphate, pH 8.0, 100 mM NaCl, and 1% OG) and was characterized on 12% SDS gel under reduced and nonreduced conditions (Fig. 1). Under reduced conditions, both a and @ chains showed increased sharpness of bands and migrated at lower molecular weight region as compared with glycosylated a and @ chains (Fig. LA). Similarly, faster migrating partially dissociated a and @ chains were observed under nonreduced conditions (Fig. 1B). A significant amount (40%) of the deglycosylated I& remained in the a/@ heterodimeric form as measured by density scanning of the native gel (Fig. IC). In contrast to the a and @ chains, the intact a/@ heterodimer of the deglycosylated IA" did not show significant mobility difference in the native gel.
To ensure complete removal of N-linked sugar residues from IA" had occurred, lectin-link Western blot analysis was performed (Fig. 2). IA* and deglycosylated IA" were run on a 12% polyacrylamide SDS gel under reduced conditions. One set ( A ) was stained with silver nitrate to visualize the a and p chains. The other set was subjected to electro-transfer onto a nitrocellulose membrane. Transferred proteins on nitrocellulose were fixed and allowed to react with biotinylated lectin. The membrane was then allowed to react with alkaline phosphatase-coupled avidin to develop color with substrate. As shown in Fig. 2B, the deglycosylated IA" lane did not show any reactivity. IA* incubated under identical conditions but in the absence of N-glycanase, as well as standard glycoprotein mix, showed strong reactivity in the DSA-biotinylated lectin and avidin system. Peptide binding to IA" or deglycosylated IA" was examined . In all peptide binding experiments, an equivalent amount of radiolabeled peptides incubated and dialyzed under identical conditions, but in the absence of class I1 molecules, were used as control, and counts appearing at the origin (usually t 2 % ) were subtracted for calculating the percent occupancy of class I1 with labeled peptide. As shown in Fig. 3 (solid bar), both IA" (column 1 ) and deglycosylated IA" (column 2 ) were capable of binding MBP(89-101) peptide. The percent of these Ia molecules occupied with peptide ranged N-Linked Oligosaccharides of MHC Class I1 from 40 to 50 in various experiments. Specificity of the binding was confirmed by either using an irrelevant peptide like OVA(323-340)Y340, which is known to bind to IAd (22), or by competing out the radiolabeled MBP(89-101) peptide with excess unlabeled MBP(89-101) peptide. In all control experiments, the deglycosylated IA" behaved similar to native I&.
Next we compared the rate of dissociation of IA" . MBP(89-101) and deglycosylated IA".MBP(89-101) complex to see whether sugar residues are critical in stabilizing the preformed complexes. Complexes of IA". MBP(89-101) and deglycosylated IA". MBP(89-101) with lz51-labeled peptides were prepared, purified, and at various times a 1-pl aliquot from the reaction mixture was loaded onto a silica gel TLC plate in triplicate and the percent of IA" or deglycosylated IA" with bound peptide was calculated. Data presented in Fig. 4A shows that the complexes of IA".MBP(89-101) were very stable at 4 and at 37 "C. However, in the case of deglycosylated IA". MBP(89-101) complex, the dissociation rate was relatively high at 37 "C as compared with 4 "C (Fig. 4B). Assuming a first order of kinetics, the slope from the inset figures was used to calculate the dissociation rate constant ( K d ) .
The IA".MBP(89-101) complexes showed low affinity to the HS-1 cells, but at the end of the 6-h incubation, the complexes were able to bind a similar number of receptors like the IA". MBP(89-101) complexes.

DISCUSSION
In this study, N-linked oligosaccharide moities of murine IA" a/@ heterodimeric molecules were enzymatically removed by asparagine amidase (N-glycanase) to examine the effect of the two N-linked oligosaccharides of the CY chain and one Nlinked oligosaccharide of the P chain of MHC class I1 molecules in antigenic peptide binding, T cell recognition, and T cell function. This enzyme catalyzes the hydrolysis of Asnlinked oligosaccharides at the P-aspartylglucosylamine bond between the innermost GlcNAc and the asparagine residue of glycoproteins. In general, the cleavage of N-linked sugar residues by N-glycanase is highly efficient when glycoproteins are fully denatured. Cleavage of N-linked oligosaccharides of native glycoproteins often results in incomplete digestion and requires 10-20 times more enzyme units. Our attempt to cleave N-linked sugar residues of murine I& under fully native conditions was totally unsuccessful even at 20-fold excess enzyme units. Addition of mild reducing agent (up to 20 mM cysteine) along with 20-fold excess enzyme and increased incubation period did not improve the cleavage of oligosaccharides of these molecules under native conditions. Because of these reasons, IA" was denatured in the presence of SDS and ME prior to the digestion. Exposure of MHC class I1 molecules to SDS had no effect on peptide binding ability of these molecules. This was shown earlier (24) and was confirmed recently in our laboratory (25), because the a/ @ heterodimeric MHC class I1 molecules and their individual polypeptide chains electroeluted from native SDS-polyacrylamide gels can equally bind antigenic peptides. Similarly, chains isolated from reduced SDS gels, where ME is added in the sample buffer, can equally bind antigenic peptide.' As expected, both the a and P chains of deglycosylated IA" when analyzed on reduced silver-stained SDS gel showed a significant shift in their molecular weight and increased band sharpness with no detectable glycosylated chains. However, no significant difference in the mobility of the a l p heterodimer was observed when IA" and deglycosylated IA" were analyzed under nonreduced 12% SDS-polyacrylamide gel electrophoresis. To further demonstrate the complete removal of all three N-linked sugar residues from IA", a highly sensitive Lectin-link Western blot analysis was performed that involves a biotin-avidin system. In this system lectins bind specifically to the sugar chains on the glycoproteins that are transferred onto nitrocellulose and easily visualized by enzyme-substrate color development. Detection of glycosylated IA" was optimized using five different biotinylated lectins: concanavalin A, Ricinus communis agglutinin (RCA), DSA, Phasseolus vulgaris erythrolectin (PHA-E), and wheat germ agglutinin. Among all these, DSA was most sensitive and worked best for detecting IA" glycosylation in our experiments. The deglycosylated IA" recovered following N-glycanase treatment under B. Nag, D. Passmore, and D. Kopa, unpublished results. the experimental condition, did not show any detectable reactivity with the DSA-biotinlalkaline phosphate-avidin system. Milligram quantities of IA" were subjected to N-glycanase treatment and the recovered deglycosylated IA" was used for various studies.
Binding of the MBP(89-101) peptide to deglycosylated IA" was examined to see the effect of oligosaccharide removal on peptide binding ability. Calculating the percent of MHC class I1 molecules with bound labeled peptide by the TLC method is highly sensitive and has been shown to correlate well with the standard dialysis method (19). In all peptide binding experiments, the deglycosylated IA" was able to bind antigenic peptide in quantities similar to the native IA" molecules. The binding data presented here suggest that the denatured MHC class I1 molecules in the presence of SDS and 2-ME are capable of binding antigenic peptide like the nonreduced and nondenatured preparations. This result is not surprising, since isolated a and @ chains or dimers of MHC class I1 molecules eluted from SDS gels have been shown to bind antigenic peptides like the native dimer molecule (24,25). Since there was no significant difference between the deglycosylated IA" and native IA" in their ability to bind antigenic peptide, we decided to compare the stability of these two complexes by measuring the dissociation kinetics. Dissociation kinetics of several different MHC class 11-peptide complexes indicate that the complexes of MHC class I1 and selected peptides are usually very stable (26)(27)(28)(29). In fact, in a long term complex stability study using human MHC class I1 molecules and antigenic peptides, we have recently observed that preformed complexes are 95-100% stable for over a period of 6 months at 4 "C.' In this study, the dissociation kinetics of deglycosylated IA". MBP To compare the T cell recognition of IA".MBP(89-101) and deglycosylated IA". MBP(89-101) complexes in vitro, binding of preformed complexes to cloned HS-1 T cells was measured. In T cell binding experiments, radiolabeled MHC class I1 was complexed with unlabeled peptides and the binding was measured at 37 "C. The rate of complex association was initially slow in the case of deglycosylated IA" . MBP(89-101) complexes compared with the native IA". MBP(89-101) complex. However at the end of a 6-h incubation, the number of bound complexes per T cell was almost identical. Both the dissociation kinetics and the T cell recognition data suggest that the N-linked sugar residues are perhaps important in stabilizing the MHC-peptide complexes and are involved in cell-cell interaction.
We have shown recently that the cloned HS-1 T cells when exposed to the complexes of IA' plus MBP(90-101) resulted in T cell nonresponsiveness to a subsequent challenge of MBP(90-101) along with fresh APC (30). A similar set of experiment was performed to determine the role of N-linked oligosaccharide components of MHC class I1 on T cell inactivation. APC-depleted HS-1 cells when incubated with either IA" .MBP(89-101) or deglycosylated IA". MBP(89-101) complexes, and challenged with an increasing concentration of MBP(89-101) peptide, became almost completely nonresponsive. Lack of a proliferative response was not the result of either cell death or loss of viability, because in various control experiments cells exposed with IA" alone, deglycosylated IA" alone, or complexes of IA" and deglycosylated IA" with MBP(1-14) responded well in the proliferation assay. To ensure that the lack of proliferative response in the case of deglycosylated IP.MBP(89-101) complexes is not due to release of bound peptide, a 10-fold molar excess of peptide MBP(89-101) was included in our control experiments that showed no effect on proliferative response. In conclusion, these results demonstrate that the N-linked sugar residues of murine IA" molecules are not essential for peptide binding, T cell recognition, or in uitro induction of T cell nonresponsiveness. Under physiological conditions, the N-linked oligosaccharides moities may play an important role in stabilizing the MHC class 11-peptide complexes on the surface of APC and perhaps are involved in cell-cell adhesion. Further studies with several different T cell clones are necessary to generalize the role of N-linked sugar residues of MHC class I1 molecules.