Localization of three distinct heparin-binding domains of laminin by monoclonal antibodies.
Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.
Skubitz
A P
AP
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis 55455.
McCarthy
J B
JB
Charonis
A S
AS
Furcht
L T
LT
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D007797:Laminin / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011863:Radioimmunoassay; D014316:Tritium
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007797:Laminin; D014316:Tritium; D006493:Heparin