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

我们根据Kunkel法用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。测定突变体DNA和野生型DNA的核苷酸序列，构建表达载体，转染COS7细胞。在western blotting中，突变蛋白的分子量比野生型蛋白小，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，tunicamycin （n -糖基化抑制剂）处理的细胞中野生型酶的分子量更小。tunicamycin处理细胞中表达的分子量与突变蛋白N76Q/N200Q/N324Q的分子量一致。突变蛋白的酶活性发生了改变，这表明与该酶相关的糖水化合物参与了酶活性的调节。虽然目前对酶活性的调节机制尚不清楚，但存在糖链影响蛋白质结构的可能性。因此，今后有必要通过晶体结构分析等进一步研究来阐明酶活性的调节机制。血浆型PAF乙酰水解酶目前除从豚鼠血浆中纯化外，还从人血浆中纯化，据报道人血浆酶是一种糖蛋白，也是一种豚鼠血浆酶。另一方面，糖链影响人肝源性细胞系HepG2分泌的血浆型PAF乙酰水解酶的活性。今后有必要详细研究糖链对酶活性的影响。

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.