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Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have different functions. Derivatization on sialic acid was designed to detect the different linkages by MALDI-TOF MS. In this study, a two-step derivatization by dimethylamine and ammonium hydroxide was improved to modify the sialic acid and made it easier to detect the different linkages of sialic acids on MALDI-TOF MS. Using this derivatization method, specific sialic acids linkages on N-glycans of protein samples such as fetuin and lactoferrin were detected. For complex cell samples, increased α2,3-linked and α2,6-linked sialic acids on bi-antennary and tri-antennary N-glycans were observed in A549 cells induced by hypoxia environment. Taken together, our two-step derivatization of sialic acids offers a simple and accurate way to detect specific linkages on N-glycans with MALDI-TOF mass spectrometer.

Glycans are one of the fundamental classes of macromolecules and are involved in a broad range of biological phenomena. A large variety of glycan structures can be synthesized depending on tissue or cell types and environmental changes. Here, we developed a comprehensive glycosylation mapping tool, termed GlycoMaple, to visualize and estimate glycan structures based on gene expression. We informatically selected 950 genes involved in glycosylation and its regulation. Expression profiles of these genes were mapped onto global glycan metabolic pathways to predict glycan structures, which were confirmed using glycomic analyses. Based on the predictions of N-glycan processing, we constructed 40 knockout HEK293 cell lines and analyzed the effects of gene knockout on glycan structures. Finally, the glycan structures of 64 cell lines, 37 tissues, and primary colon tumor tissues were estimated and compared using publicly available databases. Our systematic approach can accelerate glycan analyses and engineering in mammalian cells.

Sialidases (EC 3.2.1.18), glycosidases that cleave the linkages whereby sialic acids are attached to glycoconjugates, are found in most bacterial species. Because sialidases can convert polysialogangliosides to monosialoganglioside GM1, they have potential clinical application for treatment of human neurological and other disorders including Alzheimer's disease, Parkinson's disease, and spinal cord injury. Sialidases with high substrate specificity are desirable for more efficient GM1 production. In this study, the sialidase neuA3 gene from the non-pathogenic bacterium Streptomyces avermitilis , which is commonly used for industrial applications, was analyzed, cloned, and expressed in E. coli BL21 (DE3). Purified NeuA3 enzyme was characterized using 2′-(4-methylumbelliferyl)-α- d -N-acetylneuraminic acid (4-MUN) as a synthetic substrate. NeuA3 has a low molecular weight (∼38 kDa), showed strong stability in the presence of various divalent metal ions and temperature and pH values, preferentially cleaved α2,3- and α2,6-linked sialic acids from gangliosides, and efficiently converted crude porcine brain gangliosides to GM1. NeuA3 treatment of malignant human bladder cancer cells YTS-1 presented enhanced cell surface expression of GM1. The novel sialidase NeuA3 will be useful for functional studies of sialylated oligosaccharides and other sialoglycoconjugates, especially for studying the functions of GM1 in cancer research.

The discovery, isolation, and subsequent identification of cell membrane glycoproteins involved in the structure and function of the cell surface are becoming more and more important. Here, concanavalin A–magnetic particle conjugates were employed to isolate the special membrane glycoproteins from living HepG-2 cells. The isolated glycoproteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry as well as annotated. A total of 37 membrane glycoproteins were identified, and 25 of them were ascertained to locate in the extracellular region.

Altered expression of glycans is associated with cell-cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture.