Epithelial Genetics Group, Human Genetics Unit, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.
J Biol Chem. 2009 Dec 18;284(51):35588-96. doi: 10.1074/jbc.M109.052811.
The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha3 and LaNt alpha5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.
层粘连蛋白是一组异三聚体基底膜蛋白，在细胞黏附、迁移和组织形态发生中发挥作用。通过对层粘连蛋白编码基因的计算机分析，我们鉴定出一组来自 LAMA3 和 LAMA5 基因 5'端的新型交替剪接异构体。这些异构体与 netrins 相似，因为它们含有一个层粘连蛋白 N 端结构域，后面跟着一小段层粘连蛋白型表皮生长因子样重复序列。我们建议将这些 mRNA 的预测蛋白产物命名为 LaNt（层粘连蛋白 N 端）alpha3 和 LaNt alpha5。RT-PCR 证实了这些转录本在 mRNA 水平上的存在。此外，它们表现出不同的、组织特异性的表达谱。为了确认 LaNt alpha3 蛋白的存在，我们针对假定多肽中的独特结构域生成了一种抗体。该抗体通过免疫印迹识别预测分子量为 64 kDa 的蛋白质。此外，免疫荧光分析显示 LaNt alpha3 在上皮组织中呈现基底膜染色，并且 LaNt alpha3 定位于培养角质形成细胞的基底膜相关表面。我们还通过 RNAi 介导的敲低测试了 LaNt alpha3 的功能。表现出特定的 LaNt alpha3 敲低的角质形成细胞显示出黏附能力受损、应激抗性降低以及体外划痕伤口闭合能力降低。
