Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.

GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, shuttles lipoprotein lipase (LPL) from subendothelial spaces to the capillary lumen. An absence of GPIHBP1 prevents the entry of LPL into capillaries, blocking LPL-mediated triglyceride hydrolysis and leading to markedly elevated triglyceride levels in the plasma (i.e., chylomicronemia). Earlier studies have established that chylomicronemia can be caused by LPL mutations that interfere with catalytic activity. We hypothesized that some cases of chylomicronemia might be caused by LPL mutations that interfere with LPL's ability to bind to GPIHBP1. Any such mutation would provide insights into LPL sequences required for GPIHBP1 binding. Here, we report that two LPL missense mutations initially identified in patients with chylomicronemia, C418Y and E421K, abolish LPL's ability to bind to GPIHBP1 without interfering with LPL catalytic activity or binding to heparin. Both mutations abolish LPL transport across endothelial cells by GPIHBP1. These findings suggest that sequences downstream from LPL's principal heparin-binding domain (amino acids 403–407) are important for GPIHBP1 binding. In support of this idea, a chicken LPL (cLPL)–specific monoclonal antibody, xCAL 1–11 (epitope, cLPL amino acids 416–435), blocks cLPL binding to GPIHBP1 but not to heparin. Also, changing cLPL residues 421 to 425, 426 to 430, and 431 to 435 to alanine blocks cLPL binding to GPIHBP1 without inhibiting catalytic activity. Together, these data define a mechanism by which LPL mutations could elicit disease and provide insights into LPL sequences required for binding to GPIHBP1.

Black and white are opposites as are oxidation and reduction. Performing an oxidation, for example, of a sec-alcohol and a reduction of the corresponding ketone in the same vessel without separation of the reagents seems to be an impossible task. Here we show that oxidative cofactor recycling of NADP (+) and reductive regeneration of NADH can be performed simultaneously in the same compartment without significant interference. Regeneration cycles can be run in opposing directions beside each other enabling one-pot transformation of racemic alcohols to one enantiomer via concurrent enantioselective oxidation and asymmetric reduction employing defined alcohol dehydrogenases with opposite stereo- and cofactor-preference. Thus, by careful selection of appropriate enzymes, NADH recycling can be performed in the presence of NADP (+) recycling to achieve overall, for example, deracemisation of sec-alcohols or stereoinversion representing a possible concept for a "green" equivalent to the chemical-intensive Mitsunobu inversion.

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 ( GPIHBP1)-defi cient mice. GPI-HBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These fi ndings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating "a platform for lipolysis." More recent studies have identifi ed a second and more intriguing role for GPIHBP1-picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPI-HBP1-LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1's ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1. The outline for the lipolytic processing of lipoproteins has been established for decades ( 1, 2 ). Triglyceride-rich Abbreviations: ANGPTL, angiopoietin-like protein; BODIPY, borondipyrromethane; CHO, Chinese hamster ovary; cLPL, chicken lipoprotein lipase; DiI, 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; EL, endothelial lipase; GPI, glycosylphosphatidylinositol; GPIHBP1, glycosylphosphatidylinositol -anchored high density lipoprotein-binding protein 1; HSPG, heparan sulfate proteoglycan; Ly6, Lymphocyte Antigen 6; PIPLC, phosphatidylinositol-specifi c phospholipase C; SR-BI, scavenger receptor class B, type 1. This work was supported by a Scientist Development Award from the American Heart Association, National Offi ce (to A.P.B.), R01 HL094732 (to A.P.B.), P01 HL090553 (to S.G.Y.), and R01 HL087228 (to S.G.Y.). The authors have declared that no confl ict of interest exists. Manuscript received 28 June 2011 and in revised form

Objective-To define the ability of GPIHBP1 to bind other lipase family members and other apolipoproteins (apos) and lipoproteins. Methods and Results-GPIHBP1, a GPI-anchored lymphocyte antigen (Ly)6 protein of capillary endothelial cells, binds lipoprotein lipase (LPL) avidly, but its ability to bind related lipase family members has never been evaluated. As judged by cell-based and cell-free binding assays, LPL binds to GPIHBP1, but other members of the lipase family do not. We also examined the binding of apoAV-phospholipid disks to GPIHBP1. ApoAV binds avidly to GPIHBP1-transfected cells; this binding requires GPIHBP1's amino-terminal acidic domain and is independent of its cysteine-rich Ly6 domain (the latter domain is essential for LPL binding). GPIHBP1-transfected cells did not bind high-density lipoprotein. Chylomicrons bind avidly to GPIHBP1-transfected Chinese hamster ovary cells, but this binding is dependent on GPIHBP1's ability to bind LPL within the cell culture medium. Conclusion-GPIHBP1 binds LPL but does not bind other lipase family members. GPIHBP1 binds apoAV but does not bind apoAI or high-density lipoprotein. mice display severe hypertriglyceridemia, even on a low-fat diet, with plasma triglyceride levels of 3000 to 6000 mg/ dL. 1,3,4 Subsequent studies proved that GPIHBP1 is responsible for transporting lipoprotein lipase (LPL) into the lumen of capillaries. 5 In the absence of GPIHBP1, LPL is mislocalized to the interstitial spaces 5 and, therefore, cannot hydrolyze triglycerides in plasma lipoproteins.GPIHBP1 contains a single lymphocyte antigen 6 (Ly6) domain containing 10 cysteines, which are arranged in a characteristic spacing pattern. 6 Mutation of any of these cysteines abolishes GPIHBP1's capacity to bind LPL. 7 A second noteworthy feature of GPIHBP1-and the one that distinguishes it from other Ly6 protein family members-is a striking acidic domain at its amino terminus. This domain is highly enriched in negatively charged amino acids (21 of 26 consecutive residues in human GPIHBP1 are aspartate or glutamate). Mutating the acidic domain abolishes GPIHBP1's ability to bind LPL. 8 The binding of LPL to GPIHBP1 appears to depend on a positively charged heparin-binding domain in LPL. 9,10 Mutating the positively charged amino acids in LPL's principal heparin-binding domain eliminates its ability to bind to GPIHBP1. 8 Two other lipase family members, hepatic lipase (HL) and endothelial lipase (EL), also contain heparinbinding domains. [11][12][13][14] Whether these other heparin-binding lipases also bind to GPIHBP1 has never been assessed.Transfection of Chinese hamster ovary (CHO) pgsA-745 cells with GPIHBP1 confers the ability to bind apolipoprotein (apo) AV-phospholipid disks. 1 ApoAV contains a strong heparinbinding domain, 15 In the current study, we sought to define binding preferences for GPIHBP1. We tested the ability of other lipase family members to bind to GPIHBP1 and assessed the structural features of GPIHBP1 required for apoAV binding. In addition, we investigated th...