MHCII concentration, or MHCII conformational changes, glycosylation pattern seen in human MHCII is known to although we cannot rule out small localized structural va-ria closely mirror the overall glycosylation pattern found in cell tions that do not affect peptide ligands. glycoproteins (Néel et al., 1987), this work suggests that It is conceivable that the requirement for large native MHCII is differentially glycosylated in active IBD and cancer. complex N-glycans on MHCII for GlyAg binding is based In strong support of this interpretation, transgenic animals ex- on extending the surface area of the binding platform beyond pressing 1,2-fucosyltransferase have significant changes in that of the canonical peptide-binding groove. GlyAg and cellular glycosylation patterns and develop spontaneous c-oli peptide association with MHCII is competitive and GlyAg tis (Brown et al., 2004).The etiology of this colitis was found presentation by APCs is dependent on HLA-DM expression to be centered upon the T cell compartment rather than de- (Cobb and Kasper, 2008;Velez et al., 2009), establishing that fects in the mucosal barrier function. Remarkably, the GlyAg bound peptide must be removed before GlyAgs can bind. used in this study, PSA, has been directly implicated as being As a result, GlyAgs likely form contacts within the peptide- a key bacterial factor that contributes to the maintenance of binding groove. However, the presented form of GlyAgs ranges gastric immune balance (Mazmanian et al., 2005, 2008). Spe- in molecular mass between 3 and 10 kD (Cobb et al., 2004; cifically, PSA administered to gnotobiotic animals restores the Cobb and Kasper, 2008), which is greater than the typical TH1/TH2 balance to that of conventionally colonized animals 13–18-aa peptide found bound to MHCII. Furthermore, we (Mazmanian et al., 2005), and PSA elicits IL-10–producing have shown that the helical nature of the GlyAg conformation T cells that show remarkable efficacy in down-regulating must be maintained to facilitate MHCII binding (Kreisman inflammation in animal models of IBD (Mazmanian et al., et al., 2007). Based on the known structure of MHCII (Gauthier 2008). Because the T cells activated by PSA appear to serve an et al., 1998), it is difficult to imagine how the relatively co-n antiinflammatory/tolerogenic role in the gut, our findings strained surface area of the peptide-binding groove alone can suggest that IBD-associated changes in protein glycosylation stably associate with a helical 3–10 kD polysaccharide. Given (Campbell et al., 2001; Brown et al., 2004) could result in that complex N-glycans can be the physical size of an immun-o defects in PSA presentation, leading to a failure to induce globulin domain (30 Å x 10 Å x 10 Å; Rudd et al., 1999), protective antiinflammatory IL-10–producing T cells and an inclusion of the two conserved sites for MHCII glycosylation exacerbation of gut pathology. could potentially add up to 600 Å2 in surface area on which the GlyAg molecule could associate, although the glycan array MATERIALS AND METHODS results show that these structures alone are insufficient for Reagents and cell lines. Human Raji B cells (American Type Culture Co-l GlyAg binding. Our findings allow us to speculate that native lection) were maintained in RPMI 1640 (Invitrogen) media supplemented complex N-glycans, as well as the MHCII protein backbone, with 10% FBS, penicillin, streptomycin (pen/strep), and-mercaptoethanol form direct contacts with GlyAgs and that modification of (ME). CHO cells transfected to secrete soluble human HLA-DR2 (DR2) these N-glycans destabilizes GlyAg association by removing a were provided by K. Wucherpfennig (Dana Farber Cancer Center, Boston, physical portion of the binding site. MA; Gauthier et al., 1998) and grown in protein free medium (HyQ SFM- 4CHO; HyClone) supplemented without (untreated; UNT) and with 10 µg/ml This study represents the first time that the N-linked KF (Cayman Chemical) or 175 µM CS (Sigma-Aldrich).The inhibitors and glycans on MHCII have been shown to play an integral role media were replaced daily. Recombinant DR2 proteins expressed with and in antigen binding. However, in 1995 it was reported that alter- without drugs present in the media (UNT-DR2, CS-DR2, and KF-DR2) ing the glycosylation sites in Ed resulted in changes in thymus were purified to homogeneity (>95% purity as judged by SDS-PAGE) from selection of V7+ T cells (Ishikawa et al., 1995), suggesting that the spent culture media as previously described (Cobb et al., 2004). HEK293 glycosylation can influence the binding of a subset of TCRs, cells expressing murine TLR2 (InvivoGen) were grown in standard DME medium supplemented with 10% FBS. PSA was expressed by aB. fragilis variant depending on the V usage, although it remains unclear that expresses only PSA in the capsule (Krinos et al., 2001) and purified to whether this effect was direct (i.e., the glycans participated in homogeneity, radiolabeled, fluorescently tagged with Alexa Fluor 594, and the binding) or indirect (e.g., through structural alignment of biotinylated as previously described (Cobb et al., 2004). Biotinylated MBPp the binding surfaces). A few years later, the Mellins group r-e (residues 84–99) and OVAp (residues 323–339) were purchased from New ported that creation of a novel glycosylation site at N94 in England Peptide. mAb specific for HLA-DR (clone L243) was expressed and DRA showed severe defects in peptide antigen presentation purified from HB-55 B cell hybridoma supernatants using a recombinant protein A column (GE Healthcare) and labeled with Alexa Fluor 488 acco-rd (Guerra et al., 1998). It was determined that the new glycan ing to the manufacturer’s protocol (Invitrogen). site interfered with HLA-DM–catalyzed ligand exchange on MHCII, and thus had an indirect role in peptide binding. Flow cytometry. For in vitro and ex vivo experiments, cells were cu-l The nature of glycans expressed by a given cell is depe-n tured for 3 d in the presence and absence of 10 µg/ml KF or 175 µM CS. dent on the expression pattern of glycosyltransferases in the The inhibitors and media were replaced daily. After treatment, the cells Golgi apparatus. It is established, for example, that gastric ep-i were washed in PBS and resuspended in FACS staining buffer (PBS, thelial cells show varied glycosylation of MHCII as compared pH 7.2, 3% FBS, and 0.5% sodium azide) supplemented with either 2 µg with B cells (Barrera et al., 2002). Perhaps more significantly, oAlulesxvaulFglaruiosrle4u8c8o–acgognljuutginatiend(LP2H4A3-mL;AVbecotorr5LµagboFrIaTtCor-iceos)nfjourga3t0edmP hinasoe-n alterations in cellular protein glycosylation have been reported ice. Cells were washed twice in PBS before FACS analysis on a C6 flow in inflammatory bowel disease (IBD) and colon cancer cytometer (Accuri Cytometers). Staining primary cells from animals was patients (Campbell et al., 2001; Bodger et al., 2006). Because the performed using the same protocol. Intracellular cytokine staining was 1050 MHC class II glycosylation in presentation of glycoantigens | Ryan et al.