A r t i c l e molecular weight PSA as previously described (Cobb et al., 2004; Cobb and Kasper, 2008).We found that PSA binding to Bac-DR2 was significantly reduced compared with the fully glycosylated UNT-DR2, whereas the binding of MBPp increased with the Bac-DR2 (Fig. 4 E). We also compared UNT-DR2 with CS-DR2 and KF-DR2 and found no change in conventional peptide antigen binding, but a 75% reduction in GlyAg binding (Fig. 4, F and G). Because PSA is a highly charged antigen that is known to depend upon charges for MHCII binding (Kreisman et al., 2007; Cobb and Kasper, 2008), we treated UNT-DR2 with neuraminidase to remove negatively charged terminal sialic acids. Lectin ELISA confirmed the reduction of sialic acids (Fig. 4 H) and the neuraminidase-digested protein antigen binding was com- pared with the binding to native UNT-DR2 (Fig. 4 I). We found a small, but statistically insignificant, change in overall binding of both GlyAg and conventional peptide, demon- strating that the negative charges provided by terminal sialic Figure 4. Both native N-glycans and the HLA-DR2 protein backbone are required for PSA binding. (A and B) Flow cytometry analysis of CS- and KF-treated CHO cells were compared with UNT cells using a lectin sensitive to the presence of native complex N-glycans. Representative histograms are shown. (C and D) HLA-DR2 expressed by UNT, CS-treated, and KF-treated CHO cells was immobilized and probed using a panel of FITC-conjugated lectins to detect changes in MHCII glycosylation. The raw data showing relative concentrations of the target glycan structure (D) and fold change (E) of each lectin is shown. n = 9 per data point. (E) Quantitative binding experiments using PSA or MBPp with purified HLA-DR2 expressed in bacteria (Bac-DR2; nonglycosylated) or DR2 expressed in CHO cells (CHO-DR2; fully glycosylated). n = 4 per data point. (F and G) MBPp and PSA binding to DR2 lacking native N-glycans expressed in CHO cells grown in CS-supplemented (n = 9; F) or KF-supplemented media (n = 18; G) compared with CHO-DR2 expressed in the absence of inhibitor. (H) Lectin ELISA of neuraminidase-digested CHO-DR2. (I) Native CHO-DR2 enzymatically trimmed with (2→3,6,8,9)- neuraminidase (NA-DR2) binding to PSA or MBPp compared with untreated CHO-DR2. n = 21. (J) Alexa Fluor 488–conjugated PSA was used as ligand in a glycan-binding array containing 320 glycans (Table S1) at the Consortium for Functional Glycomics. Positive signal is typically above 20,000 U. n = 4 per glycan. All bar graphs show the mean ± SEM. JEM VOL. 208, May 9, 2011 1045