Mutation of a basic sequence in the laminin alpha2LG3 module leads to a lack of proteolytic processing and has different effects on beta1 integrin-mediated cell adhesion and alpha-dystroglycan binding.
A RRKRRQ sequence unique to the LG3 module of the laminin alpha2 chain was previously shown to be sensitive to endogenous proteolysis during the recombinant production of the tandem array alpha2LG1-3. Mutation of RQ surrounding the cleaved peptide bond did not prevent this processing and intracellular degradation. Alanine mutagenesis of three alternate basic residues, however, was shown to prevent the cleavage in alpha2LG1-3, allowing for the alpha2LG3 module to be obtained as a folded, globular fragment. The mutation did not change heparin and sulfatide binding or cell adhesion of alpha2LG1-3 which can be mediated by alpha3beta1 and alpha6beta1 integrins. It did, however, cause a 10-fold reduction in alpha-dystroglycan binding. The data favor the interpretation that binding epitopes for heparin/sulfatides, beta1 integrins and alpha-dystroglycan occupy different parts of the alpha2LG1-3 structure.
Talts
J F
JF
Max-Planck-Institut für Biochemie, Abt. Proteinchemie, Martinsried, Germany.
Timpl
R
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001667:Binding, Competitive; D002448:Cell Adhesion; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D049030:Dystroglycans; D010450:Endopeptidases / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins; D013433:Sulfoglycosphingolipids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D019012:Integrin beta1; D007797:Laminin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D013433:Sulfoglycosphingolipids; C102645:laminin alpha 2; D049030:Dystroglycans; D006493:Heparin; D010450:Endopeptidases