Identification of a lipoprotein lipase cofactor-binding site by chemical cross-linking and transfer of apolipoprotein C-II-responsive lipolysis from lipoprotein lipase to hepatic lipase.
To localize the regions of lipoprotein lipase (LPL) that are responsive to activation by apoC-II, an apoC-II peptide fragment was cross-linked to bovine LPL. Following chemical hydrolysis and peptide separation, a specific fragment of LPL (residues 65-86) was identified to interact with apoC-II. The fragment contains regions of amino acid sequence dissimilarity compared with hepatic lipase (HL), a member of the same gene family that is not responsive to apoC-II. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (residues 65-68 and 73-79) were exchanged with the corresponding human HL sequences. The chimeras consisted of an HL backbone with the suspected LPL regions replacing the corresponding HL sequences either individually (HLLPL-(65-68) and HLLPL-(73-79)) or together (HLLPLD). Similarly, LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequences (LPLHL-(77-80), LPLHL-(85-91), and LPLHLD). Using a synthetic triolein substrate, the lipase activity of the purified enzymes was measured in the presence and absence of apoC-II. Addition of apoC-II to HLLPL-(65-68) and HLLPL-(73-79) did not significantly alter their enzyme activity. However, the activity of HLLPLD increased approximately 5-fold in the presence of apoC-II compared with an increase in native LPL activity of approximately 11-fold. Addition of apoC-II to LPLHL-(77-80) resulted in approximately 10-fold activation, whereas only approximately 6- and approximately 4-fold activation of enzyme activity was observed in LPLHL-(85-91) and LPLHLD, respectively. In summary, our results have identified 11 amino acid residues in the N-terminal domain of LPL (residues 65-68 and 73-79) that appear to act cooperatively to enable substantial activation of human LPL by apoC-II.
McIlhargey
Trina L
TL
University of British Columbia McDonald Research Laboratories/iCAPTUR4E Centre, Department of Pathology and Laboratory Medicine, St. Paul's Hospital and the University of British Columbia, Vancouver, Canada.
Yang
Yingying
Y
Wong
Howard
H
Hill
John S
JS
D000595:Amino Acid Sequence; D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D008066:Lipolysis; D008071:Lipoprotein Lipase / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014304:Triolein / Q000494:pharmacology
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D003432:Cross-Linking Reagents; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014304:Triolein; D008049:Lipase; D008071:Lipoprotein Lipase