Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaα2→3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaα2→3Gal and Siaα2→6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS giycoforms in different tissues.
Journal Article Intermolecular Interaction between Glycolipids and Glycophorin on Liposomal Membranes Get access Tamao ENDO, Tamao ENDO Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of TokyoHongo, Bunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar Shoshichi NOJIMA, Shoshichi NOJIMA Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of TokyoHongo, Bunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar Keizo INOUE Keizo INOUE Department of Health Chemistry, Faculty of Pharmaceutical Sciences, The University of TokyoHongo, Bunkyo-ku, Tokyo 113 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 92, Issue 6, October 1982, Pages 1883–1890, https://doi.org/10.1093/oxfordjournals.jbchem.a134118 Published: 01 October 1982 Article history Received: 24 July 1982 Published: 01 October 1982
We found that a lectin, Datura stramonium agglutinin, induced irreversible differentiation in C6 glioma cells. The differentiated cells had long processes, a low rate of proliferation and a high content of glial fibrillary acidic protein. When the medium was replaced with Datura stramonium agglutinin-free medium after 1 h, cell proliferation continued to be inhibited. Experiments with several other lectins indicated that both recognition of linear N-acetyllactosamine repeats and recognition of multiantennary units of cell-surface glycans were required for the inhibition of C6 proliferation. Proliferation of four human glial tumour cells was also inhibited by Datura stramonium agglutinin. Further, these differentiated human glial tumour cells had long processes and a high content of glial fibrillary acidic protein similar to differentiated C6 glioma cells. Taken together, these observations suggest that Datura stramonium agglutinin may be useful as a new therapy for treating glioma without side effects.
Glycans are involved in many fundamental cellular processes such as growth, differentiation, and morphogenesis. However, their broad structural diversity makes analysis difficult. Glycomics via mass spectrometry has focused on the composition of glycans, but informatics analysis has not kept pace with the development of instrumentation and measurement techniques. We developed Toolbox Accelerating Glycomics (TAG), in which glycans can be added manually to the glycan list that can be freely designed with labels and sialic acid modifications, and fast processing is possible. In the present work, we improved TAG for large-scale analysis such as cohort analysis of serum samples. The sialic acid linkage-specific alkylamidation (SALSA) method converts differences in linkages such as α2,3- and α2,6-linkages of sialic acids into differences in mass. Glycans modified by SALSA and several structures discovered in recent years were added to the glycan list. A routine to generate calibration curves has been implemented to explore quantitation. These improvements are based on redefinitions of residues and glycans in the TAG List to incorporate information on glycans that could not be attributed because it was not assumed in the previous version of TAG. These functions were verified through analysis of purchased sera and 74 spectra with linearity at the level of R2 > 0.8 with 81 estimated glycan structures obtained including some candidate of rare glycans such as those with the N,N’-diacetyllactosediamine structure, suggesting they can be applied to large-scale analyses.
Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.
We examined the distribution of sialoglycoconjugates in the cerebellum of 9-week-old and 30-month-old rats using light microscopy and electron microscopy in combination with two lectins, Maackia amurensis lectin (MAL) for Sia(alpha)2-3Gal and Sambucus sieboldiana agglutinin (SSA) for Sia(alpha)2-6Gal. Each lectin showed characteristic staining patterns. In young adult rats, MAL stained a strongly granular layer, a weakly molecular layer, and the medullary lamina, while SSA more strongly stained the medullary lamina than the molecular and granular layers. After aging, different staining patterns were obtained. Intense SSA reactivity was observed in the granular layer and intense MAL reactivity was observed in the medullary lamina of the aged groups. The reactivity of Purkinje cells with MAL was downregulated in the aged rats. These results indicated that Sia(alpha)2-3Gal and Sia(alpha)2-6Gal were expressed in distinct regions of the rat cerebellum and that their expression patterns changed in the aged brain.
