Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site

Abstract

Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.