Sialyltransferases as specific cell surface probes of terminal and penultimate saccharide structures on living cells

Sidney W. Whiteheart, Gerald Warren Hart

Research output: Contribution to journalArticlepeer-review

Abstract

Rat liver β-galactoside α-2,6-sialyltransferase and Vibrio cholerae sialidase were used, in conjunction with CMP-N-acetyl-[3H]neuraminic acid, to probe the glycoconjugate distribution, sialylation state, and level of penultimate Galβ1-4GlcNAc residues on the surfaces of murine thymic lymphocytes. We report a detailed characterization of this sialyltransferase-mediated labeling system. Exogenous sialylation of intact cells is dependent on transferase, sugar nucleotide donor, cell number, and incubation time. Additionally, we have demonstrated that the system labeling the cell surface is noncytotoxic and nonmetabolic and is interacting with the entire cell population. Analysis of the exosialylated structures indicates that the sialyltransferase specifically produces an α2-6 linkage on N-linked oligosaccharides. Using this labeling system, we have probed the cell surface saccharide structures of murine thymocytes and demonstrated that most Galβ1-4GlcNAc residues are sialylated in the native state. However, one antigen, T200 (Ly-5), is strikingly undersialylated when compared to other cell surface glycoproteins (e.g., Thy 1.2). Upon analysis of exogenously sialylated oligosaccharides, labeled sialic acid was found almost exclusively on monosialylated structures with the remainder on bisialylated oligosaccharides. This suggests that the purified sialyltransferase is very precise in its recognition of oligosaccharides present on the surface of living thymic lymphocytes. This paper illustrates the combined uses of specific glycosidases and glycosyltransferases and how they can be employed in the detailed study of selected cell surface saccharide structures on living nucleated cells.

Original languageEnglish (US)
Pages (from-to)123-135
Number of pages13
JournalAnalytical Biochemistry
Volume163
Issue number1
DOIs
StatePublished - May 15 1987

Keywords

  • cell surface
  • glycoproteins
  • glycosyltransferases
  • radiolabeling
  • sialic acid
  • sialyltransferase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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