Identification of a heparin-binding domain in the distal carboxyl-terminal region of lipoprotein lipase by site-directed mutagenesis.
The interaction of lipoprotein lipase (LPL) with heparan sulfate proteoglycans plays an important role in the metabolism and catalytic function of the enzyme. We have used site-directed mutagenesis to replace the basic residues contained in a discontinuous charge cluster (residues Lys 321, Arg 405, Arg 407, Lys 409, Lys 415, and Lys 416) of avian LPL with asparagine. The mutant proteins were expressed in Chinese hamster ovary cells and their affinity for heparin was evaluated by heparin-Sepharose chromatography. Mutation of residues Lys 321, Arg 405, Arg 407, Lys 409, and Lys 416 resulted in a decrease in affinity for heparin. The triple mutant LPL(R405N, R407N, K409N) possessed almost no high-affinity binding. The LPL mutants showed enzymatic activities ranging between 50-100% of that seen for wild-type LPL demonstrating that the overall structure of the enzyme was not significantly altered by the mutations. Mutation of previously identified heparin-binding regions of LPL results in a relatively small decrease in heparin-binding affinity, as compared with mutations in this carboxyl-terminal region, indicating that Lys 321, Arg 405, Arg 407, Lys 409, and Lys 416 constitute the major heparin-binding domain in LPL.
Sendak
R A
RA
Division of Nutritional Science, Cornell University, Ithaca, New York 14853, USA.
Bensadoun
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001717:Birds; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D017931:DNA Primers; D006493:Heparin / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D006493:Heparin; D008071:Lipoprotein Lipase