Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

Mart Reimund,Mikael Larsson,Oleg Kovrov,Sergo Kasvandik,Gunilla Olivecrona,Aivar Lookene

doi:10.1074/jbc.m114.634626

Abstract

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.

Highlights

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is crucial for the transport and localization of lipoprotein lipase (LPL) at the vascular endothelium
What is the contribution of the two domains of GPIHPB1 to the interaction? Do the domains of GPIHBP1 act as two independent binding sites or as two regions of one binding site? Is the N-terminal domain just needed for tethering LPL to the more specific interaction with the Ly6 domain or does the N-terminal domain play a more specific role on its own? Can kinetic studies performed in vitro explain why LPL preferentially binds to GPIHBP1 and not to other polyanions like heparan sulfate? To address these questions, we have investigated the mechanism of the interaction of LPL with GPIHBP1 using surface plasmon resonance (SPR), fluorescence anisotropy, enzyme activity measurements, and a combination of chemical cross-linking with mass spectrometry
In the present study we demonstrate that LPL binds to two distinct binding sites on GPIHBP1: one within the acidic N-terminal domain and one within the Ly6 domain

Summary

Background

GPIHBP1 is crucial for the transport and localization of lipoprotein lipase (LPL) at the vascular endothelium. We provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. The mutation Q115P in the Ly6 domain of human GPIHBP1, corresponding to Q114P in the mouse sequence, is the first discovered and well characterized example [6] Both of these mutants were unable to bind LPL in cell culture experi-. The experiments were performed with mouse GPIHBP1, the Ly6 domain mutant GPIHBP1 Q114P (mouse sequence) [6], the Ly6 domain (GPIHBP1 lacking the N-terminal domain), synthetic peptides corresponding to the N-terminal domain of GPIHBP1 from mouse, human, or bovine sequence (N-terminal peptide), and with bovine LPL

Experimental Procedures

Results

Discussion