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The role of glycohydrates in the function of plasma-type PAF-acetylhydrolase.

糖水合物在血浆型 PAF-乙酰水解酶功能中的作用。

基本信息

批准号：
11672186
负责人：
KARASAWA Ken
金额：
$ 2.24万
依托单位：
Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672186/
关键词：
platelet-activating factor PAF gene transfer glycosylation PAF-acetylhydrolase PAFアセチルヒドロラーゼ

项目摘要

We prepared the mutants in which possible N-glycosylation sites (N76, N200 and N324) of guinea pig plasma-type PAF-acetylhydrolase cannot be glycosylated by displacement of each Asn residue with Gln residue according to the method of Kunkel. DNA nucleotide sequences of these mutants DNAs and wild-type DNA were determined, expression vectors were constructed, and COS7 cells were transfected with resulting DNA.The mutant proteins showed smaller molecular weights than wild-type protein in the western blotting, suggesting that this enzyme was modulated by glycosylation after translation. In addition, wild-type enzyme showed smaller molecular weight in the cells treated with tunicamycin, an inhibitor of N-glycosylation, compared with control cells. The molecular weights expressed in the cells treated with tunicamycin corresponded to those of mutant protein, N76Q/N200Q/N324Q.The enzyme activity of the mutant proteins were altered, suggesting that glycohydrates linked to this enzyme was involved in the regulation of the enzyme activity. Although the mechanism of modulation of enzyme activity remains to be determined at present, there exists possibility that sugar chains could affect the protein structure. Therefore, further study such as crystal structure analysis will be necessary to elucidate the mechanism of modulation of enzyme activity in the future.Plasma-type PAF acetylhydrolases have been hitherto purified from human plasma besides guinea pig plasma, and human enzyme has been reported to be a glycoprtein as well as guinea-pig enzyme. On the other hand, it is reported that sugar chain affects the activity of plasma-type PAF acetylhydrolase secreted from HepG2, human liver derived cell-line. It is necessary to study the effect of sugar chain on the enzyme activity in detail from now on.

根据Kunkel的方法，我们通过用Gln残基置换每个Asn残基，制备了豚鼠血浆型PAF-乙酰水解酶的可能的N-糖基化位点（N76、N200和N324）不能被糖基化的突变体。测定了突变体和野生型DNA的核苷酸序列，构建了表达载体，转染COS 7细胞，Western blotting结果显示突变体蛋白的分子量小于野生型蛋白，表明该酶在翻译后受到糖基化的调节。此外，与对照细胞相比，野生型酶在用衣霉素（一种N-糖基化抑制剂）处理的细胞中显示出较小的分子量。经衣霉素处理的细胞表达的分子量与突变蛋白N76 Q/N200 Q/N324 Q的分子量一致，突变蛋白的酶活性发生了改变，表明与该酶相连的糖基参与了酶活性的调节。虽然目前对酶活性调节的机制还不清楚，但糖链可能影响蛋白质结构。因此，进一步的研究，如晶体结构分析将是必要的，以阐明在未来的酶活性的调制机制。血浆型PAF乙酰水解酶迄今已从人血浆中纯化，除了豚鼠血浆，和人酶已被报道为糖蛋白以及豚鼠酶。另一方面，据报道糖链影响人肝源性细胞系HepG 2分泌的血浆型PAF乙酰水解酶的活性。今后有必要深入研究糖链对酶活性的影响。