TY - JOUR
T1 - Sialyltransferases as specific cell surface probes of terminal and penultimate saccharide structures on living cells
AU - Whiteheart, Sidney W.
AU - Hart, Gerald Warren
PY - 1987/5/15
Y1 - 1987/5/15
N2 - 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.
AB - 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.
KW - cell surface
KW - glycoproteins
KW - glycosyltransferases
KW - radiolabeling
KW - sialic acid
KW - sialyltransferase
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U2 - 10.1016/0003-2697(87)90102-3
DO - 10.1016/0003-2697(87)90102-3
M3 - Article
C2 - 3304006
AN - SCOPUS:0023192786
VL - 163
SP - 123
EP - 135
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -