Identification and characterization of tissue specific active sites in the laminins

层粘连蛋白是由基因上不同的α、β和γ链组成的异三聚体基底膜糖蛋白。到目前为止，已经鉴定了5个α链，3个β链和3个γ链，并且每个亚基的不同组合形成了至少15个异构体（Laminin-1-15）。层粘连蛋白具有多种生物活性，包括促进细胞粘附、生长、迁移、神经突生长和分化。我们的目标是了解层粘连蛋白在正常发育和疾病过程中的生物学作用，并将活性序列应用于包括组织再生和工程在内的治疗应用。我们的方法是使用大量合成肽和重组蛋白来定位层粘连蛋白分子上的细胞粘附位点。我们通过系统筛选673个重叠合成肽的细胞结合位点，从层粘连蛋白-1中鉴定出约20个活性序列。首先，我们重点研究了其n α链G结构域的生物学功能，并利用重组蛋白和合成肽筛选了活性位点。我们制备了包含层粘连蛋白α1、α3和α4链G结构域的多种重组蛋白，并测试了它们的细胞附着活性。一些重组蛋白表现出细胞附着和神经突生长活性。我们还合成了300多个覆盖整个G结构域的重叠肽，并通过肽包被板和肽偶联的Sepharose beads测试了它们的细胞附着活性。超过20个多肽促进细胞附着，5个多肽抑制重组蛋白介导的生物活性。一些肽被发现与整合素和syndecans相互作用。这些结果表明这些活性位点参与了层粘连蛋白α链G结构域的生物学功能。这些活性肽可能有助于确定层粘连蛋白-受体相互作用的分子机制和层粘连蛋白介导的细胞信号通路。少

Laminins are heterotrimeric basement membrane glycoproteins consisting of genetically distinct α, β, and γ chains. So far, five α, three β, and three γ chains have been identified, and at least fifteen isoforms (Laminin-1-15) are formed by various combinations of each subunit. Laminins have diverse biological activities including promotion of cell adhesion, growth, migration, neurite outgrowth, and differentiation. Our goal is to understand the biological role of the laminins in normal development and disease processes and to apply the active sequences for therapeutic application including tissue regeneration and engineering. Our approach has been to localize cell adhesion sites on the laminin molecules using a large set of synthetic peptides and recombinant proteins. We have identified about 20 active sequences from the laminin-1 using a systematic screening for cell binding sites with 673 overlapping synthetic peptides. First, we have focused on the biological functions of the lamini … More n αchain G domains and screened active sites using a recombinant protein and synthetic peptides. We prepared various recombinant proteins, comprising the G domains of laminin α1, α3 and α4 chains, and tested their cell attachment activity. Several recombinant proteins showed cell attachment and neurite outgrowth activities. We also synthesized more than 300 overlapping peptides covering the entire G domains and tested their cell attachment activity sing peptide-coated plates and peptide-conjugated Sepharose beads. More than 20 peptides promoted cell attachment and five peptides inhibited the biological activity mediated by the recombinant proteins. Some of the peptides were found to interact with integrins and syndecans. These results suggest that the active sites are involved in the biological functions of the laminin α chain G domains. These active peptides may be useful for defining of the molecular mechanism of laminin-receptor interactions and laminin-mediated cellular signaling pathways. Less