surface-localized PSA (Fig. 3 B), but not DQ-OVA (Fig. 3 D) native complex N-glycans on all proteins expressed by the signal in CS- and KF-treated cells compared with UNT, but cells F(ig. 4, A and B). To better characterize the N-glycans little difference seen within the cells (Fig. 3, B and D). present on the purified DR2 samples, a panel of lectins was The results shown in Figs. 2 and 3 collectively demonstrate used in ELISA assays. CS-DR2, KF-DR2, and UNT-DR2 that modification of N-glycans on MHCII proteins in human were captured in wells precoated with L243 mAb and then B cells reduces MHCII-dependent presentation of GlyAg, but probed with FITC-conjugated lectins to quantify the types of not conventional peptide or intact protein antigens, and that glycans present in the DR2 samples (Fig. 4, C and D). We this alteration is not caused by changes in cell viability, MHCII found no change in succinylated WGA (S-WGA) binding expression or trafficking, or GlyAg processing. and increased Con A binding, but reductions in PHA-L, WGA, GSL I, STL, and UAE I lectin binding for CS-DR2 CS and KF reduce native complex N-glycans in Chinese and KF-DR2 compared with UNT-DR2. These data indi- hamster ovary (CHO) cells expressing recombinant HLA-DR2 cate significant modification of native N-glycans (PHA-L), Defects in GlyAg presentation within the context of live APCs fucosylation (UAE I),-linked galactose residues (GSL I), and could result from several processes that may be altered with CS terminal sialic acids (WGA and STL).The retention of S-WGA and KF treatment, such as defects in HLA-DM activity or binding suggests that GlcNAc residues, which are found in cathepsin activity to cleave the invariant chain (Ii) from the the early core glycan structures (Fig. 1 B), remain. Likewise, MHCII complex.To determine whether the observed defect the increased Con A staining demonstrates retention of man- was caused by deficiencies in GlyAg binding to MHCII, nose and/or glucose residues, typical of early core structures, recombinant HLA-DR2 (DR2) was expressed in CHO cells in within the glycans. Overall, the lectin screen demonstrates the presence and absence of CS and KF.The UNT, CS, and KF that the recombinant CS-DR2 and KF-DR2 lack the native grown DR2 molecules (UNT-DR2, CS-DR2, and KF-DR2) complex N-glycans found in UNT-DR2. were purified to homogeneity using an L243 mAb affinity column as previously described (Cobb et al., 2004). MHCII binding to GlyAg requires both the protein backbone To confirm the effectiveness of CS and KF, the CHO cells and native complex N-glycans were analyzed by fluorescent lectin staining and flow cyto m- Bacteria-expressed nonglycosylated DR2 (Bac-DR2) and etry. In both cases, CS and KF significantly reduced PHA-L CHO-expressed UNT-DR2 were used in quantitative binding binding to the cell surface, demonstrating the elimination of assays with 10 µg/ml biotinylated MBPp or biotinylated low Figure 3. B cells lacking native N-glycans show internal but not surface MHCII-PSA colocalization. (A and B) Uptake of PSA (red) in UNT, CS-treated, and KF-treated Raji B cells. Intracellular colocalization with MHCII protein (green) is shown in yellow. (C and D) DQ-OVA was used as an intact protein antigen control, which fluoresces only after proteolytic cleavage (Lewis and Cobb, 2010). Representative images are shown; Bars, 10 µm. n = 8 for quantification. All bar graphs show the mean ± SEM. 1044 MHC class II glycosylation in presentation of glycoantigens | Ryan et al.