Secretion of alpha1,3-galactosyltransferase by cultured cells and presence of enzyme in animal sera.
Glycosyltransferases are normally synthesized as membrane-anchored proteins. However, we recently found that the murine enzyme UDP-Gal:Gal beta1 -->4GLcNAc (Gal to Gal) alpha1,3 galactosyltransferase (alpha1,3GT) is secreted in a soluble form into media by mouse teratocarcinoma F9 cells (Cho SK, Yeh J-C, Cho M, Cummings RD (1996) J Biol Chem 271: 3238-46). To study the biosynthesis of this enzyme and whether secretion of the soluble enzyme is a general phenomenon, a solid-phase assay was developed for the alpha1,3GT activity. A recombinant and soluble form of the murine alpha1,3GT was produced in H293 cells (H293-alpha1,3GT) to aid in optimizing the assay. Desialylated orosomucoid was used as an immobilized acceptor in coated microtiter plates. The formation of product was detected by a biotinylated human-derived anti-alpha-Gal IgG and streptavidin conjugated to either alkaline phosphatase or the recombinant bioluminescent protein aequorin. Enzyme activity was dependent on the concentrations of asialoorosomucoid, UDP-Gal, alpha1,3GT and the time of incubation. The assay was also useful in monitoring alpha1,3GT activity during enzyme enrichment procedures. Using this assay, we found that alpha1,3GT activity was present in both cell extracts and culture media of several mammalian cell lines. Enzyme activity was also present in the sera from several mammals, but activity was absent in the sera from either humans or baboons. Our results demonstrate the development of a novel assay for the alpha1,3GT and provide evidence that secretion of the enzyme is a common biological phenomenon.
Cho
S K
SK
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Yeh
J C
JC
Cummings
R D
RD
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004285:Dogs; D005700:Galactosyltransferases / Q000097:blood* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010215:Papio; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000097:blood / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C037959:beta-D-galactosyl-N-acetylglucosaminylglycopeptide alpha-1,3-galactosyltransferase