FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2136674557> ?p ?o ?g. }

• W2136674557 endingPage "38582" @default.
• W2136674557 startingPage "38576" @default.
• W2136674557 abstract "Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333–343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11–20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV. Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333–343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11–20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV. Human apolipoprotein (apo) 3The abbreviations used are: apo, apolipoprotein; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; STB, standard Tris buffer; WT, wild type.3The abbreviations used are: apo, apolipoprotein; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; STB, standard Tris buffer; WT, wild type. A-IV is a 46-kDa glycoprotein that is the largest member of the exchangeable apolipoprotein family. It is synthesized by enterocytes of the small intestine in response to lipid absorption and is secreted into circulation on the surface of chylomicrons. As chylomicrons undergo lipolysis in the plasma compartment, apoA-IV rapidly dissociates from their surface and thereafter circulates as a lipid-free protein and in association with high density lipoprotein (1.Green P.H. Glickman R.M. Riley J.W. Quinet E. J. Clin. Investig. 1980; 65: 911-919Crossref PubMed Scopus (257) Google Scholar). It has been proposed that apoA-IV evolved to play a role in chylomicron assembly or catabolism (2.Weinberg R.B. Anderson R.A. Cook V.R. Emmanuel F. Denefle P. Hermann M. Steinmetz A. J. Lipid Res. 2000; 41: 1410-1418Abstract Full Text Full Text PDF PubMed Google Scholar). However, several additional functions have been proposed, including inhibition of lipid oxidation (3.Qin X. Swertfeger D.K. Zheng S. Hui D.Y. Tso P. Am. J. Physiol. 1998; 274: H1836-H1840Crossref PubMed Google Scholar) and inflammatory processes (4.Vowinkel T. Mori M. Krieglstein C.F. Russell J. Saijo F. Bharwani S. Turnage R.H. Davidson W.S. Tso P. Granger D.N. Kalogeris T.J. J. Clin. Investig. 2004; 114: 260-269Crossref PubMed Scopus (136) Google Scholar), participation in reverse cholesterol transport (5.Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (275) Google Scholar), and regulation of food intake (6.Fujimoto K. Cardelli J.A. Tso P. Am. J. Physiol. 1992; 262: G1002-G1006Crossref PubMed Google Scholar). Because plasma apoA-IV can exist both as a component of plasma lipoproteins and as a lipid-free protein, it is possible that alternate conformations perform distinct functions. However, the structural features that mediate the conversion between these two states are largely unknown. ApoA-IV shares many features with the other exchangeable apolipoproteins, especially apoA-I and apoE. Indeed, intraexonic duplication of a primordial apoA-I gene may have led to the appearance of apoA-IV some 300 million years ago (7.Karathanasis S.K. Oettgen P. Haddad I.A. Antonarakis S.E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8457-8461Crossref PubMed Scopus (61) Google Scholar). A distinct feature of the primary sequences of the exchangeable apolipoproteins is a variable number of 22-residue amphipathic α-helical repeats, which likely confer the ability to bind to the surface of lipoprotein particles (8.Boguski M.S. Elshourbagy N. Taylor J.M. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 992-996Crossref PubMed Scopus (61) Google Scholar). ApoA-IV (376 residues) contains 13 such repeats (most punctuated by proline residues), located between residues 40 and 332. The first 39 amino acids of apoA-IV are encoded by a separate exon and contain potential weakly amphipathic helical domains that are similar to those found in globular proteins (9.Elshourbagy N.A. Walker D.W. Paik Y.K. Boguski M.S. Freeman M. Gordon J.I. Taylor J.M. J. Biol. Chem. 1987; 262: 7973-7981Abstract Full Text PDF PubMed Google Scholar). In contrast, the C terminus (residues 333–376) is predicted to be devoid of ordered secondary structure. Residues 354–367 are composed of a repeating EQQQ sequence that is not found in any other apolipoprotein (10.Weinberg R.B. J. Lipid Res. 1994; 35: 2212-2222Abstract Full Text PDF PubMed Google Scholar). Lipid-free forms of apoA-I and apoE have been shown to exhibit a compartmentalized architecture characterized by a well organized N-terminal domain and a relatively unstable C-terminal domain (11.Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (593) Google Scholar, 12.Davidson W.S. Hazlett T. Mantulin W.W. Jonas A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13605-13610Crossref PubMed Scopus (131) Google Scholar, 13.Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) that contains lipid binding sequences. Given the similarities among these apolipoproteins, we expected that apoA-IV would exhibit a similar organization. However, we found that the α-helices in apoA-IV are organized around a single large domain (14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar). Furthermore, the C-terminal third of apoA-IV not only lacked a lipid-binding domain, it appeared to actually inhibit lipid interactions. Removing the C-terminal 44 amino acids of apoA-IV (Δ333–376) resulted in a mutant that reorganized liposomes significantly faster than both WT apoA-IV and apoA-I (14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar). The goal of the present study was to identify the sequence(s) in the C-terminal region responsible for this lipid binding inhibitory effect. Materials—SDS-PAGE gels were obtained from Bio-Rad or Amersham Biosciences. Primer synthesis and DNA sequencing were performed by the University of Cincinnati DNA Core. Restriction enzymes were purchased from New England Biolabs (Beverly, MA). IgA protease (Igase) was purchased from MoBiTec. BL-21 (DE3) Escherichia coli and the pET30 vector were from Novagen (Madison, WI). Isopropyl-β-d-thiogalactoside was purchased from Fisher. His-bind resin was purchased from Novagen (Madison, WI). Centriprep centrifugal concentrators were purchased from Millipore/Amicon Bioseparations (Bedford, MA). 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was purchased from Avanti Polar Lipids (Birmingham, AL). All chemical reagents were of the highest quality available. Mutagenesis of Human ApoA-IV—Human apoA-IV DNA was ligated into the pET30 expression vector using the NcoI and HindIII sites. A cleavable N-terminal poly (His) tag was appended to facilitate purification. The C-terminal mutations were created by performing PCR-based site-directed mutagenesis (Quick-Change, Novagen) (14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar). For the Δ333–376, Δ344–376, and Δ352–376 mutants, a stop codon was inserted in place of the first amino acid to be deleted. For internal deletion mutants, complementary forward and reverse primers were designed with clamp regions on the 5′- and 3′-ends of the sequence removed. The Δ333–343 mutant DNA contained in the pET30 vector was used as the template for the double mutants (Δ1–10, 333–343, Δ1–20, 333–343, Δ1–30, 333–343, and Δ1–39, 333–343). These mutants used a forward primer that had a 3′-clamp region in which the nucleotides encoded the amino acids at position 11, 21, 31, and 40, respectively. The primer also consisted of a 5′-flap region that encoded for the NcoI site and an Igase (IgA protease) cleavage site that was used to remove the N-terminal His tag (as described below). For further details, please refer to Ref. 14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar. Point mutagenesis was performed directly in the expression vector using the Quick-Change approach as for the introduction of the stop codons. Protein Expression and Purification—Our protocol was similar to previous work with rat apoE (15.Pearson K. Liu M. Shen L. Tso P. Davidson W.S. Protein Expression Purif. 2005; 41: 447-453Crossref PubMed Scopus (4) Google Scholar). The expression vector was transformed into E. coli BL-21-competent cells and plated overnight at 37 °C. Kanamycin (30 μg/ml) was used as the selective agent. Cell colonies were picked and grown in Luria-Bertani (LB) culture media overnight in 10-ml culture tubes in a shaking incubator at 37 °C. The cells in culture media were then transferred to fresh 100-ml cultures and grown to an A600 of 0.6–0.7. At this point, isopropyl 1-thio-β-d-galactopyranoside was added for 2 h to induce overexpression of the protein. Cell pellets were collected by centrifugation and brought up in His-bind buffer along with protease inhibitors. Cells were disrupted by sonication and centrifuged, and the supernatant was filtered through a Millex 0.45-μm filter and added to His-bind columns according to the manufacturer's instructions. The protein was eluted, concentrated, and digested with IgA protease, a protease that cleaves before Thr in the sequence Ala-Pro-Arg-Pro-Pro-Thr-Pro. This removed the His tag and left a Thr-Pro at the N terminus. Next, the sample was passed over the His-bind column a second time to remove the cleaved tag. The His-bind column buffers in the purification contained 3 m guanidine. Finally, the samples were concentrated and dialyzed into standard Tris buffer (STB) (10 mm Tris-HCl, 1 mm EDTA, 150 mm NaCl, and 0.2% NaN3) for storage. DMPC Liposome Solubilization—DMPC in chloroform was dried in a borosilicate tube with a stream of nitrogen, solubilized in degassed STB, and bath-sonicated for 30 s to form multilamellar liposomes. Liposomes were placed in thermostated cuvettes at 24.5 °C, proteins were added to the liposomes at a final DMPC/protein mass ratio of 2.5:1, and the absorbance was continuously monitored at 325 nm in an Amersham Biosciences Ultraspec 4000 at 24.5 °C. Rate constants, k, were calculated for each mutant by fitting the first 5 min of data to a single exponential decay curve as a function of time (t): f(t) = a-kt using Sigma Plot 2000 (16.McLean L.R. Hagaman K.A. Biochim. Biophys. Acta. 1993; 1167: 289-295Crossref PubMed Scopus (6) Google Scholar, 17.Panagotopulos S.E. Witting S.R. Horace E.M. Hui D.Y. Maiorano J.N. Davidson W.S. J. Biol. Chem. 2002; 277: 39477-39484Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Interfacial Behavior at the Oil/Water Interface—The binding of apoA-IV at the oil/water interface was examined using a Tracker® automatic tensiometer (IT Concept) (28.Davidson W.S. Arnvig-McGuire K. Kennedy A. Kosman J. Hazlett T.L. Jonas A. Biochemistry. 1999; 38: 14387-14395Crossref PubMed Scopus (69) Google Scholar). Drops of pure triolein were injected into a sample chamber containing 25 μg/ml protein in phosphate-buffered saline buffer, and protein adsorption to the surface of the drop was monitored as the time-dependent decrease in interfacial tension. Thermal Denaturation Studies—The thermal denaturation of the lipid-free apoA-IV mutants was monitored as the change in molar ellipticity at 222 nm over a temperature range of 20–80 °C (18.Acharya P. Segall M.L. Zaiou M. Morrow J. Weisgraber K.H. Phillips M.C. Lund-Katz S. Snow J. Biochim. Biophys. Acta. 2002; 1584: 9-19Crossref PubMed Scopus (63) Google Scholar). The van't Hoff enthalpy, ΔHv, was calculated as described previously (13.Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Fluorescence Spectroscopy—Protein samples were studied at 0.1 mg/ml in STB on a Photon Technology International Quantamaster spectrometer in photon counting mode at room temperature. Excitation wavelength was 295 nm, excitation and emission band passes were 3.0 nm, and emission wavelength was monitored from 302 to 375 nm. Buffer blanks were subtracted from each of the samples. Quenching studies were performed by the addition of 0–0.2 m acrylamide; Stern-Volmer quenching constants, Ksv, were determined as described previously (19.Lehrer S.S. Biochemistry. 1971; 10: 3254-3263Crossref PubMed Scopus (1654) Google Scholar). To study the lipid association properties of human apoA-IV, we developed an efficient bacterial expression system that enables systematic deletion mutagenesis. Fig. 1 summarizes the N- and C-terminal deletion mutants that were generated for this study. The mutants were greater than 95% pure and had a size consistent with the deletions (Fig. 2). As reported previously, WT apoA-IV forms a mixture of monomers and dimers in solution with an approximate distribution of 70% dimers to 30% monomers (20.Weinberg R.B. Spector M.S. J. Biol. Chem. 1985; 260: 14279-14286Abstract Full Text PDF PubMed Google Scholar). To determine whether the deletions altered the oligomerization state of the mutants, we examined the mutant proteins by non-denaturing gel PAGE. None of the deletions significantly affected the quaternary structure of apoA-IV (data not shown).FIGURE 2SDS-PAGE analysis of WT apoA-IV and selected truncation mutants. Three-microgram samples of purified apoA-IV mutants were electrophoresed on an 18% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane 1, low molecular weight markers; lane 2, WT apoA-IV; lane 3, apoA-IV Δ344–376; lane 4, apoA-IV Δ333–376; lane 5, apoA-IV Δ333–343; lane 6, apoA-IV Δ333–337; lane 7, apoA-IV Δ338–342; lane 8, low molecular weight marker; Lane 9, apoA-IV Δ1–10,333–343; lane 10, apoA-IV Δ1–20,333–343.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Consistent with other studies (2.Weinberg R.B. Anderson R.A. Cook V.R. Emmanuel F. Denefle P. Hermann M. Steinmetz A. J. Lipid Res. 2000; 41: 1410-1418Abstract Full Text Full Text PDF PubMed Google Scholar, 21.Weinberg R.B. Ibdah J.A. Phillips M.C. J. Biol. Chem. 1992; 267: 8977-8983Abstract Full Text PDF PubMed Google Scholar), WT apoA-IV was only marginally able to reorganize the DMPC multilamellar liposomes into micellar particles that scatter less light (Fig. 3A). However, as noted in our previous study (14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar), apoA-IV Δ333–376 cleared DMPC vesicles at a dramatically faster rate than WT apoA-IV, surpassing apoA-I, which is well documented to bind lipid with higher affinity than apoA-IV (2.Weinberg R.B. Anderson R.A. Cook V.R. Emmanuel F. Denefle P. Hermann M. Steinmetz A. J. Lipid Res. 2000; 41: 1410-1418Abstract Full Text Full Text PDF PubMed Google Scholar, 22.Weinberg R.B. Spector M.S. J. Lipid Res. 1985; 26: 26-37Abstract Full Text PDF PubMed Google Scholar, 23.Weinberg R.B. Spector M.S. J. Biol. Chem. 1985; 260: 4914-4921Abstract Full Text PDF PubMed Google Scholar). As the DMPC assay measures the end result of a complex process that involves both an initial apolipoprotein-lipid interaction and a subsequent lipid reorganization (see reaction scheme in Ref. 24.Segall M.L. Dhanasekaran P. Baldwin F. Anantharamaiah G.M. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Lipid Res. 2002; 43: 1688-1700Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), we also examined the lipid affinity of WT and Δ333–376 apoA-IV using oil drop tensiometry (25.Weinberg R.B. Cook V.R. Beckstead J.A. Martin D.D. Gallagher J.W. Shelness G.S. Ryan R.O. J. Biol. Chem. 2003; 278: 34438-34444Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). This method measures the rate at which apolipoproteins in solution bind to and lower the surface tension of the more hydrophobic triolein/water interface. As such, this technique provides a biophysical model for the binding of apoA-IV to the nascent triglyceride-rich chylomicron surface. As seen in Fig. 3B, apoA-IV Δ333–376 caused a much more rapid decrease in interfacial tension compared with WT protein, similar to its behavior in the DMPC clearance assay. In fact, for each of the mutants studied, oil drop tensiometry yielded qualitatively similar results as the DMPC clearance assay (data not shown). We hypothesized that a specific sequence in the C-terminal 44 amino acids of apoA-IV mediated the lipid binding inhibitory effect. We therefore designed a series of truncation mutants to localize the active inhibitory sequence. Deletion of residues Δ352–376 had no effect on the rate of lipid binding compared with WT (data not shown). Likewise, internal deletion of the unique EQQQ domain from Δ354–367, which generates a “pig-like” apoA-IV (26.Navarro M.A. Acin S. Iturralde M. Calleja L. Carnicer R. Guzman-Garcia M.A. Gonzalez-Ramon N. Mata P. Isabel B. Lopez-Bote C.J. Lampreave F. Piniero A. Osada J. Gene (Amst.). 2004; 325: 157-164Crossref PubMed Scopus (8) Google Scholar), also had no effect on lipid binding (Fig. 4A). We next examined apoA-IV Δ344–376 because serine 343 is the terminal amino acid in chicken apoA-IV (2.Weinberg R.B. Anderson R.A. Cook V.R. Emmanuel F. Denefle P. Hermann M. Steinmetz A. J. Lipid Res. 2000; 41: 1410-1418Abstract Full Text Full Text PDF PubMed Google Scholar). Once again, this mutant was similar to WT in its lipid binding behavior (Fig. 4A). These data suggested that the lipid binding inhibitory sequence resided between residues 333 and 343. To confirm this, we examined the behavior of an internal deletion mutant, apoA-IV Δ333–343. As shown in Fig. 4B, apoA-IV Δ343–343 disrupted DMPC liposomes as fast as apoA-IV Δ333–376. A consideration of the sequence and net charge conservation of residues 333–343 across seven species revealed that residues 333–337 are highly conserved, whereas residues 338–342 are much less so (Fig. 5A). We therefore generated two shorter internal deletion mutants, apoA-IV Δ333–337, which lacked the highly conserved sequence, and apoA-IV Δ338–342, which lacked the more variable interval. Fig. 5B shows that apoA-IV Δ338–342 exhibited an intermediate binding rate. However, apoA-IV Δ333–337 was extremely efficient. In fact, this mutant was one of the fastest lipid-binding apolipoproteins we have observed. These data suggest that the most potent lipid binding inhibitory sequence in apoA-IV is located between residues 333 and 337. We previously noted that the increases in lipid binding rate displayed by the Δ333–376 and Δ271–376 deletion mutants were completely effaced by simultaneous deletion of the first 39 residues from the N terminus (14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar), suggesting that both domains can impact upon the lipid binding affinity of apoA-IV. To determine whether this phenomenon was maintained with the Δ333–343 mutant, we prepared a series of C- and N-terminal double deletion mutants. Deletion of the N-terminal 39 residues on the background of the apoA-IV Δ333–343 mutant (Δ1–39,333–343) converted the rapid binding Δ333–343 mutant to a slow lipid-binding protein, with kinetics similar to WT apoA-IV (Fig. 6). Similarly, a Δ1–20,333–343 deletion mutant also displayed slow binding. However, when only the first 10 residues were deleted from the N terminus, Δ1–10,333–343, the mutant maintained the rapid binding profile of the Δ333–343 single mutant. An internal deletion mutant, apoA-IV Δ11–20,333–343 that contained an intact N terminus, also displayed slow lipid binding kinetics (Fig. 6B). The DMPC clearance rate constants for all apoA-IV deletion mutants in this study, calculated as described under “Experimental Procedures,” are listed in TABLE ONE. The clearance rate constants for apoA-IV Δ333–376, Δ333–343, Δ333–337, and Δ1–10,333–343 were statistically different compared with WT apoA-IV. There was no difference between the clearance rate for WT apoA-IV and the Δ354–367, Δ344–376, Δ338–342, Δ1–39,333–343, and Δ1–20,333–343 mutants. Although there was no statistical difference between the clearance rate for WT apoA-IV and apoA-IV Δ338–342 over the first 5 min, it is evident from Fig. 5 that this deletion mutant was better able to reorganize lipid at longer incubation times.TABLE ONERate constants for WT and mutant apoA-IV in the DMPC clearance assayMutant proteinkak is the rate constant derived from the first 5 min of the DMPC clearance assay; means ± S.D.WT apoA-IV0.039 ± 0.012Δ354–3670.024 ± 0.005Δ344–3760.042 ± 0.017Δ333–3760.279 ± 0.080bDenotes difference of p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testΔ333–3430.171 ± 0.029bDenotes difference of p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testΔ333–3370.217 ± 0.039bDenotes difference of p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testΔ338–3420.094 ± 0.034Δ1–39,333–3430.040 ± 0.006Δ1–20,333–3430.051 ± 0.006Δ1–10,333–3430.260 ± 0.065bDenotes difference of p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testa k is the rate constant derived from the first 5 min of the DMPC clearance assay; means ± S.D.b Denotes difference of p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons test Open table in a new tab The data in TABLE ONE indicate that residues 333–337 are critical for maintaining a conformation of WT apoA-IV that is relatively inefficient at interacting with lipid surfaces. To examine the impact of the deletions on protein structure we determined their thermodynamic stability using circular dichroism spectroscopy (TABLE TWO). Thermal denaturation studies determined that the Δ344–376 deletion did not significantly alter global protein stability versus the WT, as measured by the van't Hoff enthalpy of denaturation. However, mutants that lacked the inhibitory sequence exhibited enthalpies that were slightly lower than WT apoA-IV ranging from 38 to 45 kcal/mol. We also examined the fluorescence properties of Trp-12, which has proven to be a useful measure of apoA-IV conformation (27.Weinberg R.B. Biochemistry. 1988; 27: 1515-1521Crossref PubMed Scopus (19) Google Scholar). The wavelength of maximum fluorescence (λmax) of Trp-12 in WT apoA-IV was 335 nm, indicative of a relatively hydrophobic environment (28.Davidson W.S. Arnvig-McGuire K. Kennedy A. Kosman J. Hazlett T.L. Jonas A. Biochemistry. 1999; 38: 14387-14395Crossref PubMed Scopus (69) Google Scholar) (TABLE THREE). ApoA-IV Δ344–376 had a similar λmax; however, deletion of Δ333–376 or Δ333–343 caused a significant red shift in Trp emission, indicating that the N terminus had relocated to a more polar environment. Acrylamide fluorescence quenching studies further indicated that the change in the polarity of the N terminus was accompanied by a parallel increase in aqueous accessibility to the neutral quenching agent (Fig. 7). These observations, together with the DMPC binding data, suggest that the C-terminal inhibitory sequence may maintain the N-terminal globular domain in a more compact conformation that shields Trp-12 from the aqueous milieu.TABLE TWOThermal denaturation parameters of WT and mutant apoA-IVMutant proteinΔHνDMPC association ratekcal/molaThe estimated error on these experiments is ± 0.5 kcal/mol (n = 2)WT apoA-IV48SlowΔ344–37647SlowΔ333–37645FastΔ333–34340FastΔ333–33743FastΔ338–34240SlowbThis mutant reaches an equilibrium that is similar to the “fast” lipid binding mutants (Fig. 5), but a statistical comparison of the rate in the first 5 min shows no significant difference from “slow” lipid binding mutants (TABLE ONE)Δ1–10, 333–34338FastΔ1–20, 333–34343SlowΔ271–376cThe data for these two mutants is shown for discussion purposes and was previously published in Ref. 1434FastΔ1–39, 271–376cThe data for these two mutants is shown for discussion purposes and was previously published in Ref. 1428Slowa The estimated error on these experiments is ± 0.5 kcal/mol (n = 2)b This mutant reaches an equilibrium that is similar to the “fast” lipid binding mutants (Fig. 5), but a statistical comparison of the rate in the first 5 min shows no significant difference from “slow” lipid binding mutants (TABLE ONE)c The data for these two mutants is shown for discussion purposes and was previously published in Ref. 14.Pearson K. Saito H. Woods S.C. Lund-Katz S. Tso P. Phillips M.C. Davidson W.S. Biochemistry. 2004; 43: 10719-10729Crossref PubMed Scopus (32) Google Scholar Open table in a new tab TABLE THREEFluorescence quenching parameters of WT and mutant apoA-IVMutant proteinλmaxaThe λmax is the wavelength of maximum fluorescence at 25 °C (n = 3, ± 1 S.D.)KsvbThe Ksv is the Stern-Volmer quenching constant, indicating relative exposure of the N-terminal tryptophan residue to acrylamidenmm–1WT apoA-IV334.7 ± 2.12.84 ± 0.20Δ344–376335.5 ± 0.72.81 ± 0.23Δ333–376342.6 ± 1.9cp < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons test8.78 ± 1.09cp < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testΔ333–343338.5 ± 1.0cp < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons test5.53 ± 0.02cp < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons testa The λmax is the wavelength of maximum fluorescence at 25 °C (n = 3, ± 1 S.D.)b The Ksv is the Stern-Volmer quenching constant, indicating relative exposure of the N-terminal tryptophan residue to acrylamidec p < 0.05 between WT and mutant as determined by one-way analysis of variance followed by a Tukey-Kramer multiple comparisons test Open table in a new tab Finally, we used point mutagenesis to determine whether specific residues could be responsible for the C-terminal inhibitory effect. When Ser-333 was replaced with an alanine, no effect on DMPC association was observed (Fig. 8). However, when either Phe-334 or Phe-335 was changed to alanine, the full lipid binding effect noted for the Δ333–337 mutant was recapitulated. Thus, dramatic changes in the lipid affinity of apoA-IV can be induced by the mutation of a single amino acid. It is well established that apoA-IV binds to lipid with much lower affinity than other members of the exchangeable apolipoprotein family (21.Weinberg R.B. Ibdah J.A. Phillips M.C. J. Biol. Chem. 1992; 267: 8977-8983Abstract Full Text PDF PubMed Google Scholar, 23.Weinberg R.B. Spector M.S. J. Biol. Chem. 1985; 260: 4914-4921Abstract Full Text PDF PubMed Google Scholar). It has been postulated that its distinctively weak lipid binding behavior is due to: (a) the relative hydrophilicity and low amphipathic moment of its constitutive α-helices (29.Weinberg R.B. Biochim. Biophys. Acta. 1987; 918: 299-303Crossref PubMed Scopus (18) Google Scholar), (b) the fact that most of these helices are of the Y-type, which may not be capable of deeply penetrating lipid surfaces (30.Segrest J.P. Garber D.W. Brouillette C.G. Harvey S.C. Anantharamaiah G.M. Adv. Protein Chem. 1994; 45: 303-369Crossref PubMed Google Scholar), and/or (c) the possibility that th" @default.
• W2136674557 created "2016-06-24" @default.
• W2136674557 creator A5003796011 @default.
• W2136674557 creator A5008316828 @default.
• W2136674557 creator A5020610670 @default.
• W2136674557 creator A5023512225 @default.
• W2136674557 creator A5062615822 @default.
• W2136674557 creator A5068495200 @default.
• W2136674557 creator A5080337755 @default.
• W2136674557 date "2005-11-01" @default.
• W2136674557 modified "2023-10-16" @default.
• W2136674557 title "Specific Sequences in the N and C Termini of Apolipoprotein A-IV Modulate Its Conformation and Lipid Association" @default.
• W2136674557 cites W1508973909 @default.
• W2136674557 cites W1509567174 @default.
• W2136674557 cites W1530559862 @default.
• W2136674557 cites W1585699199 @default.
• W2136674557 cites W1598788587 @default.
• W2136674557 cites W1603336438 @default.
• W2136674557 cites W1966008200 @default.
• W2136674557 cites W1971439798 @default.
• W2136674557 cites W1973241402 @default.
• W2136674557 cites W1986368984 @default.
• W2136674557 cites W1986439388 @default.
• W2136674557 cites W1989364943 @default.
• W2136674557 cites W1994543234 @default.
• W2136674557 cites W1994862909 @default.
• W2136674557 cites W1995725157 @default.
• W2136674557 cites W1997268657 @default.
• W2136674557 cites W2002022989 @default.
• W2136674557 cites W2002610788 @default.
• W2136674557 cites W2039618266 @default.
• W2136674557 cites W2045474288 @default.
• W2136674557 cites W2054413550 @default.
• W2136674557 cites W2059995793 @default.
• W2136674557 cites W2066894739 @default.
• W2136674557 cites W2104147675 @default.
• W2136674557 cites W2106452452 @default.
• W2136674557 cites W2108198268 @default.
• W2136674557 cites W2126180792 @default.
• W2136674557 cites W2134969640 @default.
• W2136674557 cites W2141163113 @default.
• W2136674557 cites W2142095688 @default.
• W2136674557 cites W2185305167 @default.
• W2136674557 cites W2185438865 @default.
• W2136674557 cites W2263771359 @default.
• W2136674557 cites W2331191058 @default.
• W2136674557 cites W2340007151 @default.
• W2136674557 cites W2344016273 @default.
• W2136674557 cites W4213009587 @default.
• W2136674557 cites W4241138499 @default.
• W2136674557 cites W98152484 @default.
• W2136674557 doi "https://doi.org/10.1074/jbc.m506802200" @default.
• W2136674557 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16159879" @default.
• W2136674557 hasPublicationYear "2005" @default.
• W2136674557 type Work @default.
• W2136674557 sameAs 2136674557 @default.
• W2136674557 citedByCount "21" @default.
• W2136674557 countsByYear W21366745572012 @default.
• W2136674557 countsByYear W21366745572013 @default.
• W2136674557 countsByYear W21366745572014 @default.
• W2136674557 countsByYear W21366745572015 @default.
• W2136674557 countsByYear W21366745572017 @default.
• W2136674557 countsByYear W21366745572019 @default.
• W2136674557 crossrefType "journal-article" @default.
• W2136674557 hasAuthorship W2136674557A5003796011 @default.
• W2136674557 hasAuthorship W2136674557A5008316828 @default.
• W2136674557 hasAuthorship W2136674557A5020610670 @default.
• W2136674557 hasAuthorship W2136674557A5023512225 @default.
• W2136674557 hasAuthorship W2136674557A5062615822 @default.
• W2136674557 hasAuthorship W2136674557A5068495200 @default.
• W2136674557 hasAuthorship W2136674557A5080337755 @default.
• W2136674557 hasConcept C104317684 @default.
• W2136674557 hasConcept C11772777 @default.
• W2136674557 hasConcept C143065580 @default.
• W2136674557 hasConcept C185592680 @default.
• W2136674557 hasConcept C2778163477 @default.
• W2136674557 hasConcept C2778918659 @default.
• W2136674557 hasConcept C2780072125 @default.
• W2136674557 hasConcept C41625074 @default.
• W2136674557 hasConcept C4733338 @default.
• W2136674557 hasConcept C55493867 @default.
• W2136674557 hasConcept C62746215 @default.
• W2136674557 hasConcept C8243546 @default.
• W2136674557 hasConcept C86803240 @default.
• W2136674557 hasConceptScore W2136674557C104317684 @default.
• W2136674557 hasConceptScore W2136674557C11772777 @default.
• W2136674557 hasConceptScore W2136674557C143065580 @default.
• W2136674557 hasConceptScore W2136674557C185592680 @default.
• W2136674557 hasConceptScore W2136674557C2778163477 @default.
• W2136674557 hasConceptScore W2136674557C2778918659 @default.
• W2136674557 hasConceptScore W2136674557C2780072125 @default.
• W2136674557 hasConceptScore W2136674557C41625074 @default.
• W2136674557 hasConceptScore W2136674557C4733338 @default.
• W2136674557 hasConceptScore W2136674557C55493867 @default.
• W2136674557 hasConceptScore W2136674557C62746215 @default.
• W2136674557 hasConceptScore W2136674557C8243546 @default.
• W2136674557 hasConceptScore W2136674557C86803240 @default.
• W2136674557 hasIssue "46" @default.

• next