Author: Herr Dr. Amoneit
Last saved by: Saritas, Sevim (Euro Fed Lipid)

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1
Aivar Lookene^1, Mart Reimund^1, Mikael Larsson^2,. Gunilla Olivecrona^2, Oleg Kovrov^1
^1 Department of Chemistry, Tallinn University of Technology, Tallinn 12618, Estonia
   ^2 Department of Medical Biosciences, Umeå University, SE-901 87 Umeå, Sweden
Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial membrane protein that transports lipoprotein lipase, the ratelimiting enzyme in the hydrolysis of plasma triglycerides, from subendothelium space to the endothelial lume. Mutations in the GPIHBP1 gene dramatically reduce the amount of the heparin-releasable lipoprotein lipase in plasma and cause massive hypertriglyceridemia.  In the present study we provide evidence that two principial parts of GPIHBP1, an acidic N-terminal domain and a central Ly6 domain, interact with lipoprotein lipase as two distinct binding sites. This conclusion is based on comparative binding studies performed with the N-terminal and the Ly6 domain of GPIHBP1, the wild type GPIHBP1 and GPIHBP1 mutant Q114P, using surface plasmon resonance, stabilization measurements and fluorescence anisotropy. While lipoprotein lipase and the N-terminal domain formed a tight but short life-time complex characterized with fast on- and off rates, the complex between lipoprotein lipase and the Ly6 domain formed slower and remained together for a longer time. Unlike the interaction of Ly6 domain with lipoprotein lipase, the interaction between lipoprotein lipase and the N-terminal domain was strongly salt-dependent. Heparin eluted lipoprotein lipase completely from the N-terminal binding site, partly from the wild type GPIHBP1 but was unable to elute the enzyme from the Ly6 domain. Lipoprotein lipase, when complexed with a synthetic peptide corresponding to the N-terminal domain of GPIHBP, retained its affinity for the Ly6 domain. Furthermore, only the fraction of lipoprotein lipase bound to the N-terminal domain was able to interact with lipoproteins.