Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N-and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.omics-based biomarker discovery | stemness | interglycomic correlations | glycoblotting | β-elimination in the presence of pyrazolone

␤ 2 -Glycoprotein I ( ␤ 2 -GPI) is a major antigen for anticardiolipin antibodies (aCL, Abs) present in patients with antiphospholipid syndrome. We recently reported that ␤ 2 -GPI specifically binds to oxidized LDL (oxLDL) and that the ␤ 2 -GPI's major ligand, oxLig-1 is 7-ketocholesteryl-9-carboxynonanoate (Kobayashi, K., E. Matsuura, Q. P.

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc) ⁄ N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG 2 better than IgG 1 , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG 2 from the udder to blood, rather than to secrete IgG 1 into colostrum.

Background: Given the biological importance of glycosphingolipids (GSLs), a widespread need exists for sensitive, rapid, and quantitative GSL-glycome analysis. Results: Rhodococcal endoglycosylceramidase (EGCase)-assisted glycan cleavage was optimized for the major GSL classes and combined with glycoblotting to unveil cellular glycomic profiles. Conclusion: Cellular GSL-glycomes were quantitatively and qualitatively characterized by newly established technique. Significance: GSL-glycomics provides a unique way to delineate/characterize cells.

␤ 2 -Glycoprotein I ( ␤ 2 -GPI) is a major antigen for antiphospholipid antibodies (Abs) present in patients with the antiphospholipid syndrome (APS). We previously reported that ␤ 2 -GPI specifically binds to oxidized low density lipoprotein (oxLDL), but not to native low density lipoprotein (LDL). In the present study, a ligand specific for ␤ 2 -GPI, oxLig-1, was purified from the extracted lipids of oxLDL. The structure of oxLig-1 was shown to be identical to that of synthesized 7-ketocholesteryl-9-carboxynonanoate by mass spectroscopy and nuclear magnetic resonance analyses. Both purified and synthesized oxLig-1 were recognized by ␤ 2 -GPI and subsequently by anti-␤ 2 -GPI auto-Abs, either in enzyme-linked immunosorbent assay (ELISA) or in ligand blot analysis. Binding of liposomes containing oxLig-1 (oxLig-1-liposomes) to mouse macrophages, J774A.1 cells, was relatively low, as compared with that of phosphatidylserine (PS)-liposomes. In contrast, binding of oxLig-1liposomes was enhanced more than 10-fold in the presence of both ␤ 2 -GPI and an anti-␤ 2 -GPI auto-Ab (WB-CAL-1), derived from (NZW x BXSB) F1 mouse, an animal APS model. Anti-␤ 2 -GPI auto-Abs derived from APS patients with episodes of arterial thrombosis were detected in ELISA, using a solid phase oxLig-1 complexed with ␤ 2 -GPI. We suggest that autoimmune atherogenesis linked to ␤ 2 -GPI interaction with oxLDL and Abs may be present in APS.

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Wild species in the genus Vigna are a great resource of tolerance to various stresses including salinity. We have previously screened the genetic resources of the genus Vigna and identified several accessions that have independently evolved salt tolerance. However, many aspects of such tolerance have remained unknown. Thus, we used autoradiography with radioactive sodium ( 22 Na + ) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to visualize and compare Na + allocation in Vigna angularis (Willd.) Ohwi & H.Ohashi (azuki bean), Vigna nakashimae (Ohwi) Ohwi & H.Ohashi, Vigna riukiuensis (Ohwi) Ohwi & H.Ohashi, Vigna luteola (Jacq.) Benth. and Vigna marina (Burm.) Merr.. The results indicated: 1) Tolerant accessions suppress Na + accumulation compared to azuki bean. 2) V. nakashimae and V. marina does so by accumulating higher amount of K + , whereas V. riukiuensis and V. luteola does so by other mechanisms. 3) V. luteola avoids salt-shedding by allocating excess Na + to newly expanded leaves. As the mechanisms of the tolerant species were different, they could be piled up in a single crop via classical breeding or by genetic engineering or genome editing.

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