A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus -: Sequence homologies with human leucine-rich α2-glycoprotein
A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus -: Sequence homologies with human leucine-rich α2-glycoprotein
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DOI：
10.1074/jbc.273.31.19469
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发表时间：
1998-07-31
影响因子：
4.8
通讯作者：
Hayashi, K
中科院分区：
文献类型：
--
作者：
Okumura, K;Ohkura, N;Hayashi, K
磷脂酶 A(2) (PLA(2)) 抑制剂 FLIP 是从中国马牧蛇 (Agkistrodon blomhoffii siniticus) 血浆中纯化得到的，是一种 160 kDa 的三聚体，具有三个 50 kDa 的亚基；它特异性抑制来自其自身毒液的基本 PLA 的酶活性（Ohkura, N.、Okuhara, H.、Inoue, S.、Ikeda, a 和 Hayashi, K. (1997) Biochem. J. 325, 527-531），在本研究中，发现 50-kDa 亚基被 N-连接碳水化合物糖基化，并且酶促去糖基化将 50-kDa 亚基的分子量降低至 39-kDa，发现 FLIP 的一个 160-kDa 三聚体与三个基本 PLA 分子形成稳定的复合物，表明一个基本 PLA 分子将以化学计量与 FLIP 的一个亚基结合。根据部分确定的亚基氨基酸序列，使用通过聚合酶链式反应制备的探针从中国mamushi肝脏cDNA文库中分离编码FLIP的cDNA。该cDNA包含一个开放阅读框，编码23个残基的信号序列，随后是一个308个残基的蛋白质，其中包含通过赖氨酰内肽酶消化亚基而衍生的所有肽的序列。成熟蛋白的分子量经计算为34,594 Da，推导的氨基酸序列包含四个潜在的N-糖基化位点。 FLIP 的序列与已知的 PLA 抑制剂的序列没有显着的同源性。但有趣的是，它与人类富含亮氨酸的 α(2)-糖蛋白（一种功能未知的血清蛋白）有 33% 的同一性。该序列最显着的特征是它包含 9 个富含亮氨酸的重复序列 (LRR)，每个重复序列有 24 个氨基酸残基，因此涵盖了超过三分之二的分子。 FLIP 中的 LRR 可能负责与基本 PLA(2) 的特异性结合，因为 LRR 被认为是参与蛋白质-蛋白质相互作用的基序。
The phospholipase A(2) (PLA(2)) inhibitor FLIP, purified from the blood plasma of Chinese mamushi snake (Agkistrodon blomhoffii siniticus), is a 160-kDa trimer with three 50-kDa subunits; and it inhibits specifically the enzymatic activity of the basic PLA, from its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, a, and Hayashi, K. (1997) Biochem. J. 325, 527-531), In the present study, the 50-kDa subunit was found to be glycosylated with N-linked carbohydrate, and enzymatic deglycosylation decreased the molecular mass of the 50-kDa subunit to 39-kDa, One 160-kDa trimer of FLIP was found to form a stable complex with three basic PLA, molecules, indicating that one basic PLA, molecule would bind stoichiometrically to one subunit of FLIP. A cDNA encoding FLIP was isolated from a Chinese mamushi liver cDNA library by use of a probe prepared by a polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a 308-residue protein, which contained the sequences of all the peptides derived by lysyl endopeptidase digestion of the subunit. The molecular mass of the mature protein was calculated to be 34,594 Da, and the deduced amino acid sequence contained four potential N-glycosylation sites. The sequence of FLIP showed no significant homology with that of the known PLA, inhibitors. But, interestingly, it exhibited 33% identity with that of human leucine-rich alpha(2)-glycoprotein, a serum protein of unknown function. The most striking feature of the sequence is that it contained nine leucine-rich repeats (LRRs), each of 24 amino acid residues and thus encompassing over two-thirds of the molecule. LRRs in FLIP might be responsible for the specific binding to basic PLA(2), since LRRs are considered as the motifs involved in protein-protein interactions.