SEARCH FOR THE DOMAIN IN LAMININ α4 CHAIN RESPONSIBLE TO ANGIOGENESIS

寻找层粘连蛋白 α4 链中负责血管生成的结构域

• 批准号: 11460154
• 负责人: KITAGAWA Yasuo
• 金额: $ 9.66万
• 依托单位: NAGOYA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460154/
• 关键词: capillary; regenerative medicine; laminin; heparin; anti-tumor drugs; extracellular matrix; angiogenesis; structure for cell-cell interaction; 基底膜; 細胞外マトリックス; 糖鎖修飾

Control of angiogenesis in connective tissues is a key for reconstructive tissue engineering. Exploration of angiogenec peptide sequence in extracellular matrix proteins is important to design drugs having anti-tumor activity. We have ever demonstrated that laminin-8 is the member of laminin family specifically secreted by endothelial cells cultured under angiogenic condition. In order to find the domain having angiogenic activity in laminin-8 (composed of α4, β1 and γ1 chains), we here molecular dissected the G domain of mouse α4 chain. G domains of the mouse laminin α1 and α4 chains consisting of its five subdomains LG1-LG5 were overexpressed in CHO cells and purified by heparin chromatography. α1LG1-LG5 and α4LG1-LG5 eluted at NaCl concentrations of 0.30 and 0.47 M, respectively. In solid phase binding assays with immobilized heparin, half-maximal concentrations of 14 (α1LG1-LG5) and 1.4 nM (α4LG1-LG5) were observed. N-glycan cleavage of α4LG1-LG5 did not affect affinity to heparin. … More The affinity of α4LG1-LG5 was significantly reduced upon denaturation with 8 M urea but could be recovered by removing urea. Chymotrypsin digestion of α4LG1-LG5 yielded high and low heparin affinity fragments containing either the α4LG2-LG3 or α4LG4-LG5 modules, respectively. Trypsin digestion of heparin-bound α4LG1-LG5 yielded a high affinity fragment of ca. 190 residues corresponding to the α4LG4 module, indicating that the high affinity binding site is contained within α4LG4. Competition for heparin binding of synthetic peptides covering the α4LG4 region with complete α4LG1-LG5 suggests that the sequence AHGRL1521 is crucial for high affinity binding. When compared with the known structure of α2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded β-sheet sandwich, which is opposite to the proposed binding sites for calcium ion, α-dystroglycan and heparansulfate. Together with recent results reported by Talts et al., (J.Biol.Chem., in press (2001)), our preliminary results suggested that self-association of laminin-8 at G domain is critical for organization of a interacting structure between endothelial cells and connective tissues. Less

控制结缔组织中的血管生成是重建组织工程的关键。探索细胞外基质蛋白中的血管形成肽序列对于设计具有抗肿瘤活性的药物具有重要意义。我们已经证明，层粘连蛋白-8是血管生成条件下培养的内皮细胞特异性分泌的层粘连蛋白家族成员。为了寻找在层粘连蛋白-8(由α-4、β-1和γ-1链组成)中具有血管生成活性的结构域，我们对小鼠α-4链的G结构域进行了分子解剖。小鼠层粘连蛋白α-1和α-4链的G区在CHO细胞中过表达，并用肝素层析纯化。α1LG1-LG5和α4LG1-LG5分别在0.30M和0.47M的氯化钠浓度下洗脱。在与固定化肝素的固相结合实验中，观察到14(α1LG1LG5)和1.4nM(α4LG1LG5)的半峰浓度。α4LG1-LG5的N-糖链切割不影响与肝素的亲和力。…α4LG1LG5经8M尿素变性后亲和力明显降低，但去除尿素后亲和力可恢复。α4LG1LG5经胰凝乳酶消化后，可获得分别含有α4LG2-LG3或α4LG4-LG5模块的高亲和力和低亲和力的肝素片段。胰酶消化肝素结合的α4LG1-LG5产生约190个残基的高亲和力片段，对应于α4LG4模块，表明高亲和力结合位点包含在α4LG4中。覆盖α4LG4区域的合成肽与完整的α4LG1-LG5竞争肝素结合，表明AHGRL1521序列是高亲和力结合的关键。与α2LG5的已知结构相比，该序列对应于14链β-Sheet三明治的转接连接链E和F，而不是所建议的钙离子、α-营养不良聚糖和硫酸乙酰肝素的结合位点。结合Talts等人最近报道的结果(J.Biol.Chem.，in Press(2001))，我们的初步结果表明，层粘连蛋白-8在G区域的自结合对于内皮细胞和结缔组织之间的相互作用结构的组织至关重要。较少

项目成果

熊谷知乃,新美友章,北川泰雄: "細胞外マトリックスー基礎と臨床-(第5章を担当)"愛智出版. 544 (2000)

Tomino Kumagai，Tomoaki Niimi，Yasuo Kitakawa：“细胞外基质-基础和临床（负责第5章）”爱知出版544（2000）。

Mojgan Azimi, Tomoaki Niimi, Naoko Yoshida, and Yasuo Kitagawa: "Differential expression of mRNAs encoding laminin chain variants during in vitro development of mouse blastocysts."Cytotechnology. 31. 183-191 (1999)

Mojgan Azimi、Tomoaki Niimi、Naoko Yoshida 和 Yasuo Kitakawa：“小鼠囊胚体外发育过程中编码层粘连蛋白链变体的 mRNA 的差异表达。”细胞技术。

Chino Kumagai, Masaki Okano, and Yasuo Kitagawa: "Tree heterotrimeric laminins produced by human keratinocytes."Cytotechnology. 33. 167-174 (2000)

Chino Kumagai、Masaki Okano 和 Yasuo Kitakawa：“由人类角质形成细胞产生的树异三聚层粘连蛋白。”细胞技术。

Yasuo Kitagawa, and Nobuko Kawaguchi: "De novo adipogenesis for reconstructive surgery."Cytotechnology. 31. 29-33 (1999)

Yasuo Kitakawa 和 Nobuko Kawaguchi：“重建手术的从头脂肪生成”。细胞技术。

Hirotake Yamaguchi, Hironobu Yamashita, Hitoshi Mori, Ikuko Okazaki, Motoyoshi Nomizu, Konrad Beck, and Yasuo Kitagawa: "High and low affinity heparin-binding sites in the G domain of the mouse laminin α4 chain."J.Biol.Chem.. 275. 29458-29465 (2000)

Hirotake Yamaguchi、Hironobu Yamashita、Hitoshi Mori、Ikuko Okazaki、Motoyoshi Nomizu、Konrad Beck 和 Yasuo Kitakawa：“小鼠层粘连蛋白 α4 链 G 结构域中的高亲和力和低亲和力肝素结合位点。”J.Biol.Chem.. 275.29458-29465 (2000)