Okazaki Ikuko, Suzuki Nobuharu, Nishi Norio, Utani Atsushi, Matsuura Hiroshi, Shinkai Hiroshi, Yamashita Hironobu, Kitagawa Yasuo, Nomizu Motoyoshi
Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.
J Biol Chem. 2002 Oct 4;277(40):37070-8. doi: 10.1074/jbc.M201672200. Epub 2002 Jul 18.
Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.
层粘连蛋白是由α、β和γ链组成的三聚体细胞外基质蛋白家族。到目前为止，已鉴定出五种不同的层粘连蛋白α链。层粘连蛋白α4链存在于层粘连蛋白-8/9中，在间充质来源的细胞中表达，如内皮细胞和脂肪细胞。此前，我们在层粘连蛋白α4链的C末端球状结构域（G结构域）中鉴定出肝素结合位点。在这里，我们重点研究了层粘连蛋白α4链G结构域的生物学功能，并使用重组蛋白和合成肽筛选了活性位点。包含整个G结构域的重组α4G蛋白促进了细胞附着活性。重组α4G的细胞附着活性被肝素完全阻断，被EDTA部分抑制。我们合成了覆盖整个G结构域的116个重叠肽，并测试了它们的细胞附着活性。20个肽显示出细胞附着活性，16个与肝素结合。我们进一步在细胞附着和肝素与重组α4G蛋白结合的竞争试验中测试了这20个活性肽的作用。促进细胞附着和肝素结合的A4G6（LAIKNDNLVYVY）、A4G20（DVISLYNFKHIY）、A4G82（TLFLAHGRLVFM）和A4G83（LVFMFNVGHKKL）显著抑制了细胞附着和肝素与重组α4G的结合。这些结果表明，这四个活性位点参与了层粘连蛋白α4链G结构域的生物学功能。此外，发现重组α4G、A4G6和A4G20与syndecan-4相互作用。这些活性肽可能有助于确定层粘连蛋白-受体相互作用和层粘连蛋白介导的细胞信号通路的分子机制。
