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. 2018 Dec 14;9:2857. doi: 10.3389/fimmu.2018.02857

Figure 1.

Figure 1

Expression of PNA-reactive glycans by germinal center B cells is correlated with downregulation of the α2,3-sialyltransferase ST3Gal1. (A) Gating strategy for analyzing tonsillar naïve (N), germinal center (GC), memory (M), and plasmablast (PB) B cells by flow cytometry. Representative fluorescence minus one (FMO) controls used for gating of CD27 are shown. (B) Analysis of peanut lectin binding to human tonsil B cells. Representative histograms of results are shown (left) as well as quantification of geometric mean fluorescence intensities (MFI) (right). (C) Schematic of synthesis of potential PNA-reactive O-linked glycans on B cells. O-glycan synthesis is initiated in the Golgi apparatus by polypeptide N-acetylgalactosamine transferases (GALNTs), which transfer a single GalNAc to select serine/threonine residues of a polypeptide backbone. The initiating GalNAc can be terminally sialylated, or further extended by C1GalT1 (C1GALT1) to form the simplest PNA-reactive epitope, a Core 1 O-glycan termed “T-antigen.” This core T-antigen moiety can be branched and elongated by other glycosyltransferases to form extended Core 2 O-glycans, which retain binding to PNA, or modified with sialic acid by the α2,3-sialyltransferase ST3Gal1 (ST3GAL1), which destroys PNA reactivity. Endogenous (or exogenous) sialidases may remove sialic acids and restore PNA binding. (D) Analysis of ST3GAL1 expression in tonsillar B cells by quantitative real-time reverse transcription PCR (qRT-PCR), sorted as in (A). Data are normalized to the housekeeping gene VCP and presented relative to naïve B cells. Data are representative of eight (B) or three (D) distinct tonsil specimens pooled from two (B) or three (D) independent experiments. Statistics were calculated using a Kruskal–Wallis test with Dunn's multiple comparisons test (B) or One-way analysis of variance (ANOVA) and Tukey's multiple comparisons test. Throughout, bars and error bars depict the mean and SEM, respectively. ns = not significant, ***p ≤ 0.001. ΔMFI, background subtracted geometric mean fluorescence intensity; GalNAc, N-acetylgalactosamine; Gal, galactose; Sia, sialic acid.