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

• Published in: The Journal of biological chemistry Vol. 271; no. 36; pp. 21906 - 21913
• Main Authors: Salinelli, S. (Baylor College of Medicine, Houston, TX.), Lo, J.Y, Mims, M.P, Zsigmond, E, Smith, L.C, Chan, L
• Format: Journal Article
• Language: English
• Published: United States 06.09.1996
• Subjects: ACTIVIDAD CATALITICA; ACTIVITE CATALYTIQUE; Animals; Apolipoprotein B-100; Apolipoproteins B; Apolipoproteins B - metabolism; Apolipoproteins E; Apolipoproteins E - metabolism; Binding Sites; CATABOLISME; CATABOLISMO; Catalysis; catalytic activity; cell lines; CHIMIORECEPTEUR; Cricetinae; Cricetulus; CULTIVO DE CELULAS; CULTURE DE CELLULE; DEGRADACION; DEGRADATION; Enzyme-Linked Immunosorbent Assay; ESTIMULO; FIBROBLASTE; FIBROBLASTOS; fibroblasts; GENERO HUMANO; genetics; GENRE HUMAIN; Humans; lipoprotein lipase; Lipoprotein Lipase - genetics; Lipoprotein Lipase - metabolism; LIPOPROTEINA LIPASA; LIPOPROTEINAS; LIPOPROTEINE; LIPOPROTEINE LIPASE; Lipoproteins, LDL; Lipoproteins, LDL - metabolism; Lipoproteins, VLDL; Lipoproteins, VLDL - metabolism; low density lipoprotein; metabolism; molecular conformation; MUTACION; MUTANT; MUTANTES; mutants; MUTATION; Point Mutation; protein degradation; Protein Structure, Tertiary; PROTEINAS; PROTEINE; QUIMIORECEPTORES; Rabbits; receptors; Receptors, LDL; Receptors, LDL - metabolism; site-directed mutagenesis; STIMULUS; Structure-Activity Relationship; Transfection; very low density lipoprotein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.36.21906

We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125I-VLDL binding to the hLPL-expressing cells, whereas rabbit p-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125I-VLDL to the same extent as excess unlabeled 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, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S447 replaced by Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S