A human polysialyltransferase directs in vitro synthesis of polysialic acid

Abstract

Polysialic acid (PSA) is a linear homopolymer of α-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., Muhlenhoff, 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 α-2,8-linked sialic acid on a glycoconjugate template containing α-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 α1-acid glycoprotein, fetuin or human α1-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 α- 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 α-2,8-1inked sialic acid to α-2,3- linked sialic acid and the polycondensation of all α-2,8-linked sialic acids, yielding PSA.