SEARCH FOR THE DOMAIN IN LAMININ α4 CHAIN RESPONSIBLE TO ANGIOGENESIS

结缔组织血管生成的控制是重建组织工程的关键。探索细胞外基质蛋白中血管生成肽序列对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是层粘连蛋白家族的成员，是在血管生成条件下培养的内皮细胞特异性分泌的。为了找到层粘连蛋白-8（由α4、β1和γ1链组成）中具有血管生成活性的结构域，我们对小鼠α4链的G结构域进行了分子解剖。小鼠层粘连蛋白α1和α4链的G结构域由其五个亚结构域LG1-LG5组成，在CHO细胞中过表达，并通过肝素层析纯化。α1LG1-LG5和α4LG1-LG5分别在NaCl浓度为0.30和0.47 M时洗脱。固定化肝素固相结合实验中，最大半浓度为14 （α1LG1-LG5）和1.4 nM （α4LG1-LG5）。α4LG1-LG5的n -聚糖裂解不影响其对肝素的亲和力。α4LG1-LG5经8 M尿素变性后亲和力显著降低，但去除尿素后可恢复亲和力。胰凝乳酶对α4LG1-LG5的酶切分别产生含有α4LG2-LG3和α4LG4-LG5模块的高和低肝素亲和片段。胰蛋白酶酶切肝素结合的α4LG1-LG5得到约190个与α4LG4模块对应的高亲和力片段，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，这表明AHGRL1521序列对于高亲和力结合至关重要。与已知α2LG5的结构相比，该序列对应于14链β-sheet三明治中连接E和F链的转位，这与钙离子、α-三聚糖酐和硫酸肝素的结合位点相反。加上Talts等人最近报告的结果，[J.Biol.Chem.]（2001）)，我们的初步结果表明，G结构域层粘连蛋白-8的自结合对于内皮细胞和结缔组织之间相互作用结构的组织至关重要。少

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