Construction and functional characterization of recombinant fusion proteins of human lipoprotein lipase and apolipoprotein CII.
The hydrolysis of triacylglycerols of chylomicrons and very low density lipoproteins by lipoprotein lipase (LPL) requires the presence of apolipoprotein (apo) CII as a cofactor. To obtain further information on the interaction of apo CII and LPL, we generated two fusion proteins consisting of the complete LPL molecule and the mature form of apo CII. The cDNAs of both proteins were either connected directly or by a segment encoding a 16-amino-acid linker peptide. The fused cDNAs were stably expressed in human embryonic kidney (HEK) 293 cells and the enzymic properties of the recombinant proteins were examined. The fusion proteins hydrolysed both emulsified long-chain (lipase) triacyglycerol substrate and a water-soluble short-chain (esterase) fatty acid ester substrate (p-nitrophenylbutyrate), regardless of whether or not they contained the linker peptide. In the absence of exogenous apo CII, the fusion proteins had up to 3.5-times higher basal activity than wild-type LPL. Similar to wild-type LPL, the fusion proteins were inhibited by 1 M NaCl, however less than wild-type LPL. A polyclonal antibody specific for apo CII impaired their ability to hydrolyse triacylglycerol emulsions. A similar effect was seen when the tetrapeptide KGEE was used as inhibitor, which corresponds to the carboxy-terminal four amino acids of apo CII.
Hoffmann
M M
MM
Institute of Biochemistry, Medical Faculty, University of Cologne, Germany.
Stoffel
W
W
D000595:Amino Acid Sequence; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D008071:Lipoprotein Lipase / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D008071:Lipoprotein Lipase