2 and and3), 3), and this binding was inhibited with heparin ( Fig. In contrast, WT LPL bound avidly to GPIHBP1 ( Figs. 2) or immunofluorescence microscopy ( Fig. Neither LPL-C418Y nor LPL-E421K bound to GPIHBP1, as judged by Western blot ( Fig. Monoclonal antibody 5F9 was used as the capture antibody LPL binding was detected with biotinylated antibody 5D2. The concentration of LPL in each fraction was determined with an ELISA ( 15). ( B) Elution profiles of WT LPL, LPL-C418Y, and LPL-E421K from a heparin–Sepharose column with a linear NaCl gradient. In control experiments, I194T and S132G mutations inhibited LPL catalytic activity, as previously reported ( 20, 21). ( A) Specific activity of WT LPL, LPL-C418Y, and LPL-E421K. Here, we identified two such LPL mutations, thereby uncovering a potential mechanism for chylomicronemia and gaining insights into LPL sequences required for binding GPIHBP1.Įffect of LPL missense mutations on enzymatic activity and binding to heparin. In the current study, we postulated the existence of a complementary class of mutations: LPL missense mutations that would prevent binding to GPIHBP1. In each case, these mutations abolished GPIHBP1’s capacity to bind LPL. GPIHBP1 missense mutations were recently shown to cause chylomicronemia in humans ( 12 – 14). An absence of GPIHBP1 abolishes the entry of LPL into capillaries, causing severe chylomicronemia and an accumulation of catalytically active LPL in the interstitial spaces ( 11). LPL is synthesized by myocytes and adipocytes and secreted into the interstitial spaces, but is then transported to the capillary lumen by GPIHBP1, a GPI-anchored protein of endothelial cells ( 10, 11). To hydrolyze the triglycerides within plasma lipoproteins, LPL must be located inside capillaries. (A more detailed description of the findings of the earlier publications is found in the SI Text.) The C418Y and E421K mutations were mysterious because neither mutation had significant effects on catalytic activity ( 7, 8). For example, two LPL missense mutations, C418Y and E421K, were identified in patients with severe chylomicronemia ( 7, 8) but are located in the carboxyl terminus of LPL, distant from the aminoterminal catalytic domain and downstream from carboxyl-terminal sequences implicated in binding lipid substrates ( 9). In a few instances, the importance and functional relevance of some mutations identified in patients with severe chylomicronemia has remained mysterious. Some mutations abolished catalytic activity, whereas others blocked LPL secretion or interfered with the formation of stable homodimers. After the cDNA for LPL was cloned, many LPL mutations causing chylomicronemia were identified ( 1, 5, 6), the majority of which were missense mutations in the aminoterminal catalytic domain. More than 50 years ago, Havel and Gordon ( 4) showed that a deficiency of LPL causes severe hypertriglyceridemia (i.e., chylomicronemia). Lipoprotein lipase (LPL) is a homodimeric enzyme required for the lipolytic processing of triglycerid-rich lipoproteins (chylomicrons and very low density lipoproteins) ( 1 – 3). Together, these data define a mechanism by which LPL mutations could elicit disease and provide insights into LPL sequences required for binding to GPIHBP1. 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. 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. These findings suggest that sequences downstream from LPL's principal heparin-binding domain (amino acids 403–407) are important for GPIHBP1 binding. Both mutations abolish LPL transport across endothelial cells by GPIHBP1. 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. Any such mutation would provide insights into LPL sequences required for GPIHBP1 binding. We hypothesized that some cases of chylomicronemia might be caused by LPL mutations that interfere with LPL's ability to bind to GPIHBP1. Earlier studies have established that chylomicronemia can be caused by LPL mutations that interfere with catalytic activity. 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). GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells, shuttles lipoprotein lipase (LPL) from subendothelial spaces to the capillary lumen.
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