Identification and characterization of tissue specific active sites in the laminins

层粘连蛋白是由α、β和γ链组成的异源三聚体，是存在于基底膜上的多功能糖蛋白。在发育早期基底膜组装过程中，层粘连蛋白是基底膜的重要组成部分。层粘连蛋白参与多种生物活动，如细胞粘附、迁移、生长、分化、肿瘤转移和血管生成。已经鉴定出15种层粘连蛋白同种异构体，并在组织和/或发育阶段特异性表达。整合蛋白、糖醛酸失调蛋白、syndecans和其他几种细胞表面分子是层粘连蛋白的细胞受体。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500多个覆盖整个G结构域的重叠肽，并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。此外，一些环肽增强了活性。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。我们将层粘连蛋白活性肽偶联到壳聚糖膜上。活性层粘连蛋白肽偶联壳聚糖膜增强了生物活性，并以细胞类型特异性的方式促进细胞粘附。我们还证明了肽-壳聚糖膜可以传递细胞，并适用于角化细胞转移到伤口床。肽-壳聚糖方法可能是一种强大的细胞移植工具，适用于各种组织和器官。

Laminins, heterotrimers composed of α,β, and γ chains, are multifunctional glycoproteins present in basement membranes. Laminins are the most important component of basement membranes during basement membrane assembly in early development. Laminins are involved in various biological activities such as cell adhesion, migration, growth, differentiation, tumor metastasis, and angiogenesis. Fifteen laminin isoforms have been identified and are tissue- and/or developmental stage-specifically expressed. Integrins, dystroglycan, syndecans, and the other several cell surface molecules are cellular receptors for laminins. 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 500 overlapping peptides covering the entire G domains and tested their cell attachment activity. 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. Additionally, some of the cyclic peptides enhanced the activity. 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. We conjugated the laminin active peptides onto a chitosan membrane. The active laminin peptide-conjugated chitosan membranes enhanced the biological activity and promoted cell adhesion in a cell-type specific manner. We also demonstrated that the peptide-chitosan membrane can deriver cells and is applicable for keratinocyte transferring to wound bed. The peptide-chitosan approach may be a powerful cell transplantation tool for various tissues and organs.