Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding.
Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL.
MacPhee
C E
CE
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville 3052, Victoria, Australia.
Hatters
D M
DM
Sawyer
W H
WH
Howlett
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D002417:Cattle; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008049:Lipase / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D008051:Lipid Bilayers; D008055:Lipids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012996:Solutions; D010740:Phospholipases; D008049:Lipase; D008071:Lipoprotein Lipase; D004134:Dimyristoylphosphatidylcholine