LIPOPROTEIN-LIPASE DOMAIN FUNCTION

Human lipoprotein lipase (LPL) monomer consists of two domains, a larg er NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a maj or determinant of heparin binding. Analyses of NH2-terminal domain fun ction were performed after site-directed mutagenesis of the putative a ctive-site serine residue, while COOH-terminal domain function was ass essed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cystein e, or glycine were transiently expressed in COS-7 cells. Mutant protei ns were synthesized and secreted at levels comparable to native LPL; h owever, none of the mutants retained enzymatic activity. The mutant wi th alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-di dodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable t o native LPL, These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of th e smaller COOH-terminal domain, LPL lipolytic and esterolytic activiti es as well as heparin binding properties were determined after reactio n with a monoclonal antibody specific for this domain. Lipolytic activ ity was inhibited by the monoclonal antibody, whereas esterolytic acti vity was only marginally affected, indicating that the LPL COOH-termin al domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, su ggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) bindi ng of antibody did not cause dimer dissociation. A model is proposed f or the two LPL domains fulfilling different roles in the lipolytic pro cess.