Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

E-Chiang Lee,Urvi Desai,Gennady Gololobov,Seokjoo Hong,Xiao Feng,Xuan-Chuan Yu,Jason Gay,Nat Wilganowski,Cuihua Gao,Ling-Ling Du,Joan Chen,Yi Hu,Sharon Zhao,Laura Kirkpatrick,Matthias Schneider,Brian P Zambrowicz,Greg Landes,David R Powell,William K Sonnenburg

doi:10.1074/jbc.m807899200

E-Chiang Lee, Urvi Desai + Show 17 more

Open Access

https://doi.org/10.1074/jbc.m807899200

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Abstract

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln(29)-His(53), which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu(32)-His(55). We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3(-/-) mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.

Highlights

lipoprotein lipase (LPL) is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed [1,2,3]
Our results showed that monoclonal antibody (mAb) 14D12 only bound to the fusion protein containing mANGPTL4 amino acids Gln24–Met73 (Fig. 1A)
We found that mAb 14D12 did not bind with the flanking overlapping peptides (Fig. S3B), indicating that mANGPTL4 amino acids Leu49 through His53 and Arg34 through Asp39 are involved in binding with mAb 14D12

Summary

Introduction

LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed [1,2,3]. These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. To fine map the epitope of mAb 14D12, a series of 10 expression cassettes encoding overlapping His-tagged SUMO-mouse ANGPTL4 fusion proteins spanning amino acids Gln24–Ser93 were generated.

Results

Conclusion