An LPL–specific monoclonal antibody, 88B8, that abolishes the binding of LPL to GPIHBP1

Abstract

For more than 50 years, it has been known that LPL, a triglyceride hydrolase secreted by myocytes and adipocytes, is crucial for the intravascular processing of triglyceriderich lipoproteins (TRLs) (1-3). For most of that time, it was assumed that LPL was attached to the heparan-sulfate proteoglycans along the lumen of blood vessels (4), but how LPL reached the lumen of blood vessels was a stubborn mystery. Within the past few years, that mystery has been solved (5, 6). Glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1), a GPI-anchored protein of capillary endothelial cells, picks up freshly secreted LPL within the interstitial spaces and shuttles it across endothelial cells to the capillary lumen (7,8). In the absence of GPIHBP1, LPL remains in the interstitial spaces and never reaches the capillary lumen, resulting in an accumulation of plasma TRLs and extremely high plasma triglyceride levels ("chylomicronemia") (8). Recent studies showed that GPIHBP1 (and GPIHBP1-bound LPL) are also crucial for the margination of TRLs along the capillary lumen, allowing triglyceride hydrolysis to proceed (9).GPIHBP1 has two main structural features-an aminoterminal acidic domain and a cysteine-rich three-fingered "LU domain" (7, 10). Recent studies have shown that the Abstract LPL contains two principal domains: an aminoterminal catalytic domain (residues 1-297) and a carboxylterminal domain (residues 298-448) that is important for binding lipids and binding glycosylphosphatidylinositolanchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues 403-438) were crucial. Here, we tested the ability of LPLspecific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues 403-438) were not sufficient for 88B8 binding; upstream sequences (residues 298-400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPI-HBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

Angiopoietin-like 4 promotes the intracellular cleavage of lipoprotein lipase by PCSK3/furin in adipocytes

“…To examine whether LPL is cleaved in vivo in white adipose tissue, we performed Western blotting for LPL in human adipose tissue using antibodies directed against the N-or C-terminal portion of human LPL (27). Both antibodies gave rise to two bands, corresponding to full-length LPL (slightly above 50 kDa), and the N-terminal or C-terminal LPL cleavage fragment at around 30 kDa or 20 -25 kDa, respectively (Fig.…”

“…In all adipocyte models, incubation with dec-CMK resulted in the almost complete disappearance of N-terminal LPL in cell cul- and C-terminal (88b8) portion of hLPL. B, Western blotting of lysates of human adipose tissue treated with EndoH or PNGase, using two different antibodies against hLPL, 88b8, and 5D2 (27). C, Western blotting of lysates of human adipose tissue of different subjects enrolled in the MONDIAL study, using an antibody against hLPL (88b8).…”

“…Next, proteins were transferred onto a polyvinylidene difluoride membrane using the Transblot Turbo System (Bio-Rad). Membranes were probed with a goat anti-mouse LPL antibody (28), mouse anti-human LPL antibodies 88b8 and 5D2 (27), a goat anti-human LPL antibody (Y-20, Santa Cruz Biotechnology), a rabbit anti-mouse HSP90 antibody (Cell Signaling Technology, catalog no. 4874), a rat anti-mouse ANGPTL4 antibody (Kairos 142-2, Adipogen), a mouse anti-mouse ␤-TUBULIN (sc-5274, Santa Cruz Biotechnology), or a rabbit anti-mouse PCSK3 (Abcam, ab183495) at 1:5000 (mLPL), 1:2000 (HSP90), or 1:1000 (PCSK3, ANGPTL4, hLPL, and ␤-TUBULIN) dilutions.…”

GPIHBP1 expression in gliomas promotes utilization of lipoprotein-derived nutrients

“…Most of the LPL in peripheral tissues (e.g., heart or brown adipose tissue) is bound to GPIHBP1 on capillaries; consequently, LPL and GPIHBP1 colocalize in tissue sections (Young et al, 2011; Davies et al, 2010; Davies et al, 2012; Allan et al, 2017a; Fong et al, 2016; Allan et al, 2017b; Allan et al, 2016). We hypothesized that GPIHBP1-expressing endothelial cells of gliomas could capture LPL.…”

“…GPIHBP1 is expressed in the capillary endothelial cells of peripheral tissues, with particularly high levels of expression in heart and brown adipose tissue (Beigneux et al, 2007; Davies et al, 2010; Fong et al, 2016). Most of the LPL within those tissues is bound to GPIHBP1 on capillaries (Beigneux et al, 2007; Davies et al, 2010; Davies et al, 2012; Allan et al, 2017a; Fong et al, 2016; Allan et al, 2017b; Allan et al, 2016), and the processing of TRLs in these tissues is robust, generating fatty acid nutrients for nearby parenchymal cells (Fong et al, 2016; Jiang et al, 2014a; He et al, 2018a). By contrast, GPIHBP1 is absent from capillaries of the brain (Young et al, 2011; Davies et al, 2010; Olafsen et al, 2010), a tissue that depends on glucose for fuel (Mergenthaler et al, 2013).…”