Kitajima K, Kuroyanagi H, Inoue S, Ye J, Troy F A, Inoue Y
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
J Biol Chem. 1994 Aug 26;269(34):21415-9.
The release of 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN, deaminoneuraminic acid) residues from their alpha-ketosidic linkage is required to determine the structural and functional role of KDN-glycoconjugates in sources as disparate as trout egg polysialoglycoproteins and human cancers. We report for the first time the isolation and characterization of a novel type of sialidase (KDNase), which specifically hydrolyzes KDN ketosidic but not N-acylneuraminyl linkages. KDNase activity was assayed using 4-methylumbelliferyl KDN (4-MU-KDN). A KDNase-producing microorganism was identified as Sphingobacterium multivorum. The affinity-purified enzyme was designated KDNase SM to denote its origin and that it was free of N-acylneuraminidase, proteolytic, and other glycosidase activities. KDNase SM activity toward 4-MU-KDN was not inhibited by the N-acylneuraminidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. KDNase SM released free KDN from naturally occurring substrates, including (KDN)GM3, KDN-glycoprotein, which bears a number of O-linked chains of KDN alpha 2-->3Gal beta 1-->3GalNAc alpha 1-->3 (KDN alpha 2-->(-->8KDN alpha 2-->)n-->6)GalNAc alpha 1-->, and the biantennary complex-type of N-glycan, KDN alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(KDN alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc. KDNase SM thus exhibited a broad linkage specificity and was able to hydrolyze the KDN residues ketosidically linked alpha 2-->3, alpha 2-->6, and alpha 2-->8. The enzyme did not release Neu5Ac or Neu5Gc from 4-MU-Neu5Ac, N-acetyl-neuraminyllactose, colominic acid, or other Sia(Neu5Ac or Neu5Gc)-containing glycoconjugates.
为了确定3-脱氧-D-甘油-D-半乳糖-2-壬酮糖酸（KDN，脱氨神经氨酸）残基在α-酮糖苷键中的结构和功能作用，需要将其从α-酮糖苷键中释放出来，这些残基存在于诸如鳟鱼卵多唾液酸糖蛋白和人类癌症等不同来源中。我们首次报道了一种新型唾液酸酶（KDN酶）的分离和特性，该酶特异性水解KDN酮糖苷键而非N-酰基神经氨酸苷键。使用4-甲基伞形酮基KDN（4-MU-KDN）测定KDN酶活性。鉴定出一种产生KDN酶的微生物为多食鞘氨醇杆菌。亲和纯化的酶被命名为KDN酶SM，以表明其来源且不含N-酰基神经氨酸酶、蛋白水解酶和其他糖苷酶活性。KDN酶SM对4-MU-KDN的活性不受N-酰基神经氨酸酶抑制剂2,3-脱氢-2-脱氧-N-乙酰神经氨酸的抑制。KDN酶SM从天然存在的底物中释放出游离KDN，这些底物包括（KDN）GM3、KDN-糖蛋白，其带有许多KDNα2→3Galβ1→3GalNAcα1→3（KDNα2→（→8KDNα2→）n→6）GalNAcα1→的O-连接链，以及双天线复合型N-聚糖KDNα2→3Galβ1→4GlcNAcβ1→2Manα1→6（KDNα2→3Galβ1→4GlcNAcβ1→2Manα1→3）Manβ1→4GlcNAcβ1→4GlcNAc。因此，KDN酶SM表现出广泛的连接特异性，能够水解以α2→3、α2→6和α2→8酮糖苷键连接的KDN残基。该酶不会从4-MU-Neu5Ac、N-乙酰神经氨酰乳糖、大肠菌素酸或其他含Sia（Neu5Ac或Neu5Gc）的糖缀合物中释放Neu5Ac或Neu5Gc。
