Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.
Beta 1,4-Galactosyltransferase is traditionally viewed as a biosynthetic component of the Golgi complex, but a portion of galactosyltransferase is also expressed on the cell surface, where it has been suggested to function as a receptor for extracellular oligosaccharide ligands. Although results from a variety of studies are consistent with a cell adhesion function for galactosyltransferase, the most rigorous test of surface galactosyltransferase function is to produce a surface galactosyltransferase-dependent phenotype in cells that normally express negligible levels of surface galactosyltransferase. In agreement with previous reports, human HeLa cells were found to express low levels of galactosyltransferase on their surface and, therefore, were stably transfected with cDNAs encoding murine galactosyltransferase. Murine galactosyltransferase was expressed both within the presumed Golgi complex and on the cell surface, as assayed by enzyme activity and with antiserum raised against the bacterially expressed murine enzyme. HeLa cell transfectants adhered more strongly to their extracellular substrates than did control transfectants, as evidenced by a flatter morphology in culture and a more rapid spreading upon plating. In contrast, cell spreading was low and similar among all cell types when plated on extracellular substrates that did not contain binding sites for galactosyltransferase. Antibodies and Fab fragments against recombinant murine galactosyltransferase inhibited the increased cell spreading characteristic of galactosyltransferase transfectants, as did soluble recombinant galactosyltransferase and a variety of galactosyltransferase perturbants. Thus, expression of heterologous galactosyltransferase produces a surface galactosyltransferase-dependent phenotype, confirming its function as a cell adhesion molecule.
Nguyen
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Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Hinton
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DA
Shur
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D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology*; D002846:Chromatography, Affinity; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008970:Molecular Weight; D009237:N-Acetyllactosamine Synthase / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014162:Transfection
D000906:Antibodies; D018076:DNA, Complementary; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D009237:N-Acetyllactosamine Synthase