Differential Glycosylation of MHC Class IIMolecules on Gastric Epithelial Cells:Implications in Local Immune Responses

Carlos Barrera, Rosario Espejo, and Victor E. Reyes

ABSTRACT: Class II major histocompatibility complex(MHC) expression is a hallmark of antigen presentingcells (APC). Human gastric epithelial cells (GEC) expressclass II MHC and this expression increases during infec-tion with Helicobacter pylori as does the number of CD4 Tcells found adjacent or in between epithelial cells. Theseobservations suggested that human GEC act as APCs. Tocharacterize and compare class II MHC complexes withthose present in conventional APC, immunoprecipitatedclass II MHC from GEC and B cells, as prototypic APC,were separated by two-dimensional electrophoresis. Al-though the composition of class II MHC from both cellphenotypes was similar, their electrophoretic mobilitydiffered. Methodical elimination of carbohydrates, eitherenzymatically with endoglycosidase-H or blocking withtunicamycin, revealed that the deviations were due to

differences in glycosylation in both cell phenotypes.When deglycosylated class II MHC � chains, � chains,and the invariant chain from both cell phenotypes weremixed and run in the same gel, the core proteins hadidentical migration patterns. Because differences in gly-cosylation of class II MHC proteins may affect peptideselection and/or recognition by T cells, the noted differ-ences in glycosylation of class II MHC expressed by GECcould be important in considering their potential role asAPC locally. Human Immunology 63, 384–393 (2002).© American Society for Histocompatibility and Immu-nogenetics, 2002. Published by Elsevier Science Inc.

KEYWORDS: glycosylation of MHC class II; glycosyl-ation of invariant chain protein; mucosal immunity; non-conventional antigen presenting cells

ABBREVIATIONSMHC major histocompatibility complexAPC antigen presenting cellsCLIP invariant chain class II MHC-associated

peptideIi invariant chainPMSF phenylmethylsulfonyl fluoride

Iad immunoadsorbants–beadsBFA Brefeldin-Aendo-H endo-�-N-acetylglucosaminidaseNEPHGE nonequilibrium pH gradientSDS sodium dodecyl sulfatePAGE polyacrylamide gel electrophoresis

INTRODUCTIONThe expression of major histocompatibility complex(MHC) class II molecules is characteristic of professionalantigen presenting cells (APCs). The role of MHC classII molecules on APC has been described as a prerequisitefor direct interaction with CD4� T cells prior to theinitiation of an immune response [1–3]. APCs effectivelyinternalize exogenous antigens, which become unfoldedand digested in the low pH intracellular compartments

[4, 5]. Then, the newly formed antigenic peptide frag-ments associate with � and � chains of MHC class II inendosomal compartments, following the removal of in-variant chain class II MHC-associated peptide (CLIP).The MHC class II peptide complex is then expressed onthe cell surface where CD4� T cells may recognize it.The expression of MHC class II molecules was recentlyidentified in cell types other than conventional APC.Among these cell types are the mucosal epithelial cells[6–8]. Expression of MHC class II and associated invari-ant chain (Ii) proteins were also observed upon stimula-tion of cultured epithelial cells with cytokines [9, 10].Mucosal epithelial cells from gastric tissues express MHCclass II molecules. This expression suggests their involve-ment in local immune responses [11]. However, little is

From the Departments of Pediatrics (C.B., R.E., V.E.R.) and Micro-biology and Immunology (C.B., V.E.R.), University of Texas MedicalBranch, Galveston, TX, USA

Address reprint requests to: Dr. Victor E. Reyes, Route C-66, Children’sHospital, 301 University Boulevard, UTMB, Galveston, TX 77555,USA; Tel: (409) 772-1750; Fax: (409) 772-1761; E-mail:vreyes@utmb.edu.

Received October 22, 2001; accepted February 6, 2002.

Human Immunology 63, 384–393 (2002)© American Society for Histocompatibility and Immunogenetics, 2002 0198-8859/02/$–see front matterPublished by Elsevier Science Inc. PII S0198-8859(02)00386-5

known about the intracellular events that influence an-tigen processing and presentation by nonconventionalAPCs, such as gastric epithelial cells (GEC). In separatestudies, we have reported that GEC express the machin-ery required for the processing of exogenous antigens[12] and the important costimulatory molecules neededfor effective T-cell stimulation [11].

The synthesis of � and � chains of MHC class II isfollowed by immediate association with a nonpolymor-phic type II glycoprotein known as the invariant chain(Ii). Then this complex (�, �, and Ii)3 is transported tothe Golgi compartments to undergo several post-tran-scriptional modifications, such as acylation [13], phos-phorylation [14], sulfation [15], and the addition and/orsubtraction of N-linked or O-linked carbohydrates [15–17]. Interestingly, only modifications in glycosylationprocesses induce significant changes in molecular weightas well as in net charge of the proteins. Glycosylation ofMHC class II provides two N-linked oligosaccharides at� chain positions (82–84 and 122–124) and a singleN-linked oligosaccharide in the � chain (19–21) [15, 16,18]. In the case of Ii chain, the glycosylation provide twoN-linked modifications [15] and two O-linked oligosac-charides [17, 19]. Studies have demonstrated that � and� chains expressed by different cell types exhibit differ-ential distribution of oligosaccharides, sialylation, andbranching patterns [13, 20]. In addition, the heteroge-neity in glycosylation of MHC class II molecules hasbeen associated with differences in their capacity to ac-tivate T lymphocytes in an Ag-specific manner [21–23],suggesting that differences in glycosylation of MHC classII molecules and Ii chain may be involved in the differ-ential capacity for peptide selection by APCs. Becausepeptide selection and interaction with T cells are essen-tial functions of class II MHC, any differences in post-translational modification that could influence thosefunctions are important to characterize. Therefore, inthese studies, we examined the glycosylation events ofMHC class II and Ii chains on GECs and compared themwith those on prototypical APCs. We blocked the gly-cosylation process by sequential deglycosylation ap-proaches to discern whether deviations in electrophoreticmobility were due to differences in carbohydrate modi-fications, the proteins core, or both.

MATERIALS AND METHODSCell Lines and Culture ConditionsA human leukocyte antigen DR (HLA-DR1) homozy-gous, Epstein-Barr virus transformed B-cell lineJESTHOM was used as a prototypical APC obtainedfrom Charles Carpenter (Bringham & Women’s Hospi-tal, Boston, MA, USA). The KATO III and N87 epithe-lial cell lines were received from ATCC (Rockville, MD,

USA). The cells were grown in RPMI 1640 (Gibco BRL,Gaithersburg, MD, USA) medium supplemented with10% fetal calf serum (FCS; Atlanta Biologicals, Atlanta,GA, USA), glutamine (1 mM), penicillin (1 U/ml), andstreptomycin (100 �g/ml).

AntibodiesAnti-human Ii monoclonal antibody BU45 was obtainedfrom The Binding Site (Birmingham, United Kingdom).The hybridoma secreting the anti-human MHC class IImonoclonal antibody IVA12 was obtained from ATCCand was used to generate antibody-containing ascitesfluid.

Flow CytometryA total of 106 GEC or JESTHOM control B cells, whichhad been grown in medium alone or supplemented withIFN-� (100 U/ml; Boehringer Mannheim, Mannheim,Germany) for 48 h, were incubated with anti-MHC classII monoclonal antibody (1 �g/ml) for 30 minutes in ice.Then cells were washed three times with ice-cold PBSand were incubated with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA,USA) for an additional 30 minutes in ice. Followingincubation, the cells were washed and fixed with 1%paraformaldehyde in PBS. The cells were then analyzedin a FACScan cytometer (Becton Dickinson, MountainView, CA, USA).

Metabolic RadiolabelingJESTHOM B cells and GEC in the log phase of growthwere incubated at a density of 107 cells/ml in the absenceof methionine and cysteine for 1 h. At the end of thedeprivation period, the cells were supplemented with theradiolabeled amino acids [35S]methionine and cysteine(0.5 mCi/108 cells; ICN Biomedicals, Inc, Irvine, CA,USA). After a 5-h incubation, the cells were washed threetimes with PBS. Then, cells were hypotonically lysedwith 10 mM Tris-HCl buffer, pH 8.1, containing10-mM leupeptin, 1-mM phenylmethylsulfonyl fluoride(PMSF), and 2-mM N-ethyl malemide (NEM). Nucleiwere removed by centrifugation at 10,000� g for 10minutes and the supernatants were centrifuged at100,000� g for 45 minutes to obtain the microsomalmembranes. Microsomal membrane proteins were solu-bilized in ice-cold, 10-mM Tris-HCl, pH 8.1, containing0.5% Triton X-100 (V/V). Unsolubilized material wasremoved by centrifugation at 100,000� g for 1 h andthen the samples were ready for immunoprecipitationstudies.

Immunoprecipitation and ElectrophoresisSolubilized membrane samples were precleared with nor-mal rabbit serum and formalinized Staphylococcus aureus

385Differential Glycosylation Patterns of MHC Class II Molecules on Nonconventional APC

Cowen strain A (Chemicon, El Segundo, CA, USA).Immunoadsorbants (Iad) beads were prepared by incu-bating 100-�l protein A-sepharose beads (Sigma Chem-ical Co., St. Louis, MO, USA) with approximately 1 �gof either anti-MHC class II (IVA-12) or anti-Ii (BU45).After 16-h incubation at 4°C with solubilized mem-branes, the Iad were washed five times with Iad buffer(50-mM Tris-HCl, pH 7.4, 150-mM NaCl, 5-mMEDTA, 0.5% Triton X-100 (V/V), and 0.02% NaN3

(W/V)) and eluted with SDS sample buffer [62.5-mMTris HCl, pH 6.8, 10% glycerol (W/V), 5% 2-mercap-toethanol (V/V), and 2.3% SDS (W/V)]. The sampleswere analyzed by one- and two-dimensional gel electro-phoresis.

Brefeldin-A (BFA) TreatmentGEC and control human B cells were treated with 1�g/ml of BFA (Sigma Chemical Co.) 2 h prior to andduring metabolic radiolabeling with [35S] methionineand cysteine for 5 h as described above. Following mem-brane lysis, the immunoprecipitated samples were ana-lyzed by one- and two-dimensional gel electrophoresis.

BFA and Endo-H TreatmentImmunoprecipitated samples from cells pretreated withBFA, were digested with endo-�-N-acetylglucosamini-dase (endo-H; Boehringer Mannheim) 1-mU in 50 �l of0.15-M sodium citrate buffer, pH 5.5, with 0.1-mMphenyl sulfonyl fluoride (PMSF). The reaction was incu-bated for 14 h at 37°C. This reaction was stopped byboiling the sample for 3 to 5 minutes, and then theproteins were analyzed by one- and two-dimensional gelelectrophoresis.

Tunicamycin TreatmentJESTHOM and KATO III cells were preincubated in thepresence of 3 �g/ml of tunicamycin (Boehringer Mann-heim) 3-h before and during the 5-h labeling period.After immunoprecipitation the samples were treatedwith 0.4-U N-glycosydase, 2.5-mU O-glycosydase, and2-mU neuraminidase. The reaction was allowed to pro-ceed for 12 h at 37°C. Boiling the samples stopped thereaction, and the proteins were detected by one- andtwo-dimensional gel electrophoresis.

Two-Dimensional PolyacrylamideGel ElectrophoresisImmunoprecipitated proteins were eluted from the pro-tein A-sepharose beads (PAS; Sigma Chemical Co.) witha 50-�l solution containing 9.5 M urea (BioRad, Her-cules, CA, USA), 2% Triton X-100 (V/V), 5% 2-mer-captoethanol (V/V), and ampholines (3.5/10; SigmaChemical Co.). The first dimension tube gels containing3.5/10 ampholines were run at 500 V for 6 h. The seconddimension was run in 12.5% polyacrylamyde-sodiumdodecyl sulfate (SDS) slab gels. After protein separation,the gels were dried and exposed to x-ray film.

RESULTSGastric Epithelial Cells Express MHC Class IIFlow cytometry analysis was performed to initially con-firm the expression of MHC class II molecules by GEC.We analyzed established gastric epithelial cell lines(KATO III and N87), which were cultured in the pres-ence of IFN � (100 U/ml) for 48 h, to upregulate MHCclass II and Ii chain expression. The presence of MHCclass II molecules was evaluated by flow cytometry with

FIGURE 1 Class II MHC expression by gastric epithelialcells. Class II MHC molecules were expressed in gastric epi-thelial cells KATO III and N87 after IFN� treatment. Forcomparison, the JESTHOM B-cell line was used as a control.The cells were stained with anti-class II MHC IVA-12 mAb.The displacement of the fluorescence intensity curve to theright indicated a positive expression level.

386 C. Barrera et al.

anti-MHC class II (IVA-12) antibody. The MHC class IIexpression was detected in the fluorescence intensity inthe GEC lines examined. As expected, JESTHOM B cellexpressed high levels of MHC class II and were used as apositive control (Figure 1).

To confirm the previous observations and to establishthat MHC class II complexes from KATO III andJESTHOM cells express identical elements, we immu-noprecipitated MHC class II molecules with anti-class IIIVA-12 antibody from metabolically labeled cells, andanalyzed them by two-dimensional electrophoresis(NEPHGE/SDS-PAGE). We observed that MHC class IImolecules on GEC, as in conventional APC, had theassociated Ii chain (Figure 2). However, when immuno-precipitated MHC class II and associated Ii from bothGEC and B cells were mixed and run on the sametwo-dimensional gels, it was apparent that the individualproteins had different electrophoretic mobilities as evi-denced by non-overlapping proteins in the gels. Similarresults were obtained with N87 gastric epithelia cellline. These apparent differences in molecular weightsuggested differences in glycosylation patterns.

Partial Deglycosylation of MHC Class II Moleculesand Ii Chain

To determine that the differences in electrophoretic mo-bility of MHC class II molecules and Ii chain proteinsfrom JESTHOM and KATO III cells were due to differ-ences in glycosylation, we treated the cells with brefel-din-A (BFA), to block post-translational modifications ofthe proteins that may occur beyond the Golgi apparatus.Following treatment, we analyzed anti-MHC class IIimmunoprecipitated samples by one-dimensional gelelectrophoresis (Figure 3, left panel). In the presence ofBFA, we observed partial deglycosylation of MHC classII complexes that lead to altered separation of the pro-teins. We confirmed these observations by analyzing theseparation of the proteins in two-dimensional gel elec-trophoresis (Figure 3, right panels). Samples fromJESTHOM and KATO III cells treated and untreatedwith BFA were examined individually and as a mixturein a single gel. Upon treatment and analysis by two-dimensional gel electrophoreis, the immunoprecipitatedproteins from the BFA-treatment cells were almost con-gruent, which suggested that differences observed in the

FIGURE 2 Microheterogeneity of class II MHC molecules. Two-dimensional gel electrophoresis of class II MHC complex frommetabolically labeled JESTHOM and KATO III demonstrates the similarities in expression of all of the components. The �, �,Ii, and different isoforms of Ii (p41, �2, �3) are presented after immunoprecipitation with anti-class II MHC IVA-12 mAb. (Aand B) Represent individual samples; (C) exhibits the differential mobility of the proteins when the samples were mixed andseparated together in a single gel.

387Differential Glycosylation Patterns of MHC Class II Molecules on Nonconventional APC

untreated samples are largely due to post-Golgi modifi-cations. To confirm the results obtained with anti-MHCclass II immunoprecipitated samples, we also immuno-precipitated Ii chain and separated the proteins by one-dimensional and two-dimensional gel electrophoresis(Figure 4, left and right panel, respectively). The Iiisoforms from both cells had differential migration whenthey were run together. Given that Ii is nonpolymorphic,differential migration of Ii from both cells suggests thereare remaining BFA-resistant oligosaccharides, whichcause a differential electrophoretic mobility of the Iiisoforms.

Removal of N-Linked Carbohydrates From MHCClass IITo assure the complete removal of N-linked carbohy-drates, we treated JESTHOM and KATO III cells withBFA followed by digestion with endo-�-N-acetylglu-cosaminidase (endo-H) enzyme. One-dimensional gelelectrophoresis of immunoprecipitated samples with theanti-MHC class II IVA-12 mAb revealed that the dif-ferences in mobility persist as evidenced by their molec-ular weight (Figure 5, left panel). To confirm thesedifferences after deglycosylation, we analyzed these sam-ples either individually or as a mixture by two-dimen-

sional gel electrophoresis (Figure 5, right panels). Thedifferences in molecular weight of the � chain coreprotein of MHC class II after double treatment werereadily apparent. These results suggested that there aresome other oligosaccharides associated with MHC class IIproteins, O-linked carbohydrates, or some endo-H resis-tant N-linked. Similar results were obtained when thesamples were immunoprecipitated with anti-Ii mAb (notshown).

Complete Deglycosylation of MHC Class II andIi ChainRight after translation of proteins in the endoplasmicreticulum, there is a carrier lipid protein (dolicol), whichis involved in the transfer of the first block of oligosac-charides to the newly synthesized peptide. To block thisinitial step during glycosylation and to be able to detectonly the core protein free of carbohydrates, we treatedJESTHOM and KATO III cells with tunicamycin. Tu-nicamycin inhibits the action of the dolicol carrier lipidprotein. After immunoprecipitation with anti-MHCclass II and anti-Ii chain mAbs, the proteins were ana-lyzed by one- and two-dimensional gel electrophoresis.The similar electrophoretic mobility of the proteins wasevident after tunicamycin treatment, suggesting com-

FIGURE 3 Partial deglysosylation of class II MHC molecules. (A) One-dimensional gel electrophoresis of JESTHOM andKATO III untreated and treated with BFA revealed the partial deglycosylation of �, �, and Ii chains after immunoprecipitationwith anti-class II MHC IVA-12 mAb. (B, C, and D) Two-dimensional gel electrophoresis of class II MHC molecules fromuntreated cells, mixture of cells allows the comparison of the protein migration. (E) BFA-treated cells exhibiting small differencesin electrophoretic mobility suggested an incomplete deglycosylation of class II MHC molecules [arrows].

388 C. Barrera et al.

plete prevention of glycosylation steps in the newlysynthesized � and � chains of MHC class II and Ii. Theseparation of the proteins at the same molecular weightwas more evident when samples from JESTHOM andKATO III treated with tunicamycin were mixed andseparated by two-dimensional gel electrophoresis on asingle gel (Figure 6, panel D). Because glycosylation of �chain as well as Ii causes them to migrate toward theacidic end with a gradual increase in molecular weight,removal of the carbohydrates led to a coalescence of �chain and Ii. In the case of cells treated with tunicamycinand immunoprecipitated with anti-Ii BU-45 mAb, wewere able to detect the proteins running at the sameelectrophoretic mobility (Figure 7, left panel). Theseobservations were confirmed by separation of the proteinsin two-dimensional gel electrophoresis (Figure 7, rightpanel). The processed forms of Ii as well as p41, whichmigrate toward the acidic end of the gel, depending onthe extent of glycosylation, disappeared. Instead, only Iiand a single p41 form were detected.

We conclude that the core proteins of MHC class IIand Ii chains from JESTHOM and KATO III cells didnot present differences, and, therefore, the differences inthe mobility of MHC class II and the Ii chain were aconsequence of differences in glycosylation patterns.These differences in glycosylation will be the focus offuture studies to determine their biologic role in localimmune responses.

DISCUSSION

The processing and transport of antigenic peptides to thecell surface for presentation to T cells are fundamentalevents in the initiation of systemic and mucosal immuneresponses. Exogenous antigens, such as bacteria or toxins,are internalized and processed via the endocytic pathway.The resulting antigenic peptides are bound by MHCclass II molecules and thus presented to CD4� T cells.Even though many major advances have been made inthe last decade in understanding the intracellular andmolecular events that take place during processing andpresentation of foreign antigens, the interactions at themucosal surface level are poorly understood. Althoughpresentation of exogenous antigens are properties dele-gated to MHC class II, the role of Ii chain also has to beconsidered during these events. Ii is required for theappropriate assembly of � and � chains of MHC class II,as well as for targeting this complex to the endosomalcompartments [15, 21]. The structural microheteroge-neity of MHC class II and Ii expressed by gastric epi-thelial cells has been the subject of studies done in orderto understand their role in mucosal immunity [9, 24].Studies have demonstrated constitutive expression ofMHC class II molecules by gastric epithelium, and thatexpression is upregulated during infection with Helico-bacter pylori. In addition to MHC class II increased levels,there was also an increased number of CD4� T cells in

FIGURE 4 Partial deglycosylation of Ii chain. (A) One-dimensional gel electrophoresis of JESTHOM and KATO III untreatedand treated with BFA revealed the partial deglycosylation of Ii proteins after immunoprecipitation with anti-IIBU45 mAb (Iimrepresents the mature form and Ii after deglycosylation). (B, C, and D) Two-dimensional gel electrophoresis of the Ii chain fromimmunoprecipitated samples of untreated cells. (E) JESTHOM and KATO III cells treated and examined together in a single gel.Unequal electrophoretic distribution of spots suggests incomplete deglycosylation of the Ii chain [see arrows on (E)].

389Differential Glycosylation Patterns of MHC Class II Molecules on Nonconventional APC

FIGURE 5 Removal of N-linked carbohydrates from class II MHC molecules. (A) One-dimensional gel electrophoresis ofJESTHOM and KATO III untreated and treated with BFA, followed by immunoprecipitation and digestion with endo-H. (B, C,and D) Two-dimensional gel electrophoresis of class II MHC from immunoprecipitation with anti-class II MHC IVA-12 mAbuntreated cells. The persistent differences in mobility observed in one-dimension were confirmed in these gels. (E) Note that thedifferences in mobility suggested that not all the N-linked carbohydrates were removed from the � and � chains followingenzymatic treatment [see arrows].

FIGURE 6 Total deglycosylation of class II MHC molecules. JESTHOM and KATO III cells were untreated and treated withtunicamycin and immunoprecipitated with anti-class II MHC IVA-12 mAb. (A) One-dimensional gel electrophoresis reveals thesame electrophoretic mobility of � and � chains. (B, C, and D) Two-dimensional gel electrophoresis panels clearly demonstratedthese differences. (E) Note the drastic reduction in molecular weight of the � and � chains compared before and after treatment[see arrows on �].

390 C. Barrera et al.

between and adjacent to the epithelium during thisinfection suggesting that there is local antigen presenta-tion in the tissue area. The unequal oligosaccharide dis-tribution on MHC class II and Ii chain from B cells andGEC may be relevant for the peptide selection and sub-sequent presentation to the CD4� T cells.

In the studies presented here, we provide direct evi-dence to confirm that the differences in mobility of MHCclass II molecules from conventional APC, theJESTHOM B-cell line, and nonconventional APC,KATO III cells, were due to differences in glycosylationpatterns. The N-linked and O-linked oligosaccharidesassociated with the �, �, and Ii complexes were removedby stepwise deglycosylation process.

The MHC class II and Ii chain proteins were analyzedby one-dimensional gel electrophoresis to observe molec-ular weight differences of these proteins. In addition, todiscern finite differences in the immunoprecipitated pro-teins, we also analyzed the proteins by two-dimensionalgel electrophoresis. The initial comparison of MHC classII and Ii complexes from metabolically labeled B cellsand GECs indicated that although the constituents of theclass II MHC were represented in both cell phenotypes,their size was different as determined in mixed samplesrun in two-dimensional gels. The immunoprecipitated

samples using anti-MHC class II and anti-Ii mAbs fromJESTHOM and KATO III cells after BFA-treatmentexhibited minimal differences in electrophoretic mobil-ity in both one and two dimensions. BFA favors theselection of mature forms of MHC class II [25] andprevents the digestion of Ii chain by endosomal proteases[26].

Our results suggested that class II MHC possess dif-ferent glycosylation branches or the existence of diverseO-linked oligosaccharides in both cell lines. In order todeglycosylate the �, �, and Ii complex we pretreated thecells with BFA, then removed the N-linked carbohy-drates by using endo-H glycosidase, as previously doneby others [25]. Following immunoprecipitation withanti-MHC class II and anti-Ii chain mAbs, the sampleswere analyzed by one and two-dimensional electrophore-sis. The migration patterns were visualized in individualgels as well as in a mixture on a single gel. The proteinbands that run at different levels after treatment repre-sent those with O-linked or possibly endo-H resistantN-linked carbohydrates. Others have shown that pre-treatment of MHC class II with BFA make �, �, and Iichains more sensitive to endo-H, than samples without it[25].

To completely prevent N-linked and O-linked oligo-

FIGURE 7 Total deglycosylation of Ii chain. JESTHOM and KATO III cells were treated with tunicamycin and immuno-precipitated with anti-Ii Bu-45 mAb. (A) One-dimensional gel electrophoresis presents the same electrophoretic mobility of Iiafter treatment (Ii and p41 represent the isoforms of Ii). (B, C, and D) Two-dimensional gel electrophoresis untreated samples.(E) A mixture of treated samples clearly illustrates these differences in migration. Note the dots are superimposed, showing thesame molecular weight after treatment [see arrows on (E)].

391Differential Glycosylation Patterns of MHC Class II Molecules on Nonconventional APC

saccharides from modifying the �, �, and Ii complex, wetunicamycin-treated the cells and this allowed us toexamine the unmodified core proteins from both cellphenotypes. In the absence of associated carbohydrates,the proteins from both cell phenotypes migrated in thesame location in two-dimensional gels, which confirmedthat the differences initially noted for class II MHC andassociated Ii chain from both cell phenotypes were due todifferences in glycosylation. Those differences in glyco-sylation may play a role during antigen presentation andimpact the subsequent T-cell activation [27]. The extraaddition of oligosaccharides can induce alterations in thesecondary structure of the proteins that could in turnaffect their functions in others studies [28]. The glyco-sylation of MHC class II and Ii chain complex is requiredfor the appropriate assembly of these molecules and fortheir biologic functions [29, 30]. Further studies have tobe done in order to examine the effect of these differencesin the presentation of antigens to T cells and, morespecifically, in the selection of the relevant antigenicpeptides during mucosal infections. Studying the molec-ular events during local infection will help us understandhow mucosal immunity functions and how to modulatethis response to design novel therapies, such as oralimmunization or tolerance induction.

ACKNOWLEDGMENTS

The authors want to thank Mardelle Susman for her assistance.This work was supported in part by grant DK50669 and by afellowship from The Clayton Foundation.

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393Differential Glycosylation Patterns of MHC Class II Molecules on Nonconventional APC