Glycomic mapping of polylactosaminoglycans, terminal disialyl and sialyl sulfo N-acetyllactosamine motifs on mammalian cells
Date Issued
2011
Date
2011
Author(s)
Wang, Shui-Hua
Abstract
Most mass spectrometry (MS)-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine (polyLacNAc) extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. To advance the frontiers of glycomics, this thesis work aims primarily to address the analytical challenges in structural characterization of polylactosaminoglycans and associated terminal modifications such as sialylation and sulfation decorating the human endothelial cells, mouse and human B cells.
Using the human endothelial cells, EA.hy926 and HUVEC, as starting materials, we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of polyLacNAc at both MS and MS/MS levels in conjunction with the use of endo-β-galactosidase and Smith degradation to identify branching motifs and initiation sites. N-glycans in EA.hy926 were found to be less sialylated and fucosylated but more extended and branched than those of HUVEC, thus demonstrating a fundamental glycomic difference. For EA.hy926, its polyLacNAc chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Extending to glycoproteomics, the Lycopersicon esculentum lectin based enrichment strategy was optimized at glycan, glycoprotein, and glycopeptide levels, leading to identification of over 40 protein carriers utilizing a two-step enrichment workflow.
For mouse B cells, the N-glycans of a B lymphoma cell line, BCL1, were found to be mostly core-fucosylated, capped with α-Gal or Neu5Gc sialic acid, and carry non-branched polyLacNAcs. In contrast, its O-glycans were based on simple core 1 structures, mono- or disialylated on both arms. Sialidase digestion, in conjunction with further MS/MS and chemical analyses, established the identity of the terminal disialyl motif as Neu5Gcα2-8Neu5Gc-, which was shown by endo-β-galatosidase digestion to be additionally present on both polyLacNAc extended and non-extended N-glycans. Fluorescent-labeling of released sialic acids coupled with fluorometric high performance liquid chromatography analysis revealed that the amount of the disialyl motif was comparable for both N- and O-glycans, and CD45 is one of the protein carriers. Gene knockdown studies provided positive correlation indicative of mouse α2,8-sialyltransferase VI (ST8sia VI) being involved in the biosynthesis of disialic acid on both N- and O-glycans. Importantly, both the expression level of ST8sia VI and the total amount of disialic acids increase during B cell differentiation.
Interestingly, sulfation was additionally found on the terminal mono- and disialylated LacNAc of the polyLAcNAc chains, as well as on the LacNAc proximal to the trimannosyl core in BCL1 although its biological relevance is at present unclear. On the other hand, similar analysis led to identification of α2,6-sialylated 6-sulfo-LacNAc epitope on both the N- and O-glycans of activated human B cells, which is known to constitute a better ligand than the non-sulfated α2,6-sialylated LacNAc for human CD22. These additional modifications of polyLacNAcs apparently complicate the simplistic interpretation of the modulating roles of galectins and Siglecs in the B cell differentiation model. The development of enabling analytical techniques sensitive enough to identify and characterize the fine structural details of the underlying polyLAcNAc is an important step towards a better understanding of the glycobiology of this and many other physiological processes.
Subjects
Mass spectrometry
polylactosaminoglycans
disialic aicd
sulfation
Type
thesis
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