Yurchenco P D, Quan Y, Colognato H, Mathus T, Harrison D, Yamada Y, O'Rear J J
Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10189-94. doi: 10.1073/pnas.94.19.10189.
A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The alpha-, beta-, and gamma-chain subunits of laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the alpha chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the beta and gamma chains, expressed separately or together, remained intracellular with formation of betabeta or betagamma, but not gammagamma, disulfide-linked dimers. Secretion of the beta and gamma chains required simultaneous expression of all three chains and their assembly into alphabetagamma heterotrimers. Epitope-tagged recombinant alpha subunit and recombinant laminin were affinity-purified from the conditioned medium of alphagamma and alphabetagamma clones. Rotary-shadow electron microscopy revealed that the free alpha subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. We conclude that the alpha chain can be delivered to the extracellular environment as a single subunit, whereas the beta and gamma chains cannot, and that the alpha chain drives the secretion of the trimeric molecule. Such an alpha-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.
建立了一种哺乳动物重组策略，以剖析基底膜层粘连蛋白组装和分泌的规则。层粘连蛋白-1的α、β和γ链亚基在几乎无背景的情况下以所有组合进行瞬时和/或稳定表达。在没有其正常伙伴的情况下，α链以完整蛋白和在卷曲螺旋结构域中被切割的蛋白形式分泌。相比之下，单独或一起表达的β链和γ链仍保留在细胞内，形成ββ或βγ，但不是γγ二硫键连接的二聚体。β链和γ链的分泌需要同时表达所有三条链并将它们组装成αβγ异源三聚体。从αγ和αβγ克隆的条件培养基中亲和纯化表位标记的重组α亚基和重组层粘连蛋白。旋转阴影电子显微镜显示，游离的α亚基是一种线性结构，包含N端和带有缩短长臂的球形结构，而三聚体具有天然层粘连蛋白典型的四臂形态。我们得出结论，α链可以作为单个亚基传递到细胞外环境中，而β链和γ链则不能，并且α链驱动三聚体分子的分泌。这种依赖α链的机制可能允许调节层粘连蛋白输出到新生基底膜中，并可能在控制基底膜形成中起重要作用。
