Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain

Abstract

A panel of 13 monoclonal antibodies to avian lipoprotein lipase (LPL) was screened for inhibition of LPL binding to primary avian adipocytes. One monoclonal antibody, designated xCAL (monoclonal antibody to chicken adipose lipoprotein lipase) 3-6a, was found to inhibit the binding of LPL to primary avian adipocytes. In solid phase assays, xCAL 3-6a inhibited the binding of LPL to both heparan sulfate and heparin. XCAL 3-6a did not inhibit the catalytic activity of the avian enzyme. The monoclonal antibody was not found to cross-react significantly with bovine lipoprotein lipase. In order to determine the location of the epitope of xCAL 3-6a on lipoprotein lipase, several avian lipoprotein lipase deletion mutants were constructed and produced as glutathione Stransferase (GST) fusion proteins in E. coli. These mutants were screened for their ability to react with xCAL 3-6a using Western blotting. The minimum continuous fragment of lipoprotein lipase that was required for reactivity contained the amino acids 310 to 450. Site-directed mutagenesis of basic residues 321, 405, 407, 409, 415, and 416 revealed that Arg 405 is necessary for the interaction of LPL with xCAL 3-6a. Additional deletions of either the amino-or carboxyl-terminal portion of the fragment containing residues 310-450 resulted in loss of antibody binding, suggesting that the epitope is a discontinuous one that is formed when the termini are brought together through protein folding. Heparin-Sepharose chromatography of wild-type LPL and a mutant LPL in which the well-characterized heparin-binding sequence (Arg 281-Lys 282-Arg 284) has been mutated was carried out in the presence and absence of xCAL 3-6a.These experiments indicate that lipoprotein lipase contains a heparin-binding domain, in addition to Arg 281-Arg 284, that can be blocked by xCAL 3-6a. -Sendak, R. A., K. Melford, A. Kao, and A. Bensadoun. Identification of the epitope of a monoclonal antibody that inhibits heparin binding of lipoprotein lipase: new evidence for a carboxyl-terminal heparin-binding domain.

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.…”

Association between Polymorphisms of Lipoprotein Lipase Gene and Chicken Fat Deposition

“…Two missense mutations were found in exons 7 and 9, which resulted in a substitution of Pro with Ala, and Gly with Asp, respectively. Ala is the 377 th amino acid and Asp is the 447 th amino acid coded by the LPL gene (Sendak, 1998). To ensure functional activity, the least continuous segment of LPL must contain amino acid from 310 th to 450 th .…”

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

“…Subsequently, fragments containing the appropriate mutation(s) were recloned into the plasmid pRc/CMV/LPL, a previously described construct (6) in which the LPL cDNA has been inserted into pRc/CMV (Invitrogen). The construction and cloning into pGEX-4T-2/LPL of LPL(R405N, R407N, K409N), LPL(R405N), LPL(R407N), LPL(K409N), and LPL(K415N, K416N) was previously described (13). Fragments containing the mutations were subcloned into pRc/CMV/LPL utilizing the BstE II site within LPL and the Not I site in the multiple cloning region of both vectors.…”

“…The fourth cluster is discontinuous, containing residues Lys 321, Arg 405, Arg 407, Lys 409, Lys 415, and Lys 416 (avian numbering), and is in the carboxyl-terminus of the protein. Recently we identified a monoclonal antibody to avian LPL that is capable of inhibiting the binding of LPL to primary avian adipocytes, heparin, and heparan sulfate (13). The epitope of this antibody contains Arg 405 and is in close proximity to this fourth predicted charge cluster, providing evidence that cluster four contributes to the binding of LPL to heparin.…”