| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2000: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1999: ¥11,300,000 (Direct Cost: ¥11,300,000)
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| Research Abstract |
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.
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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
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