Effects of Six APOA5 Variants, Identified in Patients With Severe Hypertriglyceridemia, on In Vitro Lipoprotein Lipase Activity and Receptor Binding

Abstract

Objective-The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. Methods and Results-We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -⌬139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (Ϫ25% [Pϭ0.005], Ϫ36% [PϽ0.0001], and Ϫ23% [Pϭ0.02]), respectively). ApoAV-C271, -X139, -X148, and ⌬139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. Key Words: apolipoprotein AV Ⅲ LDL-R family Ⅲ LR8 Ⅲ LRP1 Ⅲ HSPG-bound LPL P remature truncations of APOA5, Q139X, Q148X, and the IVS3ϩ3gϾc are associated with severe hypertriglyceridemia (HyperTG), 1-3 behaving as phenocopies of lipoprotein lipase (LPL) deficiency. These rare APOA5 variants do not always lead to a deficiency in circulating plasma apoAV, and carriers present with a range of apoAV levels (reviewed in 4 ). Kao et al 5 identified a common polymorphism G185C in a Taiwanese study which occurs at a minor allele frequency of 0.04 in controls but at a 6.3-fold higher frequency of 0.27 in hypertriglyceridemic patients (PϽ0.001). Conclusion-TheApoAV is present on chylomicrons, VLDL, and HDL, but not on IDL or LDL, suggesting that VLDL-containing apoAV is cleared before the lipolytic cascade. 6 One function of apoAV is to activate LPL, and Apoa5 knockout mice have 4-fold higher TG levels than wild type. [7][8][9] Whereas LPL transgenic mice can rescue Apoa5 knockout mice from HyperTG, this is not entirely reciprocal 8 suggesting that the effect of apoAV on plasma TG is dependent on heparinsulfate proteoglycan (HSPGs)-bound LPL. 7,8 In addition, apoAV-dependent TG catabolism acts by enhancing receptormediated endocytosis via members of the LDL-receptor (LDLR) family. 10,11 We have identified 3 novel APOA5 missense variants (E255G, G271C, H321L) in patients with TG levels Ͼ10 mmol/L. Although LPL and APOC2 variants had been excluded for the G271C carrier, the coding exons of LPL and APOC2 were sequenced in the 2 other probands. Because family studies were not possible, the APOA5 variants were expressed in vitro together with G185C, Q139X, Q148X. 1,2,5 In addition we designed a deletion construct ⌬139 to 147, to MethodsSee supplemental methods (available online at http://atvb.ahajournals. org) for full details. PatientsIn total 130 patients with TG levels Ͼ10 mmol/L were recruited into the study; seven patients from the UK, 28 patients from the Netherlands with LPL and APOC2 mutations excluded, and 95 p...

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

“…Even though apoA-V-LRP1 interactions were studied qualitatively, it is unlikely that major changes in receptor affi nity could have gone undetected with our ligand blot assay. Along these lines, in similar experiments, the human apoA-V mutants p.(Gln139-Leu147)del, p.Gly185-Cys, p.Glu255Gly, and p.His321Leu bound normally to the chicken LDLR family member LR8, whereas neither the N-terminally truncated apoA-V mutants, p.Gln139× and p.Gln148×, nor a variant prone to multimerization, p.Gly271Cys, were able to bind the avian receptor ( 42 ). On the other hand, the pair of basic residues R233/K234 contributes to LRP1 binding, as the double mutant R233E/ K234Q showed a 3-fold lower affi nity than wild-type apoA-V for the full-length human receptor ( 23 ).…”

“…Both free and DMPC-complexed apoA-V bind LRP1, SorLA/LR11, and sortilin ( 22 ), and impaired binding to LDLR family members might lead to hypertriglyceridemia ( 42 ). Somewhat unexpectedly in the light of these fi ndings, the three apoA-V mutants were shown to bind to LRP1 cluster II.…”

“…would compromise phospholipid binding, as demonstrated here for the p.Leu253Pro variant. The effects of apoA-V mutants on LPL activation appear to be highly variable depending on the affected residue ( 7,8,42,43 ). However, it is noteworthy that variant p.Leu253Pro not only did not activate LPL but rather inhibited apoA-V ( Fig.…”

“…I-Mutant2.0 also predicts decreased stability for this mutant ( ⌬ ⌬ G = Ϫ 0.67 kcal/mol), in contrast to an increase of almost the same magnitude for the p.Ala315-Val variant. Residue L253 is essentially buried at the interface between helices E and F in the 3D model of human The molecular mechanisms that result in severe hypertriglyceridemia in patients with APOA5 mutations remain poorly understood, and only one study to date has addressed the capacity of apoA-V mutants to interact with LDLR family members and activate LPL ( 42 ). In addition, a recent report focused on the role of residue G185 in apoA-V-mediated LPL stimulation ( 43 ).…”

APOA5 -1131T>C and APOC3 -455T>C polymorphisms are associated with an increased risk of coronary heart disease

“…APOA5 encodes APOA5, a protein consisting of 366 amino acids that is exclusively expressed in human liver tissue and enhances LPL activity (Zhou et al, 2013). The loss of LPL activity interferes with the ability of APOA5 to interact with lipids and lipoproteins, including TGs, VLDLs, and HDLs (Dorfmeister et al, 2008;Johansen et al, 2011). Furthermore, the elevation of plasma TGs is a known CHD risk factor, and APOA5 status constitutes a major risk factor owing to its activation of TG hydrolysis in the blood (Carey et al, 2010).…”