High and low affinity heparin-binding sites in the G domain of the mouse laminin alpha 4 chain

G domains of the mouse laminin alpha 1 and alpha 4 chains consisting of its five subdomains LG1-LG5 were overexpressed in Chinese hamster ovary cells and purified by heparin chromatography. alpha 1LG1-LG5 and alpha 4LG1-LG5 e luted at NaCl concentrations of 0.30 and 0.47 M, respectively. In solid pha se binding assays with immobilized heparin, half-maximal concentrations of 14 (alpha 1LG1-LG5) and 1.4 nM (alpha 4LG1-LG5) were observed. N-Glycan cle avage of alpha 4LG1-LG5 did not affect affinity to heparin. The affinity of ru4LG1-LG5 was significantly reduced upon denaturation with 8 M urea but c ould be recovered by removing urea. Chymotrypsin digestion of alpha 4LG1-LG 5 yielded high and low heparin affinity fragments containing either the alp ha 4LG4-LG5 or alpha 4LG2-LG3 modules, respectively. Trypsin digestion of h eparin-bound alpha 4LG1-LG5 yielded a high affinity fragment of about 190 r esidues corresponding to the alpha 4LG4 module indicating that the high aff inity binding site is contained within alpha 4LG4. Competition for heparin binding of synthetic peptides covering the alpha 4LG4 region with complete alpha 4LG1-LG5 suggests that the sequence AHGRL1521 is crucial for high aff inity binding. Introduction of mutation of H1518A or R1520A in glutathione S-transferase fusion protein of the alpha 4LG4 module produced in Escherich ia coli markedly reduced heparin binding activity of the wild type. When co mpared with the known structure of alpha 2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded beta-sheet sandwich , which is opposite to the proposed binding sites for calcium ion, alpha-dy stroglycan, and heparan sulfate.