Analysis of the assembly of laminin and the laminin-entactin complex with laminin chain specific monoclonal and polyclonal antibodies.
Antibodies specific for the A, B1, and B2 chains of laminin have been obtained and characterized. Lam V, a rat X mouse monoclonal antibody, was obtained by immunizing Lewis rats with the extracellular matrix derived from the mouse endodermal line M1536-B3. The antibody was shown to recognize a conformation-sensitive epitope present on the A chain of laminin. The antibody exhibited high avidity for native laminin and uncomplexed newly synthesized laminin A chains. cDNA clones in the vector lambda-gt11 containing sequences for the B1 and B2 chains of laminin were shown to synthesize beta-galactosidase fusion proteins in the host cells induced with IPTG. The fusion protein F3 contained amino acid residues 822-1765 of the B1 chain of mouse laminin, and the fusion protein E4 contained 219 amino acids at the carboxyl terminus of the B2 chain of rat laminin. These two fusion proteins were used to obtain rabbit polyclonal antibodies which were characterized for their specificity and ability to immunoprecipitate laminin and the B chains of laminin. The chain-specific antibodies were used to analyze the assembly and processing of laminin in the mouse endodermal cell line M1536-B3. The results indicated that the covalent assembly of the A and B chains of laminin was initiated as early as 3 min after labeling cells. At this time point uncomplexed A chain of laminin could be observed even though there was an excess of B1 and B2 chains. As early as 4 min after labeling monomeric, dimeric, and oligomeric forms of the B chains of laminin were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
C
C
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Friedman
R
R
Chung
A E
AE
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001485:Basement Membrane / Q000378:metabolism*; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000378:metabolism*; D015151:Immunoblotting; D007797:Laminin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007797:Laminin; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C029967:nidogen; D001616:beta-Galactosidase