Brandenberger R, Chiquet M
Department of Biophysical Chemistry, University of Basel, Switzerland.
J Cell Sci. 1995 Sep;108 ( Pt 9):3099-108. doi: 10.1242/jcs.108.9.3099.
Laminin isolated from chick heart is composed of several heterotrimeric variants of 800 and 700 kDa. Here, we used monoclonal antibodies against chick laminin to purify different laminin isoforms from this mixture. Antibody 8D3 specifically removed laminin containing alpha 2 chain from chick heart laminin preparations, leaving behind 700 kDa variants. Using antibody C4 against the laminin beta 2 chain, alpha 2 chain containing variants were further separated into alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin, respectively. Laminins containing alpha 2 chain and recognized by antibody 8D3 are cross-shaped molecules. Their expression during embryogenesis is tightly regulated. In 5-day embryos staining with monoclonal antibody 8D3 is restricted to the dermamyotome. Older embryos (8 days) express alpha 2 chain containing variants at myotendinous junction primordia of skeletal muscle, and only late in development these variants are generally expressed in skeletal and heart muscle basement membranes. The 700 kDa laminin variants contain beta 1, beta 2, and gamma 1 subunits affiliated with an immunologically distinct, shorter alpha x chain and appear to be T-shaped in the electron microscope. Whereas laminins with an alpha 2 subunit bind to heparin, variants with the novel alpha x chain do not. Experiments using cultured sympathetic neurons showed that laminins with alpha x chain are less potent than alpha 2 chain containing variants in promoting neurite outgrowth. In contrast, sympathetic neurons cannot discriminate between alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin substrates, respectively, and show identical high rates of neurite formation.
从鸡心脏中分离出的层粘连蛋白由几种800 kDa和700 kDa的异源三聚体变体组成。在此，我们使用抗鸡层粘连蛋白的单克隆抗体从该混合物中纯化不同的层粘连蛋白异构体。抗体8D3能特异性地从鸡心脏层粘连蛋白制剂中去除含α2链的层粘连蛋白，留下700 kDa的变体。使用针对层粘连蛋白β2链的抗体C4，含α2链的变体进一步分别分离为α2β1γ1和α2β2γ1层粘连蛋白。含α2链且能被抗体8D3识别的层粘连蛋白是十字形分子。它们在胚胎发育过程中的表达受到严格调控。在5天的胚胎中，用单克隆抗体8D3染色仅限于皮肌节。较老的胚胎（8天）在骨骼肌的肌腱连接原基处表达含α2链的变体，并且只有在发育后期这些变体才普遍在骨骼肌和心肌基底膜中表达。700 kDa的层粘连蛋白变体包含与免疫上不同的较短αx链相关的β1、β2和γ1亚基，在电子显微镜下似乎呈T形。含α2亚基的层粘连蛋白能与肝素结合，而含新型αx链的变体则不能。使用培养的交感神经元进行的实验表明，含αx链的层粘连蛋白在促进神经突生长方面比含α2链的变体效力更低。相比之下，交感神经元无法分别区分α2β1γ1和α2β2γ1层粘连蛋白底物，并且显示出相同的高神经突形成率。
