Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies.
The objective of this study was to investigate how a conformational change in lipoprotein lipase (LPL) affects its molecular functions. Monoclonal antibodies (MAbs) were raised against purified bovine milk lipoprotein lipase. MAb 5D2 bound to human and bovine LPL both before and after denaturation of LPL. MAb 5F9 also recognized LPL from both species, but only after denaturation of the antigen, suggesting that a conformational change led to exposure of a previously hidden epitope. The MAbs were used in two sandwich enzyme-linked immunosorbent assays (ELISAs). One ELISA used the same MAb (5D2) to coat the plate and detect the bound antigen. This ELISA thus required the same epitope to be present in duplicate for detection (as would be the case with a dimeric antigen). The second ELISA used MAb 5F9 to coat the plate and MAb 5D2 to detect the antigen. This ELISA detected LPL only after it had been denatured. By measuring the same sample before and after denaturation with guanidine hydrochloride (GuHCl) in the 5F9 ELISA, and subtracting one from the other, a measure of native LPL was obtained. In inactivation experiments using human LPL, activity and the measure of LPL mass obtained in the 5D2 ELISA decreased and were related inversely to the measured mass obtained in the 5F9 ELISA which increased, indicating that loss of activity is closely linked to dimer dissociation and loss of native conformation. The effect of conformation and dimeric structure on LPL-heparin interaction was studied by heparin-Sepharose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
D000328:Adult; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D000939:Epitopes; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008875:Middle Aged; D011487:Protein Conformation; D011489:Protein Denaturation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D006493:Heparin; D008071:Lipoprotein Lipase