Molecular mechanism of laminin variants expression

序列信息表明，A链（merosin、kalinin A或A’）、B1链（s-laminin或kalinin B1）和B2链（kalinin B2）结构域I和II区七核苷酸重复体之间的疏水和离子相互作用是形成各种层粘连蛋白同工型的基本机制。然而，我们对层粘连蛋白亚基的组装、替换和细胞内分选的体内分析表明，其他细胞因子参与了这些过程。牛主动脉内皮细胞（BAEC；产生AB1B2和A'B1B2）、胚胎癌F9细胞（F9；产生AB1B2）或人角化细胞用[^<35>S]甲硫氨酸标记，用交联试剂：二硫代-双琥珀酰酰丙酸酯（DSP）孵育，细胞裂解液在还原和非还原条件下免疫沉淀，SDS电泳分析。非还原电泳显示A、A′、B1、B2和B1B2二聚体的单体形式明显减少，表明未组装的片段与细胞蛋白交联。BAEC免疫沉淀还原电泳除层粘连蛋白亚基外，还显示80、60和50 kDa三个主要条带。F9细胞和角质形成细胞的相同实验显示，条带分别为100 kDa和90、70和50 kDa。二维电泳显示，在非还原电泳中，单体A、B1、B2和B1B2二聚体有许多不同的斑点。这一结果与许多伴侣分子与层粘连蛋白亚基的每个单体或二聚体形式相关联的假设是一致的。

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.