Pavlova N V, Yuziuk J A, Nakagawa H, Kiso M, Li S C, Li Y T
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
J Biol Chem. 1999 Nov 5;274(45):31974-80. doi: 10.1074/jbc.274.45.31974.
KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a sialic acid analog, has been found to be widely distributed in nature. Despite the structural similarity between KDN and Neu5Ac, alpha-ketosides of KDN are refractory to conventional sialidases. We found that the hepatopancreas of the oyster, Crassostrea virginica, contains two KDN-cleaving sialidases but is devoid of conventional sialidase. The major sialidase, KDN-sialidase, effectively cleaves alpha-ketosidically linked KDN and also slowly cleaves the alpha-ketosides of Neu5Ac. The minor sialidase, KDNase, is specific for alpha-ketosides of KDN. We were able to separate these two KDN-cleaving enzymes using hydrophobic interaction and cation-exchange chromatographies. The rate of hydrolysis of 4-methylumbelliferyl-alpha-KDN (MU-KDN) by KDN-sialidase is 30 times faster than that of MU-Neu5Ac in the presence of 0.2 M NaCl, whereas in the absence of NaCl this ratio is only 8. KDNase hydrolyzes MU-KDN over 500 times faster than MU-Neu5Ac and is not affected by NaCl. KDN-sialidase purified to electrophoretically homogeneous form was found to have a molecular mass of 25 kDa and an isoelectric point of 8.4. One of the three tryptic peptides derived from KDN-sialidase contains the consensus motif, SXDXGXTW, that has been found in all conventional sialidases. Kinetic analysis of the inhibition of the hydrolysis of MU-KDN and MU-Neu5Ac by 2, 3-dehydro-2-deoxy-KDN (KDN2-en) and 2,3-dehydro-2-deoxy-(Neu5Ac2-en) suggests that KDN-sialidase contains two separate active sites for the hydrolysis of KDN and Neu5Ac. Both KDN-sialidase and KDNase effectively hydrolyze KDN-G(M3), KDNalpha2-->3Gal beta1-->4Glc, KDNalpha2-->6Galbeta1-->4Glc, KDNalpha2-->6-N-acetylgalactosaminitol, KDNalpha2-->6(KDNalpha2-->3)N-acetylgalactosaminitol, and KDNalpha2-->6(GlcNAcbeta1-->3)N-acetylgalactosaminitol. However, only KDN-sialidase also slowly hydrolyzes G(M3), Neu5Acalpha2-->3Galbeta1-->4Glc, and Neu5Acalpha2-->6Galbeta1-->4Glc. These two KDN-cleaving sialidases should be useful for studying the structure and function of KDN-containing glycoconjugates.
2-酮基-3-脱氧-D-甘油-D-半乳糖壬糖酸（KDN）是一种唾液酸类似物，已发现其在自然界中广泛分布。尽管KDN与N-乙酰神经氨酸（Neu5Ac）结构相似，但KDN的α-酮糖苷对传统唾液酸酶具有抗性。我们发现，弗吉尼亚牡蛎（Crassostrea virginica）的肝胰腺含有两种可切割KDN的唾液酸酶，但不含传统唾液酸酶。主要的唾液酸酶，即KDN-唾液酸酶，能有效切割α-酮糖苷键连接的KDN，也能缓慢切割Neu5Ac的α-酮糖苷。次要的唾液酸酶，即KDN酶，对KDN的α-酮糖苷具有特异性。我们能够通过疏水相互作用色谱法和阳离子交换色谱法分离这两种可切割KDN的酶。在0.2 M NaCl存在下，KDN-唾液酸酶对4-甲基伞形酮基-α-KDN（MU-KDN）的水解速率比MU-Neu5Ac快30倍，而在无NaCl时，该比例仅为8。KDN酶对MU-KDN的水解速度比对MU-Neu5Ac快500倍以上，且不受NaCl影响。纯化至电泳纯形式的KDN-唾液酸酶分子量为25 kDa，等电点为8.4。从KDN-唾液酸酶衍生的三个胰蛋白酶肽段之一包含在所有传统唾液酸酶中都发现的共有基序SXDXGXTW。对2,3-脱氢-2-脱氧-KDN（KDN2-en）和2,3-脱氢-2-脱氧-（Neu5Ac2-en）抑制MU-KDN和MU-Neu5Ac水解的动力学分析表明，KDN-唾液酸酶含有两个分别用于水解KDN和Neu5Ac的活性位点。KDN-唾液酸酶和KDN酶都能有效水解KDN-G(M3)、KDNα2→3Galβ1→4Glc、KDNα2→6Galβ1→4Glc、KDNα2→6-N-乙酰半乳糖胺醇、KDNα2→6(KDNα2→3)N-乙酰半乳糖胺醇和KDNα2→6(GlcNAcβ1→3)N-乙酰半乳糖胺醇。然而，只有KDN-唾液酸酶也能缓慢水解G(M3)、Neu5Acα2→3Galβ1→4Glc和Neu5Acα2→6Galβ1→4Glc。这两种可切割KDN的唾液酸酶对于研究含KDN的糖缀合物的结构和功能应该是有用的。
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