Original Article

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

Correspondence to: André Bensadoun.

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 S-transferase (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. J. Lipid Res. 1998. 39: 633–646.

Supplementary key words: deletion mutant analysis, adipocyte binding, in vitro mutagenesis, heparin-Sepharose chromatography

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