A r t i c l e N-glycans on cellular glycoproteinFsi(g. 1 B). On the fourth the metabolic state of the cells were assessed by measuring day, the cells were provided fresh drug and either biotinylated ATP production (Fig. 2 E). No reduction in cellular metabolic myelin basic protein peptide (MBPp) or biotinylated glyc-o activity (i.e., ATP production) was seen between UNT and antigen PSA from B. fragilis and allowed to incubate for an- inhibitor-treated cells. Finally, it has been previously reported other 24 h.The MHCII-antigen complexes were subsequently that PSA and other GlyAgs require intracellular processing immunoprecipitated with mAb against MHCII (clone L243) via a nitric oxide–dependent pathway. To ensure that the and probed with streptavidin to quantify the presented anti- treated Raji B cells processed PSA normally, full-length- ra gen. We found no difference in the amount of MBPp pre- diolabeled PSA was given to the cells, and then isolated from sented by the cells treated with either inhibitor F(ig. 2,A and B) the endosomal compartments after overnight incubation. The when compared with untreated cells (UNT), yet cells treated endosomal PSA was then passed through an analytical mole-c with CS or KF showed significant reductions in the amount ular sieve column to measure the molecular size. We found of GlyAg presented (Fig. 2, A and B). that all cells equally endocytosed and processed PSA into two Given that CS and KF affect all N-glycosylated proteins molecular mass populations (Fig. 2 F, arrows) as previously in the cell, we used identically treated Raji B cells in flow cyto- published (Cobb et al., 2004). metry experiments to measure surface-localized MHCII mol-e CS- and KF-treated Raji B cells were also compared with cules. No difference was detected in the surface concentration UNT cells by confocal microscopy. The cells were given -in of MHCII (Fig. 2, C and D).We also used FITC-conjugated hibitor for 3 d before incubation with either AlexaFluor594- lectins to stain the cells and found the expected reductions in conjugated PSA (red) or DQ-OVA (shown in red), which is Phaseolus vulgaris leucoagglutinin (PHA-L), PHA-E, and WGA fluorescent only after proteolytic processing into peptides lectin staining with concomitant increases in Con A staining (Lewis and Cobb, 2010). After 24 h of antigen exposure, the in CS- and KF-treated populations (Fig. 2, C and D), suggest- cells were fixed with 4% paraformaldehyde, stained with ing that the glycans present had increased mannose composi- Alexa Fluor 488– or Alexa Fluor 633–conjugated MHCII mAb tions. To ensure that CS and KF did not affect cell viability, (shown in green for both), and viewed by confocal micros- copy (Fig. 3,A and C). We found no detectable difference in MHCII staining as a function of drug treatment. In addition, the presence of bright DQ-OVA signal at the cell surface in- dicates that there was no significant difference in OVA processing or surface localization. In contrast, a reduction in surface colocalization of PSA in cells treated with CS or KF (lack of yellow colocalization signal in overlay images) was observed. Using a series of images (n = 8), we analyzed the pixel intensity distribution within each cell for each antigen. We found highly significant reductions (P < 0.01) in JEM VOL. 208, May 9, 2011 Figure 2. B cells lacking native N-glycans show reduced PSA presentation. (A and B) Coimmuno- precipitations (coIP) with -MHCII mAb from CS- and KF-treated Raji B cells show reduced PSA (CS, P < 0.001; KF, P < 0.01), but not MBP peptide (P > 0.05), presentation compared with untreated (UNT) controls. n = 12 for all PSA; n = 15 for all MBPp. (C and D) Flow cytometry of UNT, CS-treated (C), and KF-treated (D) Raji B cells using lectin staining to detect the glycophenotype of the cell surface. Representative histograms are shown; n = 4 for each data point. (E) ATP production of UNT, CS-treated, and KF-treated Raji B cells. n = 3 for each data point. (F) [3H]-labeled PSA isolated from endosomal compart- ments was analyzed from UNT, CS-treated, and KF-treated Raji B cells and compared with intact, full-length PSA control (PSA cont.) by size exclusion HPLC using a Superose 12 column. The arrow indicates low molecular mass PSA. Representative elution profiles are shown. All bar graphs show the mean ± SEM. 1043