Site-directed mutagenesis of apolipoprotein CII to probe the role of its secondary structure for activation of lipoprotein lipase.
Apolipoprotein CII (apoCII) is a necessary activator for lipoprotein lipase (LPL). We had identified four residues (Tyr-63, Ile-66, Asp-69, and Gln-70), presumably contained in an alpha-helix, as a potential binding site for LPL. We have now used structure prediction, mutagenesis, and functional assays to explore the functional role of the secondary structure in this part of apoCII. First, mutants were generated by replacements with proline residues to disturb the helical structure. Activation by mutant G65P was reduced by 30%, whereas mutant S54P retained activation ability. Mutants V71P and L72P should be located outside the LPL-binding site, but V71P was totally inactive, whereas activation by L72P was reduced by 65%. Insertion of alanine after Tyr-63, changing the position of the putative LPL-binding site in relation to the hydrophobic face of the alpha-helix, also severely impeded the activation ability, and a double mutant (Y63A/I66A) was completely inactive. Next, to investigate the importance of conserved hydrophobic residues in the C-terminal end of apoCII, Phe-67, Val-71, Leu-72, and Leu-75 were exchanged for polar residues. Only F67S showed dramatic loss of function. Finally, fragment 39-62, previously claimed to activate LPL, was found to be completely inactive. Our data support the view that the helical structure close to the C-terminal end of apoCII is important for activation of LPL, probably by placing residues 63, 66, 69, and 70 in an optimal steric position. The structural requirements for the hydrophobic face on the back side of this helix and further out toward the C terminus were less stringent.
Shen
Yan
Y
Department of Medical Biosciences, Physiological Chemistry, Umeå University, SE-90 187 Umeå, Sweden.
Lookene
Aivar
A
Zhang
Liyang
L
Olivecrona
Gunilla
G
D000595:Amino Acid Sequence; D000818:Animals; D053304:Apolipoprotein C-II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002914:Chylomicrons / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary
D053304:Apolipoprotein C-II; D002914:Chylomicrons; D008081:Liposomes; D008071:Lipoprotein Lipase