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Apolipoprotein A-V N-terminal Domain Lipid Interaction Properties in Vitro Explain the Hypertriglyceridemic Phenotype Associated with Natural Truncation Mutants

2009; Elsevier BV; Volume: 284; Issue: 48 Linguagem: Inglês

10.1074/jbc.m109.040972

1083-351X

Kasuen Mauldin, Vincent Raussens, Trudy M. Forte, Robert O. Ryan,

Peroxisome Proliferator-Activated Receptors

The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1–146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1–146) forms reconstituted high density lipoproteins 15–17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1–146) yielded an α-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1–146) α-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1–146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1–146) α-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1–146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1–146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects. The N-terminal 146 residues of apolipoprotein (apo) A-V adopt a helix bundle conformation in the absence of lipid. Because similarly sized truncation mutants in human subjects correlate with severe hypertriglyceridemia, the lipid binding properties of apoA-V(1–146) were studied. Upon incubation with phospholipid in vitro, apoA-V(1–146) forms reconstituted high density lipoproteins 15–17 nm in diameter. Far UV circular dichroism spectroscopy analyses of lipid-bound apoA-V(1–146) yielded an α-helix secondary structure content of 60%. Fourier transformed infrared spectroscopy analysis revealed that apoA-V(1–146) α-helix segments align perpendicular with respect to particle phospholipid fatty acyl chains. Fluorescence spectroscopy of single Trp variant apoA-V(1–146) indicates that lipid interaction is accompanied by a conformational change. The data are consistent with a model wherein apoA-V(1–146) α-helices circumscribe the perimeter of a disk-shaped bilayer. The ability of apoA-V(1–146) to solubilize dimyristoylphosphatidylcholine vesicles at a rate faster than full-length apoA-V suggests that N- and C-terminal interactions in the full-length protein modulate its lipid binding properties. Preferential association of apoA-V(1–146) with murine plasma HDL, but not with VLDL, suggests that particle size is a determinant of its lipoprotein binding specificity. It may be concluded that defective lipoprotein binding of truncated apoA-V contributes to the hypertriglyceridemia phenotype associated with truncation mutations in human subjects. INTRODUCTIONThe helix bundle motif is a common molecular architecture in proteins (1.Kamtekar S. Hecht M.H. FASEB J. 1995; 9: 1013-1022Crossref PubMed Scopus (103) Google Scholar). Exchangeable apolipoproteins (apo) 4The abbreviations used are: apoapolipoproteinHTGhypertriglyceridemiaDMPCdimyristoylphosphatidylcholineNTN terminusCTC terminusHDLhigh density lipidATR-FTIRattenuated total reflectance Fourier transformed infrared spectroscopyVLDLvery low density lipidPL-Cphospholipase CTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineTGtriacylglycerol. are known to adopt this conformation, which supports their dual existence in alternate lipid-free and lipid-bound states. Classic examples of the helix bundle structure include the N-terminal (NT) domains of apoE (2.Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (594) Google Scholar) and apoA-I (3.Ajees A.A. Anantharamaiah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar) as well as apolipophorin III (4.Breiter D.R. Kanost M.R. Benning M.M. Wesenberg G. Law J.H. Wells M.A. Rayment I. Holden H.M. Biochemistry. 1991; 30: 603-608Crossref PubMed Scopus (231) Google Scholar). In the case of apoE and apoA-I, the helix bundle motifs are present within the context of a larger protein structure. In each of these examples the bundle exists as an up-and-down series of amphipathic α-helices wherein the hydrophobic face of each helical segment orients toward the interior of the bundle. At the same time, the polar face of the amphipathic helices is directed toward the exterior of the bundle. In this way the globular structure is stabilized by hydrophobic helix-helix interactions and is conferred with water solubility through projection of polar and charged amino acid side chains toward the aqueous milieu. Upon interaction with lipid surfaces, the helix bundle is postulated to unfurl, adopting an extended open conformation that promotes interaction between the hydrophobic faces of amphipathic helices and the lipid surface. Essentially, lipid binding of helix bundle apolipoproteins substitutes helix-helix contacts in the bundle for helix-lipid contacts that stabilize the lipid-bound state.In 2001 a new apolipoprotein, termed apoA-V, was reported that profoundly affects plasma TG levels (5.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. Science. 2001; 294: 169-173Crossref PubMed Scopus (794) Google Scholar, 6.van der Vliet H.N. Sammels M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Structural studies revealed that apoA-V is a two-domain protein (7.Beckstead J.A. Wong K. Gupta V. Wan C.P. Cook V.R. Weinberg R.B. Weers P.M. Ryan R.O. J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and that its N-terminal 146 residues adopt a helix bundle structure in the absence of lipid (8.Wong K. Beckstead J.A. Lee D. Weers P.M. Guigard E. Kay C.M. Ryan R.O. Biochemistry. 2008; 47: 8768-8774Crossref PubMed Scopus (13) Google Scholar). Truncated apoA-V proteins in this size range have been reported in human subjects with severe hypertriglyceridemia (HTG) (9.Marçais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. Durlach V. Moulin P. J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (139) Google Scholar, 10.Priore Oliva C. Pisciotta L. Li Volti G. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (164) Google Scholar). One of these truncation mutants, Q139X apoA-V, does not associate with VLDL or HDL in circulation (9.Marçais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. Durlach V. Moulin P. J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (139) Google Scholar). By contrast, full-length apoA-V is found on both of these lipoprotein classes in normolipidemic subjects (11.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (180) Google Scholar). Insofar as these individuals had no other common mutations known to cause HTG, it is likely that a lipid-binding defect in truncated apoA-V is associated with the HTG phenotype. To address this mechanistically, the lipid interaction properties of recombinant apoA-V(1–146) were investigated. The results obtained provide a molecular explanation for the correlation between naturally occurring C-terminal (CT) truncations in apoA-V and HTG.DISCUSSIONExchangeable apolipoproteins can transfer among lipoproteins in plasma and, in the process, likely exist in a lipid-poor state. The helix bundle motif is postulated to facilitate this exchange by promoting apolipoprotein solubility in both polar and nonpolar environments. The size of potential lipid substrate particles is an important factor regulating apolipoprotein transfer in the circulation (25.Connelly P.W. Kuksis A. Biochim. Biophys. Acta. 1981; 666: 80-89Crossref PubMed Scopus (28) Google Scholar). ApoA-V is an exchangeable apolipoprotein that in humans, is found on chylomicrons, VLDL, and HDL (11.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (180) Google Scholar). Presumably, apoA-V transfers among the different lipoprotein populations, as proposed by Nelbach et al. (21.Nelbach L. Shu X. Konrad R.J. Ryan R.O. Forte T.M. J. Lipid Res. 2008; 49: 572-580Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The NT domain of apoA-V adopts a helix bundle conformation in the absence of lipid, and this may facilitate exchange between lipoprotein particles. In the present studies, we characterized the ability of this domain to bind to particles of various lipid compositions and sizes, including DMPC vesicles, modified LDL, and HDL and VLDL from apoa5−/− mice, in an effort to gain insight into its intrinsic lipid binding properties.Compared with other well studied exchangeable apolipoproteins, full-length apoA-V is unique in that it is not soluble at neutral pH in a lipid-free state (6.van der Vliet H.N. Sammels M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). When bound to lipid, however, apoA-V is soluble at physiological pH. This suggests that during transfer between lipoprotein particles in circulation, apoA-V may exist in a lipid-poor rather than lipid-free state. The ability of apoA-V(1–146) to form discoidal complexes with phospholipid in the size range of nascent HDL particles may be physiologically relevant because apoA-V exchange between lipoprotein particles in circulation may require transient existence in a lipid core-depleted particle.Upon lipid interaction, it appears that apoA-V(1–146) undergoes a conformational change, as judged by an increase in α-helix secondary structure content and altered solvent exposure of reporter Trp residues. The increase in helix content upon lipid interaction is similar to that of other exchangeable apolipoproteins, such as apoA-I (26.Wald J.H. Krul E.S. Jonas A. J. Biol. Chem. 1990; 265: 20037-20043Abstract Full Text PDF PubMed Google Scholar). The amphipathic helix bundle motif in the lipid-free state adopts a globular conformation wherein the hydrophobic faces of its helices orient toward the center of the bundle (27.Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). Upon lipid association, the protein is predicted to adopt an open conformation, where the hydrophobic faces of its amphipathic helices interact directly with the lipid surface (27.Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). Linear infrared dichroism experiments of apoA-V(1–146)·DMPC disks are consistent with a model wherein the helices orient perpendicular with respect to the DMPC bilayer fatty acyl chains. This indicates that, in the lipid-bound state, apoA-V(1–146) adopts a belt-like conformation around the perimeter of the particle, as described for other exchangeable apolipoproteins (28.Raussens V. Narayanaswami V. Goormaghtigh E. Ryan R.O. Ruysschaert J.M. J. Biol. Chem. 1995; 270: 12542-12547Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 29.Raussens V. Fisher C.A. Goormaghtigh E. Ryan R.O. Ruysschaert J.M. J. Biol. Chem. 1998; 273: 25825-25830Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 30.Raussens V. Drury J. Forte T.M. Choy N. Goormaghtigh E. Ruysschaert J.M. Narayanaswami V. Biochem. J. 2005; 387: 747-754Crossref PubMed Scopus (29) Google Scholar, 31.Narayanaswami V. Maiorano J.N. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. J. Biol. Chem. 2004; 279: 14273-14279Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 32.Martin D.D. Budamagunta M.S. Ryan R.O. Voss J.C. Oda M.N. J. Biol. Chem. 2006; 281: 20418-20426Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 33.Silva R.A. Schneeweis L.A. Krishnan S.C. Zhang X. Axelsen P.H. Davidson W.S. J. Biol. Chem. 2007; 282: 9713-9721Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar).The lipid-induced conformational change in apoA-V(1–146) was studied using a panel of single Trp variants. Each single Trp variant reported on a specific region within apoA-V(1–146). Certain Trp residues were predicted to reside in a linker region between amphipathic helices or on the polar face of an amphipathic helix, whereas the others were predicted to reside on the nonpolar face of amphipathic helices. In keeping with these predictions, only variants with Trp predicted to reside on the hydrophobic face of amphipathic helices showed a blue shift in the wavelength of maximum fluorescence emission between alternate lipid-free and lipid-bound states. This suggests that, when presented with a suitable lipid surface, the helix bundle opens, exposing the bundle interior.To further characterize conformational adaptations in apoA-V(1–146), the relative exposure of various regions within the NT domain were investigated as a function of lipid binding. In Trp fluorescence quenching studies, it was observed that single Trp apoA-V(1–146) variants with their Trp predicted to be on the nonpolar face of amphipathic helices gave rise to the lowest Ksv values, suggesting that these regions of the protein maintain close contact with the lipid surface. For example, Trp97 is predicted to reside in a linker region between two amphipathic helices. Consistent with this, Trp97 apoA-V(1–146) fluorescence emission was highly quenched by acrylamide but less so by KI. This could be due to electrostatic repulsion of KI by negatively charged residues located near Trp97.The ability of apoA-V(1–146) to initiate contact with lipid surfaces is suggested by phospholipid vesicle solubilization studies comparing truncated and full-length apoA-V. The CT domain of apoA-V has been previously shown to avidly bind lipid (7.Beckstead J.A. Wong K. Gupta V. Wan C.P. Cook V.R. Weinberg R.B. Weers P.M. Ryan R.O. J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Interestingly, despite the absence of the CT domain, apoA-V(1–146) solubilizes DMPC at a faster rate than full-length apoA-V. This suggests that N- and C-terminal domain interactions in the intact protein modulate the lipid binding properties of apoA-V. This may be similar to interactions in apoA-I. In this apolipoprotein, the CT domain initiates lipid-binding, whereas the NT helix bundle opens up to stabilize the lipid-associated state (34.Ji Y. Jonas A. J. Biol. Chem. 1995; 270: 11290-11297Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar).Although DMPC solubilization assays characterize apolipoprotein-induced phospholipid vesicle disruption and reorganization, PL-C-modified LDL provides a means to assess binding to spherical lipoprotein substrates. PL-C activity generates apolipoprotein-binding sites by hydrolyzing phosphatidylcholine moieties present in the surface monolayer (20.Liu H. Scraba D.G. Ryan R.O. FEBS Lett. 1993; 316: 27-33Crossref PubMed Scopus (78) Google Scholar). Conversion of phosphatidylcholine into diacylglycerol destabilizes the lipoprotein particle and promotes aggregation (35.Suits A.G. Chait A. Aviram M. Heinecke J.W. Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 2713-2717Crossref PubMed Scopus (160) Google Scholar). Apolipoproteins protect LDL against PL-C-induced aggregation by forming a stable binding interaction with the modified particles (20.Liu H. Scraba D.G. Ryan R.O. FEBS Lett. 1993; 316: 27-33Crossref PubMed Scopus (78) Google Scholar). ApoA-V(1–146) binding to PL-C-treated LDL was intermediate with respect to other apolipoproteins examined. This finding is consistent with the stability properties of these apolipoproteins. At physiological pH apoA-V(1–146) is less stable than apoE3 NT (guanidine HCl denaturation midpoint of 2.0 m versus 2.5 m, respectively) (8.Wong K. Beckstead J.A. Lee D. Weers P.M. Guigard E. Kay C.M. Ryan R.O. Biochemistry. 2008; 47: 8768-8774Crossref PubMed Scopus (13) Google Scholar, 36.Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar) yet is more stable than apoA-I (1 m guanidine HCl denaturation midpoint) (37.Reijngoud D.J. Phillips M.C. Biochemistry. 1982; 21: 2969-2976Crossref PubMed Scopus (68) Google Scholar). Thus, it appears that the intrinsic stability of helix bundle apolipoproteins in solution correlates directly with the ability to bind newly created sites on a spherical lipoprotein substrate (8.Wong K. Beckstead J.A. Lee D. Weers P.M. Guigard E. Kay C.M. Ryan R.O. Biochemistry. 2008; 47: 8768-8774Crossref PubMed Scopus (13) Google Scholar).The ability of apoA-V(1–146) to associate with physiologically relevant lipoproteins was assessed using VLDL and HDL isolated from apoa5 knock-out mice. Importantly, apoA-V(1–146) failed to associate with VLDL and associated sparingly with HDL. This finding is intriguing in light of the report that, unlike full-length apoA-V, apoA-V(1–146) also fails to associate with intracellular lipid droplets (38.Shu X. Ryan R.O. Forte T.M. J. Lipid Res. 2008; 49: 1670-1676Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). In considering the sizes of various lipid substrates, VLDL and lipid droplets are considerably larger than HDL or other lipid particles employed in this study. The data indicate that apoA-V(1–146) can associate with smaller lipid substrate particles to a limited degree but apparently lacks the ability to interact with larger lipid particles. One explanation could be related to the tighter packing of phospholipid molecules on the surface of larger particles because of their decreased radius of curvature, as compared with smaller particles (39.Tajima S. Yokoyama S. Yamamoto A. J. Biol. Chem. 1983; 258: 10073-10082Abstract Full Text PDF PubMed Google Scholar, 40.Wetterau J.R. Jonas A. J. Biol. Chem. 1982; 257: 10961-10966Abstract Full Text PDF PubMed Google Scholar). Tighter packing of phospholipid polar head groups enveloping a lipid core could interfere with the ability of apoA-V(1–146) to access hydrophobic surfaces and initiate binding. This aspect is obviated in the case of PL-C-modified LDL but not in the binding experiments with VLDL and HDL. In the latter case, differences in surface lipid composition and/or protein content could also influence binding of apoA-V(1–146). Regardless, it is apparent that, with natural lipoprotein substrates in vitro, apoA-V(1–146) binding is defective. Because this is not due to an intrinsic inability to bind lipid, it suggests that the CT of apoA-V modulates the lipid interaction properties of the NT domain.Naturally occurring apoA-V truncations, including Q139X (9.Marçais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. Durlach V. Moulin P. J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (139) Google Scholar), Q148X (10.Priore Oliva C. Pisciotta L. Li Volti G. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (164) Google Scholar), and Q97X (41.Priore Oliva C. Tarugi P. Calandra S. Pisciotta L. Bellocchio A. Bertolini S. Guardamagna O. Schaap F.G. Atherosclerosis. 2006; 188: 215-217Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), have been reported in human subjects and are associated with severe hypertriglyceridemia. The distribution of apoA-V in ultracentrifugally isolated lipoprotein classes was evaluated in several carriers of the Q139X truncation mutation and demonstrated that the truncated form of the protein does not associate with plasma lipoproteins and is found only in the lipid-poor d > 1.21 g/ml fraction. Because the Q139X apoA-V mutation nomenclature includes the 23-amino acid signal peptide, the mature protein is actually 116 amino acids in length. Although apoA-V(1–146) is longer than these natural mutants, binding studies with natural lipoproteins recapitulate observations in human plasma of individuals carrying these mutant forms of apoA-V.The present findings provide a potential explanation for the HTG observed in patients with truncated apoA-V. The lack of a CT domain alters lipid binding activity such that truncated apoA-V fails to effectively bind to circulating lipoproteins, particularly TG-rich particles. Current hypotheses suggest apoA-V interactions with heparan-sulfate proteoglycans (42.Lookene A. Beckstead J.A. Nilsson S. Olivecrona G. Ryan R.O. J. Biol. Chem. 2005; 280: 25383-25387Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 43.Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) and/or glycosyl phosphatidylinositol high density lipoprotein-binding protein-1 (44.Beigneux A.P. Davies B.S. Gin P. Weinstein M.M. Farber E. Qiao X. Peale F. Bunting S. Walzem R.L. Wong J.S. Blaner W.S. Ding Z.M. Melford K. Wongsiriroj N. Shu X. de Sauvage F. Ryan R.O. Fong L.G. Bensadoun A. Young S.G. Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) indirectly enhances lipoprotein lipase activity to facilitate hydrolysis of VLDL- associated TG. The data from Nilsson et al. (45.Nilsson S.K. Lookene A. Beckstead J.A. Gliemann J. Ryan R.O. Olivecrona G. Biochemistry. 2007; 46: 3896-3904Crossref PubMed Scopus (89) Google Scholar) indicate apoA-V also serves as a ligand for endocytic receptors of the LDL receptor family, where it is possible that apoA-V may have an important role in clearance of VLDL remnants. The putative binding site on apoA-V for lipoprotein lipase activity enhancement and cell surface molecule interactions resides within the CT domain of the protein. The absence of this binding site most likely contributes to defective hydrolysis and clearance of TG-rich lipoproteins. In any case, association of apoA-V with TG-rich lipoproteins is presumably required for manifestation of these effects. If a CT truncated apoA-V is unable to bind larger, TG-rich lipoproteins, then the resulting apoA-V-deficient particles could potentially have an increased plasma residence time, contributing to HTG. Thus, it may be that defective lipid binding arising from the lack of a CT domain precludes binding to circulating lipoproteins, thereby preventing potential TG lowering effects attributed to full-length apoA-V. INTRODUCTIONThe helix bundle motif is a common molecular architecture in proteins (1.Kamtekar S. Hecht M.H. FASEB J. 1995; 9: 1013-1022Crossref PubMed Scopus (103) Google Scholar). Exchangeable apolipoproteins (apo) 4The abbreviations used are: apoapolipoproteinHTGhypertriglyceridemiaDMPCdimyristoylphosphatidylcholineNTN terminusCTC terminusHDLhigh density lipidATR-FTIRattenuated total reflectance Fourier transformed infrared spectroscopyVLDLvery low density lipidPL-Cphospholipase CTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineTGtriacylglycerol. are known to adopt this conformation, which supports their dual existence in alternate lipid-free and lipid-bound states. Classic examples of the helix bundle structure include the N-terminal (NT) domains of apoE (2.Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (594) Google Scholar) and apoA-I (3.Ajees A.A. Anantharamaiah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar) as well as apolipophorin III (4.Breiter D.R. Kanost M.R. Benning M.M. Wesenberg G. Law J.H. Wells M.A. Rayment I. Holden H.M. Biochemistry. 1991; 30: 603-608Crossref PubMed Scopus (231) Google Scholar). In the case of apoE and apoA-I, the helix bundle motifs are present within the context of a larger protein structure. In each of these examples the bundle exists as an up-and-down series of amphipathic α-helices wherein the hydrophobic face of each helical segment orients toward the interior of the bundle. At the same time, the polar face of the amphipathic helices is directed toward the exterior of the bundle. In this way the globular structure is stabilized by hydrophobic helix-helix interactions and is conferred with water solubility through projection of polar and charged amino acid side chains toward the aqueous milieu. Upon interaction with lipid surfaces, the helix bundle is postulated to unfurl, adopting an extended open conformation that promotes interaction between the hydrophobic faces of amphipathic helices and the lipid surface. Essentially, lipid binding of helix bundle apolipoproteins substitutes helix-helix contacts in the bundle for helix-lipid contacts that stabilize the lipid-bound state.In 2001 a new apolipoprotein, termed apoA-V, was reported that profoundly affects plasma TG levels (5.Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. Science. 2001; 294: 169-173Crossref PubMed Scopus (794) Google Scholar, 6.van der Vliet H.N. Sammels M.G. Leegwater A.C. Levels J.H. Reitsma P.H. Boers W. Chamuleau R.A. J. Biol. Chem. 2001; 276: 44512-44520Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Structural studies revealed that apoA-V is a two-domain protein (7.Beckstead J.A. Wong K. Gupta V. Wan C.P. Cook V.R. Weinberg R.B. Weers P.M. Ryan R.O. J. Biol. Chem. 2007; 282: 15484-15489Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and that its N-terminal 146 residues adopt a helix bundle structure in the absence of lipid (8.Wong K. Beckstead J.A. Lee D. Weers P.M. Guigard E. Kay C.M. Ryan R.O. Biochemistry. 2008; 47: 8768-8774Crossref PubMed Scopus (13) Google Scholar). Truncated apoA-V proteins in this size range have been reported in human subjects with severe hypertriglyceridemia (HTG) (9.Marçais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. Durlach V. Moulin P. J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (139) Google Scholar, 10.Priore Oliva C. Pisciotta L. Li Volti G. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (164) Google Scholar). One of these truncation mutants, Q139X apoA-V, does not associate with VLDL or HDL in circulation (9.Marçais C. Verges B. Charrière S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. Fruchart-Najib J. Fruchart J.C. Durlach V. Moulin P. J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (139) Google Scholar). By contrast, full-length apoA-V is found on both of these lipoprotein classes in normolipidemic subjects (11.O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (180) Google Scholar). Insofar as these individuals had no other common mutations known to cause HTG, it is likely that a lipid-binding defect in truncated apoA-V is associated with the HTG phenotype. To address this mechanistically, the lipid interaction properties of recombinant apoA-V(1–146) were investigated. The results obtained provide a molecular explanation for the correlation between naturally occurring C-terminal (CT) truncations in apoA-V and HTG.