Nakayama J, Fukuda M
Glycobiology Program, La Jolla Cancer Research Foundation, California 92037, USA.
J Biol Chem. 1996 Jan 26;271(4):1829-32. doi: 10.1074/jbc.271.4.1829.
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.
多唾液酸（PSA）是一种由α-2,8连接的唾液酸残基组成的线性同聚物，其表达受发育调控，并调节神经粘附分子N-CAM的粘附特性。最近，编码多唾液酸转移酶（仓鼠酶为PST-1，人酶为PST）的仓鼠和人cDNA被克隆出来，通过使用人cDNA证明，N-CAM中PSA的表达促进神经突生长（中山，J.，福田，M.N.，弗雷德特，B.，兰施特，B.，和福田，M.（1995年）美国国家科学院院刊，92，7031-7035；埃克哈特，M.A.，米伦霍夫，M.，贝瑟，A.，库普曼，J.，弗罗施，M.，和杰拉迪-沙恩，R.（1995年）自然373，715-718）。尽管这些研究表明PST-1和PST在培养细胞中合成PSA，但并未表明它们能在含有α-2,3连接唾液酸的糖缀合物模板上催化α-2,8连接唾液酸的缩聚反应。在这里，我们证明PST在体内形成PSA与N-CAM的存在无关。然后，我们开发了一种体外PSA合成测定法，使用N-CAM以外的糖蛋白作为受体，可溶性PST作为酶源。将与蛋白A融合产生的可溶性PST与大鼠α1-酸性糖蛋白、胎球蛋白或人α1-酸性糖蛋白作为受体，与供体底物CMP-[14C]NeuNAc一起孵育。特别是，胎球蛋白与可溶性PST孵育产生了一种高分子量产物，该产物对PSA特异性内切神经氨酸酶敏感。在孵育前从受体胎球蛋白中去除α-2,3连接的唾液酸时，不会形成多唾液酸化产物。这些结果表明，单一酶PST alone可以催化将第一个α-2,8连接的唾液酸添加到α-2,3连接的唾液酸上，并催化所有α-2,8连接的唾液酸的缩聚反应，从而产生PSA。
