Identification and characterization of tissue specific active sites in the laminins

层粘连蛋白是由α，β和γ链组成的杂三聚体，是存在于基底膜上的多功能糖蛋白。层粘连蛋白是发育早期基底膜组装过程中最重要的基底膜成分。层粘连蛋白参与多种生物学活动，如细胞黏附、迁移、生长、分化、肿瘤转移和血管生成。已经鉴定了15种层粘连蛋白亚型，它们是组织和/或发育阶段特异表达的。整合素、营养不良多糖、合成酶和其他几种细胞表面分子是层粘连蛋白的细胞受体。我们制备了由层粘连蛋白α1、α3和α4链的G结构域组成的各种重组蛋白，并检测了它们的细胞黏附活性。几种重组蛋白具有细胞附着和轴突生长活性。我们还合成了500多个覆盖整个G结构域的重叠多肽，并测试了它们的细胞黏附活性。有20多个多肽促进细胞贴壁，5个多肽抑制重组蛋白介导的生物活性。一些多肽被发现与整合素和整合素相互作用。此外，一些环肽增强了该酶的活性。这些结果表明，活性部位参与了层粘连蛋白α链G结构域的生物学功能。这些活性多肽可能有助于确定层粘连蛋白-受体相互作用的分子机制以及层粘连蛋白介导的细胞信号通路。我们将层粘连蛋白活性多肽连接到壳聚糖膜上。活性层粘蛋白多肽偶联壳聚糖膜以细胞类型特异性的方式提高了生物活性，促进了细胞的黏附。我们还证明了多肽-壳聚糖膜可以分化细胞，适用于角质形成细胞转移到创面床上。多肽-壳聚糖方法可能是一种强大的组织器官细胞移植工具。

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.