Identification of a heparin-binding domain in the distal carboxyl-terminal region of lipoprotein lipase by site-directed mutagenesis

Abstract

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 heparinbinding domain in LPL.-Sendak, R. A., and A. Bensadoun. Identification of a heparin-binding domain in the distal carboxyl-terminal region of lipoprotein lipase by site-directed mutagenesis.

Characterization of the lipolytic activity of endothelial lipase

“…Like LPL and HL, EL also functions in the plasma compartment, and our initial report showed that overexpression of EL in mice profoundly altered plasma lipoprotein levels. The putative heparin-binding (6)(7)(8) and hydrophobic regions implicated in lipid or lipoprotein binding (9)(10)(11)(12) that are present in LPL and HL are highly conserved in EL, suggesting that EL, like LPL and HL, is anchored to the luminal endothelial surface via heparan sulfate proteoglycans where it can interact directly with lipoproteins. Initial reports indicated that EL had phospholipase activity (4,5) but the enzymatic activity was not characterized further.…”

Novel compound heterozygous mutations for lipoprotein lipase deficiency: a G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5′ splice site mutation of intron 8

“…This aberrant joint inevitably resulted in a frameshift that generated a stop codon. This predicts production of the truncated LPL molecule lacking amino acid sequences of Val 370 -Gly 448 (e.g., Val 370 -Lys 414 and Arg 420 -Gly 448 ), which are a part of the carboxyl C-terminal domain (residues 313-448 in exons 7 to 9) containing the substrate-binding (33) and heparin-binding regions (34). Essentially, the truncated LPL molecule is presumed to be catalytically inactive due to a lack of the essential region for interaction with substrates such as chylomicrons and VLDL.…”

Binding of hepatic lipase to heparin: identification of specific heparin-binding residues in two distinct positive charge clusters

“…Cells were maintained in a humidified incubator at 5% CO 2 . The CHO cells were stably transfected with the RHL DNA constructs using Lipo-fectamine™ according to a previously described procedure (22).…”

“…Four regions of positive charge were identified: these have been subjected to site-directed mutagenesis revealing that they may play a role in the heparin binding of the protein. Cluster 1 (avian sequence numbering): Arg 281, Lys 282, and Arg 284 (19)(20)(21) and Cluster 4: Lys 321, Arg 405, Arg 407, Arg 409, and Lys 416 (22) were shown to be involved in heparin binding while conflicting results were obtained for Cluster 2: residues 296-302 (20,21). Cluster 3 (residues 147-151, human numbering) was not found to play a role in heparin binding (20).…”

“…To date, the regions of HL that are essential for the binding of the enzyme to heparin have not been identified. The heparin-binding regions of lipoprotein lipase (LPL), on the other hand, have been extensively studied (19)(20)(21)(22). LPL and HL are members of the triglyceride lipase superfamily that share extensive sequence homology, but differ significantly in substrate specificity, optimal hydrolysis conditions, cofactor requirements, and heparinbinding affinity.…”