Shin-Yi Yu

We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on permethylated di- and multiply sulfated glycans will survive in nanoESI conditions to allow detection of multiply charged intact molecular ions, and more comprehensive MS/MS can be performed in an automated fashion at higher sensitivity, compared with MALDI-MS/MS. Parallel higher energy collision dissociation (HCD) and ion trap collision induced dissociation (CID)-based MS2, coupled with product-dependent MS3 in data dependent acquisition mode proved to be highly productive when applied to resolve and identify the isomeric sulfated glycan structures. In-house glycomic data mining software, GlyPick, was developed and used to automate the downstream process of identification and relative quantification of target sulfated glycotopes based on summed intensity of their diagnostic MS2 ions extracted from thousands of HCD-MS2 and/or CID-MS2 data. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.

The emergence of zebrafish Danio rerio as a versatile model organism provides the unique opportunity to monitor the functions of glycosylation throughout vertebrate embryogenesis, providing insights into human diseases caused by glycosylation defects. Using a combination of chemical modifications, enzymatic digestion and mass spectrometry analyses, we establish here the precise glycomic profiles of eight individual zebrafish organs and demonstrate that the protein glycosylation and glycosphingolipid expression patterns exhibits exquisite specificity. Concomitant expression screening of a wide array of enzymes involved in the synthesis and transfer of sialic acids shows that the presence of organ-specific sialylation motifs correlates with the localized activity of the corresponding glycan biosynthesis pathways. These findings provide a basis for the rational design of zebrafish lines expressing desired glycosylation profiles.

Despite well-recognized biological importance, mass spectrometry (MS)-based glycomic identification of sulfo-, sialylated terminal glyco-epitopes on the N-glycans of various immune cell types remains technically challenging and rarely reported. Previous studies with monoclonal antibody have implicated a regulated expression of 6-sulfo-α2-6-sialyl LacNAc on B cells in peripheral lymph nodes and the circulating peripheral blood lymphocytes but its occurrence on leukemia cells or lymphomas have not been critically addressed. In this study, we have extended our previously developed MS-based sulfoglycomic platform by incorporating additional complementary analytical approaches in order to achieve a high sensitivity mapping and relative quantification of the detected sulfated glycotopes down to the level of defining their sialyl linkages. We showed that discovery mode sulfoglycomics and precise location of sulfate were best achieved by multimode MS analyses of fractionated, permethylated sulfated N-glycans. On the other hand, the relative degree of sulfation on individual N-glycans could be more efficiently inferred from the respective extracted ion chromatograms of native, non-sulfated and sulfated target N-glycans in single LC–MS/MS runs. The GlcNAc-6-O-sulfated α2-6-sialyl LacNAc, which constitutes the higher affinity ligand for the human inhibitory co-receptor of B cells, CD22, was found to be commonly carried on a range of complex type N-glycans from human CD19⁺ and CD4⁺ lymphocytes. We further showed that its occurrence on the most abundant α2-6-disialylated biantennary structure from the peripheral blood mononuclear cells of patients diagnosed as B-cell chronic lymphocytic leukemia varied within ±2-fold abundance from the mean value determined for isolated CD19+ lymphocytes and cultured B-CLL cells.

The addition of sulfate to glycan structures can regulate their ability to serve as ligands for glycan-binding proteins. Although sulfate groups present on the monosaccharides glucosamine, uronate, N-acetylglucosamine and N-acetylgalactosamine are recognized by defined receptors that mediate important functions, the functional significance of galactose-6-O-sulfate (Gal6S) is not known. However, in vitro studies using synthetic glycans and sulfotransferase overexpression implicate Gal6S as a binding determinant for the lymphocyte homing receptor, L-selectin. Only two sulfotransferases have been shown to generate Gal6S, namely keratan sulfate galactose 6-O-sulfotransferase (KSGal6ST) and chondroitin 6-O-sulfotransferase-1 (C6ST-1). In the present study, we use mice deficient in KSGal6ST and C6ST-1 to test whether Gal6S contributes to ligand recognition by L-selectin in vivo. First, we establish that KSGal6ST is selectively expressed in high endothelial venules (HEVs) in lymph nodes and Peyer's patches. We also determine by mass spectrometry that KSGal6ST generates Gal6S on several classes of O-glycans in peripheral lymph nodes. Furthermore, KSGal6ST, but not C6ST-1, is required for the generation of the Gal6S-containing glycan, 6,6′-disulfo-3′sLN (Siaα2→3[6S]Galβ1→4[6S]GlcNAc) or a closely related structure in lymph node HEVs. Nevertheless, L-selectin-dependent short-term homing of lymphocytes is normal in KSGal6ST-deficient mice, indicating that the Gal6S-containing structures we detected do not contribute to L-selectin ligand recognition in this setting. These results refine our understanding of the biological ligands for L-selectin and introduce a mouse model for investigating the functions of Gal6S in other contexts.

In an effort to prime our mass spectrometry (MS)-based sulfoglycomic mapping platform technology for facile identification of sulfated lacdiNAc (GalNAcβ1-4GlcNAcβ1-), we have re-examined the N-glycans of bovine thyroid stimulating hormone. We showed that MALDI-MS mapping of permethylated glycans in negative ion mode can give an accurate representation of the sulfated glycans and, through MS/MS, diagnostic ions can be derived that we can collectively define the presence of a terminal sulfated lacdiNAc moiety at high sensitivity. Based on these ions, which can also be produced by nanoESI-MSⁿ, we demonstrated that the glycome of an ovarian carcinoma cell line, RMG-1, comprises a high abundance of sulfated lacdiNAc epitopes carried on multiantennary complex type N-glycans alongside fucosylated, sialylated and/or sulfated lacNAc antennae. This represents the first report of a natural glycomic occurrence of sulfated lacdiNAc on a cell line, as opposed to other better-characterized presence on secreted glycoproteins from a handful of sources. It is anticipated that with improved methods of detection such as that developed in this work, we are likely to identify a wider occurrence of sulfated lacdiNAc and be able to more accurately delineate the regulatory mechanism dictating the choice of a cell type in synthesizing sulfated, sialylated, fucosylated and/or non-substituted lacdiNAc.

High endothelial venule (HEV)‐like vessels have been observed in gastric B cell lymphoma of mucosa‐associated lymphoid tissue type (MALT lymphoma), as well as in its preceding lesion, chronic Helicobacter pylori gastritis. Previously we reported that glycans on HEV‐like vessels in the latter lesion served as L‐selectin ligands, although their function is unclear. We have investigated sialyl Lewis X (sLeX)‐related glycoepitopes and found that MECA‐79⁻/HECA‐452⁺/NCC‐ST‐439⁺ HEV‐like vessels preferentially mark gastric MALT lymphoma compared to chronic H. pylori gastritis. We then constructed CHO cell lines expressing potential MECA‐79⁻/HECA‐452⁺/NCC‐ST‐439⁺ glycans, as well as other sLeX‐type glycans, on CD34 and evaluated L‐selectin binding to those cells, using L‐selectin–IgM chimera binding and lymphocyte adhesion assays. L‐selectin–IgM chimeras bound to CHO cells expressing 6‐sulpho‐sLeX attached to core 2‐branched O‐glycans with or without 6‐sulpho‐sLeX attached to extended core 1 O‐glycans, but only marginally to other CHO cell lines. By contrast, CHO cells expressing 6‐sulpho‐sLeX attached to extended core 1 and/or core 2‐branched O‐glycans, as well as non‐sulphated sLeX attached to core 2‐branched O‐glycans, showed substantial lymphocyte binding, while binding was negligible on lines expressing 6‐sulpho‐ and non‐sulphated sLeX attached to N‐glycans and non‐sulphated sLeX attached to extended core 1 O‐glycans. These results indicate that MECA‐79⁻/HECA‐452⁺/NCC‐ST‐439⁺ glycans, specifically, 6‐sulpho‐ and non‐sulphated sLeXs attached to core 2‐branched O‐glycans, expressed on HEV‐like vessels in gastric MALT lymphoma function as L‐selectin ligands and likely contribute to H. pylori‐specific T cell recruitment in the progression of gastric MALT lymphoma. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.