Laboratory of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan.
Biochemistry. 2012 Jun 19;51(24):4950-8. doi: 10.1021/bi300214g. Epub 2012 Jun 4.
Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1β1. However, the antibodies to integrin α1 and β1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1β1 seems to modulate neurite elongation on the short arm of α5.
层粘连蛋白 α5 是一种细胞外基质蛋白，包含多个结构域，参与多种生物学过程，如胚胎发生和肾功能。在这项研究中，我们使用重组蛋白和合成肽来鉴定 α5 短臂区域内对神经突生长活性至关重要的氨基酸残基。α5 的短臂包含三个球形结构域（LN、L4a 和 L4b）和三个杆状元件（LEa、LEb 和 LEc）。用 293 细胞产生的与 Fc 标签融合的 α5 短臂重组蛋白来检测 PC12（嗜铬细胞瘤）细胞黏附和神经突生长活性。尽管它没有细胞附着活性，但重组蛋白促进了神经突生长。为了缩小与神经突生长活性相关的区域，我们在 293 细胞中产生了两种截断的重组蛋白。缺乏 L4a 和 LEb 的重组蛋白失去了活性。此外，我们合成了 78 个部分重叠的肽段，代表 L4a 和 LEb 的大部分氨基酸序列。这些肽段中，A5-76 [鼠层粘连蛋白 α5 928-939（TSPDLFRLVFRY）在 L4a] 表现出神经突生长活性。突变研究表明，Phe(933)和 Arg(934)参与了神经突生长活性。此外，使用抗整合素单克隆抗体的抑制实验表明，α5 短臂上的神经突生长部分由整合素 α1β1 介导。然而，针对整合素 α1 和 β1 的抗体不能抑制 A5-76 肽上的神经突伸长。这些结果表明，除了与 L4a 结构域的活性位点的细胞相互作用外，整合素 α1β1 的结合似乎调节 α5 短臂上的神经突伸长。
