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• W2090843520 abstract "The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136–150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic α-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the α-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation. The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136–150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic α-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the α-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation. Apolipoprotein E (apoE) 1The abbreviations used are: apoE, apolipoprotein E; DMPC, 1,2-dimyristoyl phosphatidylcholine; HSPG, heparan sulfate proteoglycan; LDLR, low density lipoprotein receptor; LRP, LDLR-related protein; SPR, surface plasmon resonance; WT, wild type; CD, circular dichroism. is a key protein regulating lipid transport in human plasma and brain (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar, 2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 3Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (277) Google Scholar, 4Strittmatter W.J. Bova Hill C. Curr. Opin. Lipidol. 2002; 13: 119-123Crossref PubMed Scopus (67) Google Scholar). It mediates hepatic clearance of remnant lipoproteins as a high affinity ligand for the low density lipoprotein receptor (LDLR) family, including LDLR, LDLR-related protein (LRP), and cell-surface heparan sulfate proteoglycans (HSPGs) (5Cooper A.D. J. Lipid Res. 1997; 38: 2173-2192Abstract Full Text PDF PubMed Google Scholar). In the liver, HSPGs act in concert with LRP to complete the interaction of remnant particles with LRP in a process known as the HSPG-LRP pathway, in which apoE initially interacts with HSPG on the cell surface and is then transferred to the LRP for internalization (6Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The ability of apoE to interact with members of the LDLR family and with HSPG can also be significant for cell signaling events (7Swertfeger D.K. Hui D.Y. Front. Biosci. 2001; 6: D526-D535Crossref PubMed Google Scholar). Binding of apoE to LRP activates cAMP-dependent protein kinase A and inhibits platelet-derived growth factor-stimulated migration of smooth muscle cells (8Zhu Y. Hui D.Y. J. Biol. Chem. 2003; 278: 36257-36263Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Inhibition of smooth muscle cell proliferation by apoE is, on the other hand, mediated by its binding to HSPG (9Swertfeger D.K. Hui D.Y. J. Biol. Chem. 2001; 276: 25043-25048Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In addition, the interaction of apoE with HSPG has been implicated in neuronal growth and repair and, consequently, is involved in the progression of late onset familial Alzheimer's disease (10Ji Z.S. Pitas R.E. Mahley R.W. J. Biol. Chem. 1998; 273: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Mahley R.W. Rall Jr., S.C. Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1344) Google Scholar, 12Bazin H.G. Marques M.A. Owens III, A.P. Linhardt R.J. Crutcher K.A. Biochemistry. 2002; 41: 8203-8211Crossref PubMed Scopus (19) Google Scholar). ApoE comprises a single polypeptide chain of 299 amino acid residues and contains two independently folded functional domains, a 22-kDa N-terminal domain (residues 1–191) and a 10-kDa C-terminal domain (residues 222–299) (2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 13Saito H. Lund-Katz S. Phillips M.C. Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (188) Google Scholar). The N-terminal domain is folded into a four-helix bundle of amphipathic α-helices and contains the LDLR binding region (around residues 136–150 in helix 4) (14Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (601) Google Scholar, 15Narayanaswami V. Ryan R.O. Biochim. Biophys. Acta. 2000; 1483: 15-36Crossref PubMed Scopus (159) Google Scholar). The C-terminal domain also contains amphipathic α-helices that are involved in binding to lipoprotein particles (2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar, 16Westerlund J.A. Weisgraber K.H. J. Biol. Chem. 1993; 268: 15745-15750Abstract Full Text PDF PubMed Google Scholar, 17Segrest J.P. Garber D.W. Brouillette C.G. Harvey S.C. Anantharamaiah G.M. Adv. Protein Chem. 1994; 45: 303-369Crossref PubMed Google Scholar). Both the N- and C-terminal domains contain a heparin binding site (18Cardin A.D. Hirose N. Blankenship D.T. Jackson R.L. Harmony J.A. Sparrow D.A. Sparrow J.T. Biochem. Biophys. Res. Commun. 1986; 134: 783-789Crossref PubMed Scopus (90) Google Scholar, 19Weisgraber K.H. Rall Jr., S.C. Mahley R.W. Milne R.W. Marcel Y.L. Sparrow J.T. J. Biol. Chem. 1986; 261: 2068-2076Abstract Full Text PDF PubMed Google Scholar). The N-terminal domain site is located between residues 136 and 147, overlapping with the LDLR binding region (20Dong J. Peters-Libeu C.A. Weisgraber K.H. Segelke B.W. Rupp B. Capila I. Hernaiz M.J. LeBrun L.A. Linhardt R.J. Biochemistry. 2001; 40: 2826-2834Crossref PubMed Scopus (88) Google Scholar, 21Libeu C.P. Lund-Katz S. Phillips M.C. Wehrli S. Hernaiz M.J. Capila I. Linhardt R.J. Raffai R.L. Newhouse Y.M. Zhou F. Weisgraber K.H. J. Biol. Chem. 2001; 276: 39138-39144Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), whereas the C-terminal site involves basic residues around lysine 233 (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Although both sites are functional in the separated fragments, only the N-terminal site is available for interaction in both the lipid-free and lipidated states of the intact apoE molecule (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In humans, apoE is a polymorphic protein with three major isoforms, apoE2, apoE3, and apoE4, each differing by cysteine and arginine at positions 112 and 158 (11Mahley R.W. Rall Jr., S.C. Annu. Rev. Genomics Hum. Genet. 2000; 1: 507-537Crossref PubMed Scopus (1344) Google Scholar). ApoE3, the most common form, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites. Both apoE3 and apoE4 bind with equally high affinity to the LDLR, but apoE2 has defective binding to the LDLR and is associated with type III hyperlipoproteinemia (23Mahley R.W. Huang Y. Rall Jr., S.C. J. Lipid Res. 1999; 40: 1933-1949Abstract Full Text Full Text PDF PubMed Google Scholar). ApoE4 is associated with an increased risk for coronary artery disease and is a major risk factor for Alzheimer's disease (3Weisgraber K.H. Mahley R.W. FASEB J. 1996; 10: 1485-1494Crossref PubMed Scopus (277) Google Scholar, 24Davignon J. Gregg R.E. Sing C.F. Arteriosclerosis. 1988; 8: 1-21Crossref PubMed Google Scholar, 25Strittmatter W.J. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4725-4727Crossref PubMed Scopus (447) Google Scholar). A cluster of arginine and lysine residues located on the polar face of the fourth amphipathic α-helix in the N-terminal bundle represents the binding site for cell-surface receptors (2Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Despite the apparent accessibility of these basic residues, interaction of apoE with lipid is necessary for its high affinity binding to the LDLR (26Innerarity T.L. Pitas R.E. Mahley R.W. J. Biol. Chem. 1979; 254: 4186-4190Abstract Full Text PDF PubMed Google Scholar). Recent studies indicate that lipid binding induces opening of the helix bundle in the N-terminal domain (27Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 28Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar); this increases exposure of lysines 143 and 146 to the aqueous phase and thereby enhances interaction with acidic elements of the LDLR (29Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Disruption of the amphipathic α-helix spanning residues 140–150 abolishes LDLR binding, indicating that this structural motif in apoE is critical for function (30Zaiou M. Arnold K.S. Newhouse Y.M. Innerarity T.L. Weisgraber K.H. Segall M.L. Phillips M.C. Lund-Katz S. J. Lipid Res. 2000; 41: 1087-1095Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast to binding to the LDLR, the stringency for binding of apoE to the LRP or HSPG appears to be less severe. Lipid association of apoE is not required for binding to the LRP (31Narita M. Holtzman D.M. Fagan A.M. LaDu M.J. Yu L. Han X. Gross R.W. Bu G. Schwartz A.L. J. Biochem. (Tokyo). 2002; 132: 743-749Crossref PubMed Scopus (40) Google Scholar) or HSPG (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 32Shuvaev V.V. Laffont I. Siest G. FEBS Lett. 1999; 459: 353-357Crossref PubMed Scopus (22) Google Scholar), although the same apoE domain spanning residues 136–150 is involved in the binding (21Libeu C.P. Lund-Katz S. Phillips M.C. Wehrli S. Hernaiz M.J. Capila I. Linhardt R.J. Raffai R.L. Newhouse Y.M. Zhou F. Weisgraber K.H. J. Biol. Chem. 2001; 276: 39138-39144Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 33Croy J.E. Brandon T. Komives E.A. Biochemistry. 2004; 43: 7328-7335Crossref PubMed Scopus (72) Google Scholar). ApoE2 that is highly defective in LDLR binding activity (<2% of normal apoE3 activity) has significant binding activity to LRP (40–50% of apoE3) (23Mahley R.W. Huang Y. Rall Jr., S.C. J. Lipid Res. 1999; 40: 1933-1949Abstract Full Text Full Text PDF PubMed Google Scholar) and HSPG (50–90% of apoE3) (6Mahley R.W. Ji Z.S. J. Lipid Res. 1999; 40: 1-16Abstract Full Text Full Text PDF PubMed Google Scholar). The detailed molecular features that control high affinity binding of apoE to the LRP and HSPG are not yet defined fully. Previously, we have characterized the effect of point mutations of the basic residues present in the heparin binding sites on the equilibrium parameters defining interaction of apoE with heparin (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To predict the distribution of apoE to different receptors, such as LDLR, LRP, and HSPG, however, it is also important to establish the kinetics of apoE-receptor association and dissociation because receptor/ligand interactions at the cell surface do not occur at equilibrium. In this study, surface plasmon resonance (SPR) measurements were employed to determine the affinity and kinetics of the interaction of engineered apoE molecules in the lipid-free and lipidated states with heparin. Using this approach, we provide novel insights into the mechanism of apoE/heparin interaction. Materials—1,2-Dimyristoyl phosphatidylcholine (DMPC) was purchased from Avanti Polar Lipids (Pelham, AL), and stock solutions were stored in chloroform:methanol (2:1) under nitrogen at –20 °C. Brain natriuretic peptide and β-mercaptoethanol were from Sigma. Ultrapure guanidine HCl was from ICN Pharmaceuticals (Costa Mesa, CA). Porcine intestinal mucosa heparin (average molecular weight of 13,500–15,000) and its biotin conjugate were purchased from Calbiochem. Biotinylated heparin was dissolved in water and extensively dialyzed to remove any contaminating free biotin before use. Expression and Purification of Proteins—The full-length human apoE2, apoE3, apoE4 and their 22-kDa, 12-kDa, and 10-kDa fragments were expressed and purified as described previously (34Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 40723-40729Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). All mutants of apoE were made using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The cDNA was ligated into a thioredoxin fusion expression vector pET32a(+) (Novagen, Madison, WI) and transformed into the Escherichia coli strain BL21 star (DE3) (Invitrogen). The resulting thioredoxin-apoE fusion proteins were expressed and purified as described previously (28Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). For the apoE3 22-kDa (L141K/K143L/K146L/L148K) mutant that was expressed as insoluble protein, the proteins were solubilized from the cell pellet using 6 m urea (30Zaiou M. Arnold K.S. Newhouse Y.M. Innerarity T.L. Weisgraber K.H. Segall M.L. Phillips M.C. Lund-Katz S. J. Lipid Res. 2000; 41: 1087-1095Abstract Full Text Full Text PDF PubMed Google Scholar). The apoE preparations were at least 95% pure as assessed by SDS-PAGE. In all experiments, the apoE sample was freshly dialyzed from 1% β-mercaptoethanol and 6 m guanidine HCl solution into Tris buffer (10 mm Tris-HCl, 150 mm NaCl, 0.02% NaN3, 1 mm EDTA, pH 7.4) before use. The disulfide-linked apoE4 22-kDa mutant was dialyzed from 6 m guanidine HCl solution to maintain interhelical disulfide bonding (27Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). DMPC complexes with the apoE3 22-kDa variants were prepared as described previously (29Lund-Katz S. Zaiou M. Wehrli S. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. J. Biol. Chem. 2000; 275: 34459-34464Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). SPR Measurements—Experiments were performed on Biacore-X and -2000 SPR instruments (Biacore, Inc., Uppsala, Sweden). A streptavidin SA sensor chip was pretreated with three consecutive 5-μl injections of 50 mm NaOH in 1 m NaCl to remove nonspecifically bound contaminants. For immobilization of heparin on a SA chip, an injection of biotinylated heparin (10 μg/ml) in Tris buffer was made at a flow rate of 5 μl/min followed by a 10-μl injection of 2 m NaCl. Typically, 30–200 resonance units of heparin were immobilized, and the effects of mass transport were not significant because of the low surface density of ligand (35Myszka D.G. Curr. Opin. Biotechnol. 1997; 8: 50-57Crossref PubMed Scopus (430) Google Scholar). An untreated flow cell was used as a control. For kinetic measurement of apoE interaction with heparin, a 30-μl injection of the apoE sample was passed over the sensor surface at a flow rate of 10 μl/min. At the end of the sample plug, the same buffer was passed over the sensor surface to facilitate dissociation. After 3 min of dissociation time, the sensor surface was regenerated for the next sample using a 10-μl pulse of 2 m NaCl. The resultant sensorgrams were analyzed using BIAevaluation software (version 4.1). The response curves of various analyte concentrations were globally fitted to either the 1:1 Langmuir model or the two-state binding model described by the following equation (36Karlsson R. Falt A. J. Immunol. Methods. 1997; 200: 121-133Crossref PubMed Scopus (498) Google Scholar,37Lipschultz C.A. Li Y. Smith-Gill S. Methods. 2000; 20: 310-318Crossref PubMed Scopus (79) Google Scholar), A+B↔kd1ka1AB↔kdka2ABx(Eq. 1) where the equilibrium constants of each binding step are K1 = ka1/kd1 and K2 = ka2/kd2, and the overall equilibrium binding constant is calculated as KA = K1(1 + K2) and Kd = 1/KA. In this model, the analyte (A) binds to the ligand (B) to form an initial complex (AB) and then undergoes subsequent binding or conformational change to form a more stable complex (ABx). For the apoE 10- and 12-kDa fragments, binding responses in the steady-state region of the sensorgrams (Req) were also plotted against analyte concentration (C) to determine the overall equilibrium binding affinity. The data were subjected to nonlinear regression fitting according to the following equation, Req=CRmax/(C+Kd)(Eq. 2) where Rmax is the maximum binding response, and Kd is the dissociation constant. Circular Dichroism (CD) Spectroscopy—Far UV CD spectra were recorded from 195 to 250 nm at 25 °C using a Jasco J-820 spectropolarimeter. After dialyzing from 1% β-mercaptoethanol and 6 m guanidine HCl solution, the apoE sample was diluted to 25 μg/ml in Tris buffer (pH 7.4) to obtain the CD spectrum. For the apoE/heparin mixture sample, apoE was mixed with heparin (apoE:heparin ratios of 0.5–2 w/w) prior to the measurement. The results were corrected by subtracting the buffer base line or a blank sample containing an identical concentration of heparin. The α-helix content was calculated from the molar ellipticity at 222 nm, [θ]222, according to the following equation (38Morrisett J.D. David J.S. Pownall H.J. Gotto Jr., A.M. Biochemistry. 1973; 12: 1290-1299Crossref PubMed Scopus (251) Google Scholar,39Acharya 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 (65) Google Scholar).% α-helix=([θ]222+3000)/(36000+3000)×100(Eq. 3) Analytical Procedures—Protein concentrations were determined by either the Lowry procedure (40Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) or the absorbance coefficient at 280 nm. Phospholipid concentrations were determined with an enzymatic assay kit (Wako Chemicals, Richmond, VA). Comparison of ApoE Binding to Immobilized Heparin and Heparin-Sepharose Gel—We have previously characterized the effects of point mutations of lysines in the N- and C-terminal heparin binding sites on the equilibrium binding to heparin using heparin-Sepharose gel (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In this study, we made SPR measurements for the apoE/heparin interaction to examine the real-time binding kinetics. Fig. 1A shows sensorgrams of binding of apoE C-terminal fragments to the immobilized heparin on a sensor chip. Because binding equilibrium was achieved during the injection of the 10- and 12-kDa fragments of apoE, we determined binding isotherms from the relationship between the equilibrium binding response (Req) and protein concentration (Fig. 1B). These isotherms were similar to those obtained using heparin-Sepharose gel (Fig. 1C), and calculated Kd values from both methods were comparable (for example, Kd for the apoE 10-kDa fragment/heparin interaction are 29 and 76 μg/ml from Fig. 1, B and C, respectively); these results validate the SPR method for studies of the apoE/heparin interaction (41Osmond R.I. Kett W.C. Skett S.E. Coombe D.R. Anal. Biochem. 2002; 310: 199-207Crossref PubMed Scopus (111) Google Scholar). Kinetic Analysis of Heparin Binding to Full-length ApoE3 and Its 22- and 10-kDa Fragments—Fig. 2 shows a typical sensorgram for the binding of full-length apoE to heparin. The kinetic data were not fitted well by a 1:1 Langmuir binding model, as indicated by the large value of the goodness of fit (χ2 = 100) (Fig. 2, inset). However, significantly improved fit (χ2 = 7.4) was obtained using a two-state binding model, indicating that binding of apoE to heparin involves either a sequential two-step process or some conformational change (36Karlsson R. Falt A. J. Immunol. Methods. 1997; 200: 121-133Crossref PubMed Scopus (498) Google Scholar, 37Lipschultz C.A. Li Y. Smith-Gill S. Methods. 2000; 20: 310-318Crossref PubMed Scopus (79) Google Scholar). The response curve of heparin binding of the apoE3 22-kDa fragment also showed two-state binding kinetics (Fig. 3A). Changing the injection time revealed that the dissociation rate was progressively decreased after longer injection (contact) time (Fig. 3A, inset), indicating that the stability of the initial apoE-heparin complex increases over time (36Karlsson R. Falt A. J. Immunol. Methods. 1997; 200: 121-133Crossref PubMed Scopus (498) Google Scholar, 37Lipschultz C.A. Li Y. Smith-Gill S. Methods. 2000; 20: 310-318Crossref PubMed Scopus (79) Google Scholar). Such an effect of contact time on dissociation rate was not seen in the apoE 10-kDa binding to heparin, because the contribution of the second binding step to the overall process was very small (Fig. 3B). Table I summarizes the kinetic rate constants and the derived affinity constants for full-length apoE3 and its 22- and 10-kDa fragments obtained using the two-state binding model. There was good agreement in the Kd values for full-length apoE3 between our data and a previous report (32Shuvaev V.V. Laffont I. Siest G. FEBS Lett. 1999; 459: 353-357Crossref PubMed Scopus (22) Google Scholar) but some discrepancy for the apoE3 22-kDa fragment (21Libeu C.P. Lund-Katz S. Phillips M.C. Wehrli S. Hernaiz M.J. Capila I. Linhardt R.J. Raffai R.L. Newhouse Y.M. Zhou F. Weisgraber K.H. J. Biol. Chem. 2001; 276: 39138-39144Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), because all previous studies applied the 1:1 Langmuir binding model to the kinetic data. In addition, the Kd value for the 10-kDa fragment in Table I (15 μg/ml) is similar to that obtained by the steady-state analysis (29 μg/ml from Fig. 1B), further validating the two-state binding model.Fig. 3SPR sensorgrams of binding of apoE3 22-kDa (A) and 10-kDa (B) fragments (30 μg/ml) to heparin. The experimental binding data (•) were fitted with the two-state binding model. Each component was shown as initial complex (AB) and transferred complex (AB). The insets show the effect of increased injection time on the stability of the apoE-heparin complex. a,30s; b,60s; c, 120 s; d, 180 s. RU, resonance xunits.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IKinetic parameters for binding of lipid-free apoE3 and its fragments to heparinapoEka1kd1ka2kd2K1K2Kd105m-1 s-110-2 s-110-3 s-110-3 s-1105m-1μmapoE39.8 ± 3.59.2 ± 3.27.2 ± 1.02.2 ± 0.61073.30.022 ± 0.013apoE3 22-kDa0.067 ± 0.0268.4 ± 1.912 ± 23.2 ± 0.70.793.92.6 ± 1.310-kDa1.6 ± 1.130 ± 211.1 ± 0.46.7 ± 0.75.20.21.6 ± 0.8 Open table in a new tab Effects of Heparin Binding on the Secondary and Tertiary Structure of ApoE—To examine the possibility that the conformational change in apoE occurs during binding to heparin, far UV CD measurements were employed to evaluate the secondary structure of apoE. As shown in Fig. 4, no change in the spectra of the apoE 22-kDa fragment was observed in the absence or presence of heparin. The α-helix contents derived from the molar ellipticity at 222 nm were in the range of 47–49% up to a heparin: apoE weight ratio of 2 (Fig. 4, inset), indicating that there is no change in the secondary structure in apoE upon heparin binding. The four-helix bundle of the apoE 22-kDa fragment is known to open upon lipid binding (27Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 28Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2001; 276: 40949-40954Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To further confirm that any such conformational change in apoE is not involved in the two-state binding to heparin, we tested the heparin binding of the triple interhelical disulfide-linked apoE4 22-kDa mutant in which the opening of the four-helix bundle is completely restricted (27Lu B. Morrow J.A. Weisgraber K.H. J. Biol. Chem. 2000; 275: 20775-20781Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). SPR sensorgrams of heparin binding of this mutant still exhibited two-state binding kinetics (data not shown), indicating that a conformational change, such as the opening of the helix bundle, was not involved in the two-state binding for the apoE/heparin interaction. Effects of Lysine Mutations in the Heparin Binding Sites of ApoE on the Binding Kinetics—To explore the molecular mechanism of the two-state heparin binding of apoE, we used mutants with substitutions at lysines 146 and 233; these residues are located in the N- and C-terminal heparin binding sites, respectively, and contribute to an ionic interaction with heparin (21Libeu C.P. Lund-Katz S. Phillips M.C. Wehrli S. Hernaiz M.J. Capila I. Linhardt R.J. Raffai R.L. Newhouse Y.M. Zhou F. Weisgraber K.H. J. Biol. Chem. 2001; 276: 39138-39144Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). As shown in Fig. 5, large decreases in binding responses were observed with the apoE3 K146E mutant, whereas the apoE3 K233E mutant exhibited responses similar to the wild type (WT), consistent with the previous observations using heparin-Sepharose gel in which only Lys-146 plays a dominant role in heparin binding of full-length apoE (22Saito H. Dhanasekaran P. Nguyen D. Baldwin F. Weisgraber K.H. Wehrli S. Phillips M.C. Lund-Katz S. J. Biol. Chem. 2003; 278: 14782-14787A" @default.
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• W2090843520 title "Two-step Mechanism of Binding of Apolipoprotein E to Heparin" @default.
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