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Molecular and biological study on sialidase

唾液酸酶的分子生物学研究

基本信息

批准号：
02671018
负责人：
UDA Yutaka
金额：
$ 1.28万
依托单位：
Niigata College of Pharmacy
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.Two species of cDNAs for human alpha-N-acetylgalactosaminidase were isolated from a human fibroblast cDNA library. The two species differ each other by a 70 bp insertion in the coding region. Transient expression study in COS cells demonstrated that only the cDNA without the 70 bp insertion expressed alpha-N-acetylgalac … More tosaminidase activity. Analysis of mRNA species utilizing polymerase chain reaction revealed that the majority of the mRNA does not contain the 70 bp insertion, and the mRNA containing the 70 bp insertion is present only in a minor amount in human brain.The activation mechanism of human placental sialidase was studied. We observed that not only the crude enzyme preparation but also the partially purified preparation of sialidase was activated by the incubation at 37ﾟC in pH 4.8 medium. The activation inhibited by amastatin which is an inhibitor of aminopeptidase A and leucine aminopeptidase. The partially purified sialidase preparation used for the activation released leucine and glutamic acid but not arginine, lysine and alanine from methylcoumarlamide(MCA) derivatives of amino acid. Zinc ion significantly depressed the activation of sialidase and also inhibited the hydrolysis of leucine-MCA. The activation of sialidase seems to be caused by an endogeneous protease, probably aminopeptidase.Sialidase isolated from human placenta is associated with several protein components, which are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. as a sialidase protein; this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposine A,C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie(gamma1). Less

在与纯化的人胎盘唾液酸酶组分相关的蛋白质的cDNA的分子克隆过程中，我们发现表观分子量为46 kDa的蛋白质之一实际上是α-N-乙酰氨基半乳糖苷酶。pcD-HS 1204全长cDNA编码358个氨基酸，前17个残基代表推定的信号肽。预测的氨基酸序列与人α-半乳糖苷酶A和酵母α-半乳糖苷酶具有惊人的同源性。从人成纤维细胞cDNA文库中分离到两种人α-N-乙酰氨基半乳糖苷酶的cDNA。这两个物种在编码区有一个70 bp的插入，彼此不同。在COS细胞中的瞬时表达研究表明，只有没有70 bp插入的cDNA才表达α-N-乙酰半乳糖 ...更多信息 tosaminidase活性利用聚合酶链反应分析mRNA种类，发现人脑中大部分mRNA不含70 bp插入片段，含70 bp插入片段的mRNA仅占少数。我们观察到，不仅粗酶制剂，而且部分纯化的唾液酸酶制剂通过在37 ℃下在pH 4.8的培养基中孵育而被活化。氨肽酶A和亮氨酸氨肽酶抑制剂amastatin可抑制该活化。用于活化的部分纯化的唾液酸酶制剂从氨基酸的甲基香豆酰胺（MCA）衍生物释放亮氨酸和谷氨酸，但不释放精氨酸、赖氨酸和丙氨酸。锌离子显著抑制唾液酸酶的活性，也抑制亮氨酸-MCA的水解。唾液酸酶的激活似乎是由一种内源性蛋白酶引起的，可能是氨肽酶。从人胎盘中分离的唾液酸酶与几种蛋白质组分相关，这些蛋白质组分被认为在唾液酸酶的分离过程中形成聚集的复合物。其中一种60 kDa的蛋白质最近被Potier等人鉴定为唾液酸酶蛋白;该蛋白质也与抗鞘脂激活蛋白原抗体交叉反应。我们已经分离出这种蛋白质，并从以下证据中鉴定出它是免疫球蛋白G的重链组分，而不是唾液酸酶或鞘脂激活蛋白原的衍生物。在凝胶过滤HPLC上，唾液酸酶活性和60 kDa蛋白彼此清楚地分离。60 kDa的蛋白质不仅与针对人saposine A、C和D的抗体发生交叉反应，而且还与单独的第二抗体（山羊抗兔免疫球蛋白G抗体）发生交叉反应。该60 kDa蛋白与抗人免疫球蛋白G抗体发生强烈交叉反应。该60 kDa蛋白质N末端起的最初15个氨基酸的序列与免疫球蛋白G重链蛋白Tie（gamma 1）的序列相同。少