Site-directed mutagenesis of a putative heparin binding domain of avian lipoprotein lipase.
Lipoprotein lipase (LPL) binds to heparin and heparan sulfate proteoglycans. We have employed site-directed mutagenesis to dissect one of the proposed heparin binding domains of avian LPL, which contains the sequence Arg-Lys-Asn-Arg (amino acids 281-284). Various single, double, and triple mutants of chicken LPL were constructed in order to alter the positive charge of this region. The mutant and wild-type cDNAs were subcloned into an expression vector, pRc/CMV, and expressed in Chinese hamster ovary cells. In general, the LPL mutants with a decrease in regional positive charge showed a decrease in affinity for heparin and heparan sulfate proteoglycans. The greatest effect was seen with the triple mutant, LPL 5G, in which all of the positively charged amino acids were altered to neutral residues. On a heparin-Sepharose column, LPL 5G eluted at 0.96 M NaCl compared with 1.35 M for wild-type LPL. This mutant also had the lowest specific activity with 1.5 mu eq fatty acid/micrograms/h for the cell-associated pool and with no detectable activity in the media. Wild-type cells, however, produced a lipase with a specific activity of 12.4 and 13.1 mu eq fatty acid/micrograms/h for cell-associated and media lipase pools, respectively. LPL 5G also showed a decrease in affinity for the heparan sulfate proteoglycans on the cell surface of Chinese hamster ovary cells. In conclusion, the region of avian LPL between Arg281 and Arg284 does appear to be involved in heparin-binding; however, additional regions must be involved since binding was not completely abolished. In addition, specific activity of the cell-associated and secreted LPL is correlated to affinity of the enzyme for heparan sulfate chains.
Berryman
D E
DE
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
Bensadoun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D019812:Heparan Sulfate Proteoglycans; D006493:Heparin / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011509:Proteoglycans / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D019812:Heparan Sulfate Proteoglycans; D006493:Heparin; D006497:Heparan Sulfate; D008071:Lipoprotein Lipase; D009838:Oligodeoxyribonucleotides; D011509:Proteoglycans; D011994:Recombinant Proteins