A human polysialyltransferase directs in vitro synthesis of polysialic acid.
Polysialic acid (PSA) is a linear homopolymer of alpha-2,8-linked sialic acid residues whose expression is developmentally regulated and modulates the adhesive property of the neural adhesion molecule, N-CAM. Recently, hamster and human cDNAs encoding polysialyltransferase (PST-1 for the hamster enzyme and PST for the human enzyme) were cloned, and by using the human cDNA it was demonstrated that the expression of PSA in N-CAM facilitates neurite outgrowth (Nakayama, J., Fukuda, M.N., Fredette, B., Ranscht, B., and Fukuda, M. (1995) Proc. Natl. Acad. Sci. U.S.A., 92, 7031-7035; Eckhardt, M.A., Mühlenhoff, M., Bethe, A., Koopman, J., Frosch, M., and Gerardy-Schahn, R. (1995) Nature 373, 715-718.) Although these studies demonstrated that PST-1 and PST synthesize PSA in cultured cells, it was not shown that they could catalyze the polycondensation of alpha-2,8-linked sialic acid on a glycoconjugate template containing alpha-2,3-linked sialic acid. Here we demonstrate that PSA formation by PST is independent from the presence of N-CAM in vivo. We then develop an in vitro assay of PSA synthesis using glycoproteins other than N-CAM as acceptors and a soluble PST as an enzyme source. The soluble PST, produced as a chimeric protein fused with protein A, was incubated with rat alpha 1-acid glycoprotein, fetuin or human alpha 1-acid glycoprotein as acceptors together with the donor substrate CMP-[14C]NeuNAc. Incubation of fetuin with the soluble PST, in particular, resulted in a high molecular weight product that was susceptible to PSA-specific endoneuraminidase. Polysialylated products were not formed when alpha-2,3-linked sialic acid was removed from the acceptor fetuin before incubation. These results establish that a single enzyme, PST, alone can catalyze both the addition of the first alpha-2,8-linked sialic acid to alpha-2,3-linked sialic acid and the polycondensation of all alpha-2,8-linked sialic acids, yielding PSA.
Nakayama
J
J
Glycobiology Program, La Jolla Cancer Research Foundation, California 92037, USA.
Fukuda
M
M
D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids / Q000096:biosynthesis*; D012795:Sialoglycoproteins / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D000509:alpha-Fetoproteins / Q000378:metabolism
D017931:DNA Primers; D019006:Neural Cell Adhesion Molecules; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D012795:Sialoglycoproteins; D000509:alpha-Fetoproteins; C021319:polysialic acid; C046778:CMP-N-acetylneuraminate-poly-alpha-2,8-sialosyl sialyltransferase; D012799:Sialyltransferases