STRUCTURE-FUNCTION RELATIONSHIP OF LIPOPROTEIN LIPASE-MEDIATED ENHANCEMENT OF VERY-LOW-DENSITY LIPOPROTEIN BINDING AND CATABOLISM BY THE LOW-DENSITY-LIPOPROTEIN RECEPTOR - FUNCTIONAL IMPORTANCE OF A PROPERLY FOLDED SURFACE LOOP COVERING THE CATALYTIC CENTER

We examined the structure-function relationship of human lipoprotein l ipase (hLPL) in its ability to enhance the binding and catabolism of v ery low density lipoproteins (VLDL) in COS cells. Untransfected COS ce lls did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradat ion of the VLDL (a property of LPL that we call bridge function). Hepa rin pretreatment and a monoclonal antibody ID7 that blocks LDL recepto r-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL fro m a Type III hyperlipidemic subject did not bind. However, LDL did not reduce I-125-VLDL binding to the hLPL-expressing cells, whereas rabbi t beta-VLDL was an effective competitor. By contrast, LDL reduced upta ke and degradation of I-125-VLDL to the same extent as excess unlabele d VLDL or beta-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T , and S172G, gave normal enhancement of VLDL binding and catabolism, w hereas the partially active mutant S172D had markedly impaired capacit y for the process; thus, there is no correlation between bridge functi on and lipolytic activity. A naturally occurring genetic variant hLPL, S447-->Ter, has normal bridge function. The catalytic center of LPL i s covered by a 21-amino acid loop that must be repositioned before a l ipid substrate can gain access to the active site for catalysis. We st udied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatica lly inactive mutant with the loop replaced by a pancreatic lipase loop ; and C216S/G239S, an enzymatically inactive mutant with the pair of C ys residues delimiting the loop substituted by Ser residues) and a con trol double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH a nd LPL-cP) and the control double Cys mutant C418S/C438S gave normal e nhancement of VLDL binding and catabolism, whereas the third loop muta nt, C216S/C239S, was completely inactive. We conclude that although ca talytic activity and the actual primary sequence of the loop of LPL ar e relatively unimportant (wild-type LPL loop and pancreatic lipase loo ps have little sequence similarity), the intact folding of the loop, f lanked by disulfide bonds, must be maintained for LPL to express its b ridge function.