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 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.

Highlights

  • A panel of 13 monoclonal antibodies to avian lipoprotein lipase (LPL) was screened for inhibition of LPL binding to primary avian adipocytes

  • In solid phase assays with heparan sulfate and heparin immobilized on enzyme-linked immunosorbent assay (ELISA) plates, monoclonal antibody to chicken adipose lipase (xCAL) 3-6a inhibited the binding of LPL to both heparin and heparan sulfate (Fig. 3)

  • We have generated a monoclonal antibody against avian LPL that inhibits binding of LPL to adipocytes (Fig. 1 and Fig. 2)

Read more

Summary

Introduction

A panel of 13 monoclonal antibodies to avian lipoprotein lipase (LPL) was screened for inhibition of LPL binding to primary avian adipocytes. 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. Lipoprotein lipase (LPL), a key enzyme in lipid metabolism, catalyzes the hydrolysis of triglycerides in very low density lipoproteins and chylomicrons [1, 2] This catalysis takes place at the luminal surface of the capillary endothelium where LPL is anchored by heparan sulfate chains [3, 4]. Identification of the region(s) of LPL involved in heparan sulfate binding will lead to an increased understanding of how the interaction of LPL with the capillary endothelium and liver parenchymal cells affects the biological activity and metabolism of the enzyme.

Methods
Results
Conclusion

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.