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Molecular mechanism of laminin variants expression

层粘连蛋白变体表达的分子机制

基本信息

批准号：
03454063
负责人：
KITAGAWA Yasuo
金额：
$ 4.03万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

Sequence information showed that interchain hydrophobic- and ionic interactions among heptad repeats in domain I and II of the A (merosin, kalinin A or A'), B1 (s-laminin or kalinin B1) and B2 (kalinin B2) chains are the basic mechanism for the formation of various laminin isoforms. However, our in vivo analysis of the assembly, replacement and intracellular sorting of laminin subunits suggested that additional cellular factors are involved in these process.Bovine aortic endothelial cells (BAEC ; producing AB1B2 and A'B1B2), embryonal carcinoma F9 cells (F9 ; producing AB1B2) or human keratinocytes were labelled with [^<35>S]methionine, incubated with a cross-linking reagent : dithio-bis-succinimidylpropionate (DSP) and cell lysate was immunoprecipitated and analyzed by SDS electrophoresis under reducing and nonreducing conditions.Nonreducing electrophoresis showed marked reduction of monomeric forms of A, A', B1 and B2 and B1B2 dimer, suggesting that the unassembled froms were cross-linked with cellular proteins. Reduced electrophoresis of immunoprecipitate from BAEC showed three major bands of 80, 60 and 50 kDa in addition to laminin subunits. Same experiment with F9 cells and keratinocytes showed a band of 100 kDa and bands of 90, 70 and 50 kDa, respectively. Two dimensional electrophoresis showed many spots of monomeric A, B1, B2 and B1B2 dimer mirgating differently in nonreducing electrophoresis. This result was consistent with the hypothesis that many molecules of chaperones are associated with each monomeric or dimeric form of laminin subunits.

序列信息显示，A（merosin，kalinin A或A '）、B1（s-层粘连蛋白或kalinin B1）和B2（kalinin B2）链的结构域I和II中的七肽重复序列之间的链间疏水和离子相互作用是形成各种层粘连蛋白同种型的基本机制。牛主动脉内皮细胞（BAEC ;产生AB 1B 2和A ′ B1 B2）、胚胎癌F9细胞（F9 ;产生AB 1B 2）或人角质形成细胞用[1 <35>S]甲硫氨酸标记，与交联剂一起孵育：二硫代双琥珀酰亚胺丙酸酯（DSP）和细胞裂解液在还原和非还原条件下进行免疫沉淀和SDS电泳分析，非还原电泳显示A、A ′、B1和B2单体以及B1 B2二聚体的显著减少，表明未组装的形式与细胞蛋白交联。BAEC的免疫沉淀物的还原电泳显示除了层粘连蛋白亚基之外的80、60和50 kDa的三个主要条带。用F9细胞和角质形成细胞进行的相同实验分别显示100 kDa的条带和90、70和50 kDa的条带。双向电泳显示，在非还原电泳中，单体A、B1、B2和B1 B2二聚体出现许多不同的斑点。这一结果与许多分子伴侣与层粘连蛋白亚基的每个单体或二聚体形式相关的假设一致。