Identification of a major heparin and cell binding site in the LG4 module of the laminin alpha 5 chain.
The G domain of the laminin alpha chains consists of five homologous G modules (LG1-5) and has been implicated in various biological functions. In this study, we identified an active site for cell and heparin binding within the laminin alpha5 G domain using recombinant proteins and synthetic peptides. Recombinant LG4, LG5, and LG4-5 modules were generated using a mammalian expression system. The LG4 and LG4-5 modules were highly active for cell binding, whereas the LG5 module alone showed only weak binding. Heparin inhibited cell binding to the LG4-5 module, whereas no inhibition was observed with EDTA or antibodies against the integrin beta(1) subunit. These results suggest that the LG4-5 module interacts with a cell surface receptor containing heparan sulfate but not with integrins. Solid-phase assays and surface plasmon resonance measurements demonstrated strong binding of the LG4 and LG4-5 modules to heparin with K(D) values in the nanomolar range, whereas a 16-fold lower value was determined for the LG5 module. Treatment with glycosidases demonstrated that N-linked carbohydrates on the LG5 module are complex-type oligosaccharides. The LG4-5 module, devoid of N-linked carbohydrates, exhibited similar binding kinetics toward heparin. Furthermore, cell binding was unaffected by removal of N-linked glycosylation. To localize active sites on the LG4 module, various synthetic peptides were used to compete with binding of the tandem module to heparin and cells. Peptide F4 (AGQWHRVSVRWG) inhibited binding, whereas a scrambled peptide of F4 failed to compete binding. Alanine replacements demonstrated that one arginine residue within F4 was important for cell and heparin binding. Our results suggest a critical role of the LG4 module for heparan sulfate-containing receptor binding within the laminin alpha5 chain.
Nielsen
P K
PK
Molecular Biology Section, Craniofacial Developmental Biology and Regeneration Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
Gho
Y S
YS
Hoffman
M P
MP
Watanabe
H
H
Makino
M
M
Nomizu
M
M
Yamada
Y
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D017931:DNA Primers; D004492:Edetic Acid / Q000737:chemistry; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D019012:Integrin beta1 / Q000276:immunology; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019012:Integrin beta1; D007797:Laminin; D011994:Recombinant Proteins; C097043:laminin alpha5; D006493:Heparin; D004492:Edetic Acid