Brown J C, Wiedemann H, Timpl R
Max-Planck-Institut für Biochemie, Martinsried, Germany.
J Cell Sci. 1994 Jan;107 ( Pt 1):329-38. doi: 10.1242/jcs.107.1.329.
Two isoforms of laminin were extracted from human placenta by neutral buffer containing EDTA, copurified through several steps and finally separated by Mono Q anion exchange chromatography. One variant consisted of disulphide-linked 340, 230 and 190 kDa subunits, which were identified by immunoblotting as Am, B1e and B2e chains. In the other variant, the B1e chain was replaced by B1s of 180 kDa. After rotary shadowing, both variants showed a similar cross-shaped structure. The nidogen content of these laminins was substoichiometric and variable (3-70%), indicating loss by endogenous proteolysis. Yet both human isoforms were able to bind mouse nidogen with an affinity (Kd approximately 0.5 nM) comparable to that of AeB1eB2e laminin from a mouse tumour. Since the binding site is known to be contributed by a single EGF-like motif of the B2e chain, this demonstrates that activity of this site is independent of chain assembly. Binding activity of both isoforms to collagen IV and the heparan sulphate proteoglycan perlecan was correlated to the nidogen content and could be enhanced by adding nidogen. Binding to heparin was only partial and heparin did not inhibit perlecan binding. This indicated a crucial role for nidogen in mediating the integration of these laminin isoforms into basement membranes. Variant AmB1sB2e showed calcium-dependent binding to fibulin-1, while only a little activity was found for AmB1eB2e. Both isoforms promoted adhesion and spreading of several cell lines. Adhesion could be completely inhibited by antibodies to the integrin beta 1 subunit but not, or only weakly, by antibodies against beta 3, alpha 2, alpha 3, alpha 5 and alpha 6 subunits. No inhibition was observed with an Arg-Gly-Asp-containing peptide.
通过含有乙二胺四乙酸（EDTA）的中性缓冲液从人胎盘中提取了两种层粘连蛋白异构体，经过几步共纯化，最后通过Mono Q阴离子交换色谱法进行分离。一种变体由通过二硫键连接的340 kDa、230 kDa和190 kDa亚基组成，通过免疫印迹鉴定为αm、β1e和β2e链。在另一种变体中，180 kDa的β1s取代了β1e链。旋转阴影处理后，两种变体均呈现出相似的十字形结构。这些层粘连蛋白的巢蛋白含量低于化学计量且可变（3%-70%），表明存在内源性蛋白水解导致的损失。然而，两种人源异构体都能够与小鼠巢蛋白结合，其亲和力（解离常数Kd约为0.5 nM）与来自小鼠肿瘤的αeβ1eβ2e层粘连蛋白相当。由于已知结合位点由β2e链的单个表皮生长因子（EGF）样基序提供，这表明该位点的活性与链组装无关。两种异构体与IV型胶原和硫酸乙酰肝素蛋白聚糖核心蛋白聚糖的结合活性与巢蛋白含量相关，并且可以通过添加巢蛋白来增强。与肝素的结合只是部分结合，肝素并不抑制核心蛋白聚糖的结合。这表明巢蛋白在介导这些层粘连蛋白异构体整合到基底膜中起关键作用。变体αmβ1sβ2e显示出对纤连蛋白-1的钙依赖性结合，而αmβ1eβ2e只有少量活性。两种异构体都促进了几种细胞系的黏附和铺展。黏附可以被整合素β1亚基的抗体完全抑制，但不能被针对β3、α2、α3、α5和α6亚基的抗体抑制，或者只能被微弱抑制。含精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp）的肽未观察到抑制作用。
