Identification and characterization of tissue specific active sites in the laminins

层粘连蛋白中组织特异性活性位点的鉴定和表征

• 批准号: 14370735
• 负责人: NOMIZU Motoyoshi
• 金额: $ 6.53万
• 依托单位: Tokyo University of Pharmacy and Life Sciences (2004)Hokkaido University (2002-2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370735/
• 关键词: basement membrane; laminin; integrin; syndecan; chitosan; peptide; 創傷治癒

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.

层粘连蛋白是由 α、β 和 γ 链组成的异三聚体，是存在于基底膜中的多功能糖蛋白。层粘连蛋白是早期发育中基底膜组装过程中基底膜最重要的组成部分。层粘连蛋白参与细胞粘附、迁移、生长、分化、肿瘤转移、血管生成等多种生物活性。已鉴定出十五种层粘连蛋白亚型，并且是组织和/或发育阶段特异性表达的。整合素、肌营养不良聚糖、多配体聚糖和其他几种细胞表面分子是层粘连蛋白的细胞受体。我们制备了多种重组蛋白，包括层粘连蛋白α1、α3和α4链的G结构域，并测试了它们的细胞附着活性。几种重组蛋白显示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 种肽促进细胞附着，5 种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多聚糖相互作用。此外，一些环肽增强了活性。这些结果表明活性位点参与了层粘连蛋白α链G结构域的生物学功能。这些活性肽可用于定义层粘连蛋白-受体相互作用的分子机制和层粘连蛋白介导的细胞信号传导途径。我们将层粘连蛋白活性肽缀合到壳聚糖膜上。活性层粘连蛋白肽缀合的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。我们还证明肽-壳聚糖膜可以衍生细胞并适用于角质形成细胞转移至创面床。肽-壳聚糖方法可能是各种组织和器官的强大细胞移植工具。

项目成果

Identification of heparin binding sites in the laminin α2 chain LG4-5 module.

层粘连蛋白 α2 链 LG4-5 模块中肝素结合位点的鉴定。

• 发表时间: 2005
• 期刊: Peptide Science 2004
• 作者: Nakayma M;Goto TM;Sugimoto M;Nishimura T;Shinagawa T;Ohno S;Amano M;Kaibuchi K;Yutaka Momotaら;Nobuharu Suzukiら;Ohno S;Akane Kondoら;Takashi Yoshimuraら
• 通讯作者: Takashi Yoshimuraら

Cyclic peptides from the loop region of the laminin alpha 4 chain LG4 module show enhanced biological activity over linear peptides.

• DOI: 10.1021/bi049009u
• 发表时间: 2004-10
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: F. Yokoyama;N. Suzuki;M. Haruki;N. Nishi;S. Oishi;N. Fujii;A. Utani;H. Kleinman;M. Nomizu

Laminin‐1 peptide‐conjugated chitosan membranes as a novel approach for cell engineering

• DOI: 10.1096/fj.02-0564fje
• 发表时间: 2003-05
• 期刊: The FASEB Journal
• 作者: M. Mochizuki;Y. Kadoya;Y. Wakabayashi;Kozue Kato;I. Okazaki;M. Yamada;Taku Sato;N. Sakairi

Shingo Kasai: "Multifunctional peptide fibrils for biomedical materials"Biopolymers. 76. 27-33 (2004)

Shingo Kasai：“用于生物医学材料的多功能肽原纤维”生物聚合物。

Masanori Yamada: "Ile-Lys-Val-Ala-Val(IKVAV)-containing laminin α1 chain peptides form amyloid-like fibrils"FEBS Letters. 530. 48-52 (2002)

Masanori Yamada：“含有 Ile-Lys-Val-Ala-Val (IKVAV) 的层粘连蛋白 α1 链肽形成淀粉样原纤维”FEBS Letters 530. 48-52 (2002)。