Functional analyses of human apolipoprotein CII by site-directed mutagenesis: identification of residues important for activation of lipoprotein lipase.
Apolipoprotein CII (apoCII) activates lipoprotein lipase (LPL). Seven residues, located on one face of a model alpha-helix spanning residues 59-75, are fully conserved in apoCII from ten different animal species. We have mutated these residues one by one. Substitution of Ala(59) by glycine, or Thr(62) and Gly(65) by alanine did not change the activation, indicating that these residues are outside the LPL-binding site. Replacement of Tyr(63), Ile(66), Asp(69), or Gln(70) by alanine lowered the affinity for LPL and the catalytic activity of the LPL-apoCII complex. For each residue several additional replacements were made. Most mutants retained some activating ability, but replacement of Tyr(63) by phenylalanine or tryptophan and Gln(70) by glutamate caused almost complete loss of activity. All mutants bound to liposomes with similar affinity as wild-type apoCII, and they also bound with similar affinity to LPL in the absence of hydrolyzable lipids. However, the inactive mutants did not compete with wild-type apoCII in the activation assay. Therefore, we conclude that the productive apoCII-LPL interaction may be dependent on substrate molecules. In summary, our data demonstrate that residues 63, 66, 69, and 70 are of special importance for the function of apoCII, but no single amino acid residue is absolutely crucial.
Shen
Yan
Y
Department of Medical Biosciences, Umeå University, Umeå SE-90 187, Sweden.
Lookene
Aivar
A
Nilsson
Solveig
S
Olivecrona
Gunilla
G
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D050356:Lipid Metabolism; D008071:Lipoprotein Lipase / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D017931:DNA Primers; D011994:Recombinant Proteins; D008071:Lipoprotein Lipase