High and Low Affinity Heparin-binding Sites in the G Domain of the Mouse Laminin α4 Chain

Hirotake Yamaguchi,Hironobu Yamashita,Hitoshi Mori,Ikuko Okazaki,Motoyoshi Nomizu,Konrad Beck,Yasuo Kitagawa

doi:10.1074/jbc.m003103200

Hirotake Yamaguchi, Hironobu Yamashita + Show 5 more

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https://doi.org/10.1074/jbc.m003103200

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Abstract

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 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 (alpha 1LG1-LG5) and 1.4 nm (alpha 4LG1-LG5) were observed. N-Glycan cleavage of alpha 4LG1-LG5 did not affect affinity to heparin. The affinity of alpha 4LG1-LG5 was significantly reduced upon denaturation with 8 m urea but could be recovered by removing urea. Chymotrypsin digestion of alpha 4LG1-LG5 yielded high and low heparin affinity fragments containing either the alpha 4LG4-LG5 or alpha 4LG2-LG3 modules, respectively. Trypsin digestion of heparin-bound alpha 4LG1-LG5 yielded a high affinity fragment of about 190 residues corresponding to the alpha 4LG4 module indicating that the high affinity 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 affinity binding. Introduction of mutation of H1518A or R1520A in glutathione S-transferase fusion protein of the alpha 4LG4 module produced in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared 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-dystroglycan, and heparan sulfate.

Highlights

Basement membranes are sheet-like extracellular matrices underlying epithelial and endothelial cells, surrounding muscle cells, adipocytes, and peripheral nerve axons, and acting as supportive architecture for the cells to proliferate, differentiate, and migrate
When compared with other laminin G domains so far studied [15,16,17,18,19,20,21], ␣4 G domain shows the highest affinity to heparin. ␣4LG1–LG5 eluted from a heparin column at a NaCl concentration of 470 mM (Fig. 3C) whereas different LG modules of ␣1 and ␣2 chains, and the whole ␣1 G domain were eluted at an ionic strength of 140 –360 mM [15, 17, 19]
In a solid phase binding assay using immobilized heparin, the concentrations of ␣4LG1–LG5 and ␣1LG1–LG5 required for half-maximal binding were 1.4 and 14 nM, respectively (Fig. 4)

Summary

Introduction

Basement membranes are sheet-like extracellular matrices underlying epithelial and endothelial cells, surrounding muscle cells, adipocytes, and peripheral nerve axons, and acting as supportive architecture for the cells to proliferate, differentiate, and migrate These matrices contain one or more members of the laminin family as a major component. Laminin-1, -2, and -4 form polymers by reversible self-assembly of monomers at short arms in a calcium ion- and temperature-dependent manner with a critical concentration of assembly of 70 –140 nM (9 –11) This polymerization of the cross-shaped laminins has a central role in forming the meshwork architecture of basement membranes. Laminin ␣4 lacks the N-terminal short arm and shows an expression pattern distinct from that of the full size ␣ chains It is expressed in the cell of mesenchymal origin such as endothelial cells (24 –27) and mouse 3T3-L1 adipocytes [28, 29]. Heparin Binding of Laminin ␣4G Domain the expression was localized mainly in mesenchymal cells of lung, cardiac, and skeletal muscle fibers, and immunohistology detected laminin ␣4 antigen in capillary basement membranes as well as perineurium [30]

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