The Heparin/Heparan Sulfate-binding Site on Apo-serum Amyloid A
1999; Elsevier BV; Volume: 274; Issue: 11 Linguagem: Inglês
10.1074/jbc.274.11.7172
ISSN1083-351X
AutoresJohn B. Ancsin, Robert Kisilevsky,
Tópico(s)Skin and Cellular Biology Research
ResumoSerum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24–103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77–103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 Å between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (β-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775–783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143–147), respectively. Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24–103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77–103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 Å between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (β-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775–783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143–147), respectively. In response to tissue injury or infection, activated macrophages secrete cytokines (interleukin-1 and -6 and tumor necrosis factor) that induce liver synthesis of a number of acute-phase (AP) 1The abbreviations used are: AP, acute-phase; HS, heparan sulfate; HDL, high density lipoprotein; GAG, glycosaminoglycan; AA amyloid, inflammation-associated amyloid; RP-HPLC, reversed phase-high performance liquid chromatography; CR, Congo Red; RT, retention time; LDL, low density lipoprotein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; h, human; m, mouse; DS, dermatan sulfate; CS, chondroitin sulfate; HA, hyaluronan proteins (1Steel D.M. Whitehead A.S. Immunol. Today. 1994; 15: 81-88Abstract Full Text PDF PubMed Scopus (868) Google Scholar, 2Kushner I. Rzewnicki D.L. Husby G. Clinical Rheumatology. Bailliere Tindall Ltd., London1994: 513-530Google Scholar). The function of most of the AP proteins is unknown, but it is widely accepted that their purpose is to enhance host survival by neutralizing infectious agents, contributing to tissue repair, and restoring homeostasis. One of these AP proteins is a novel HDL apolipoprotein, called serum amyloid A (apoSAA), which is encoded by a multigene family conserved from fish to humans (3Husby G. Marhaug G. Dowton B. Sletten K. Sipe J.D. Amyloid Int. J. Exp. Clin. Invest. 1994; 1: 119-137Crossref Scopus (183) Google Scholar, 4Uhlar C.M. Burgess C.J. Sharp P.M. Whitehead A.S. Genomics. 1994; 19: 228-235Crossref PubMed Scopus (125) Google Scholar, 5Jensen L.E. Hiney M.P. Shields D.C. Uhlar C.M. Lindsay A.J. Whitehead A.S. J. Immunol. 1997; 158: 384-392PubMed Google Scholar). Maximum transcription rates are reached for two of the four known apoSAA isoforms (apoSAA1 and apoSAA2) 3 h after AP induction (6Lowell C.A. Stearman R.S. Morrow J.F. J. Biol. Chem. 1986; 261: 8453-8461Abstract Full Text PDF PubMed Google Scholar, 7Goldberger G. Bing D.H. Sipe J.D. Rits M. Colten H.R. J. Immunol. 1987; 138: 3967-3971PubMed Google Scholar, 8Brissette L. Young I. Narindrasorasak S. Kisilevsky R. Deeley R. J. Biol. Chem. 1989; 264: 19327-19332Abstract Full Text PDF PubMed Google Scholar). Their concentration in plasma increases 500–1000-fold (from 1–5 μg/ml up to 1 mg/ml) within 18–24 h and returns to near normal levels within 5–7 days of a single inflammatory stimulus (9McAdam K.P.W.J. Sipe J.D. J. Exp. Med. 1976; 144: 1121-1127Crossref PubMed Scopus (145) Google Scholar). ApoSAA is found mainly associated with HDL3 (10Benditt E.P. Eriksen N. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4025-4028Crossref PubMed Scopus (320) Google Scholar, 11Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar), where it has been postulated to modulate cholesterol transport during the AP response (12Kisilevsky R. Subrahmanyan L. Lab. Invest. 1992; 66: 778-785PubMed Google Scholar, 13Banka C.L. Yuan T. DeBeer M.C. Kindy M. Curtiss L.K. DeBeer F.C. J. Lipid Res. 1995; 36: 1058-1065Abstract Full Text PDF PubMed Google Scholar). But several other functions have also been proposed for apoSAA including immune regulation, as a chemoattractant and as an inhibitor of fever induction, platelet activation, and neutrophil oxidative burst (3Husby G. Marhaug G. Dowton B. Sletten K. Sipe J.D. Amyloid Int. J. Exp. Clin. Invest. 1994; 1: 119-137Crossref Scopus (183) Google Scholar). ApoSAA was originally identified by its cross-reactivity with antisera raised against peptides isolated from inflammation-associated amyloid (AA amyloid) (14Levin M. Pras M. Franklin E.C. J. Exp. Med. 1973; 138: 373-380Crossref PubMed Scopus (145) Google Scholar, 15Husby G. Natvig J.B. Michaelsen T.E. Sletten K. Host H. Nature. 1973; 244: 362-364Crossref PubMed Scopus (23) Google Scholar) which is a pathological tissue deposit associated with chronic inflammatory diseases. Amyloid is a generic term describing the primarily extracellular accumulation of fibrillar protein deposits that have unique tinctorial and structural properties and that cause the disruption of tissue architecture and function (16Sipe J.D. Crit. Rev. Clin. Lab. Sci. 1994; 31: 325-354Crossref PubMed Scopus (191) Google Scholar,17Magnus J.H. Stenstad T. Amyloid Int. J. Exp. Clin. Invest. 1997; 4: 121-134Crossref Scopus (30) Google Scholar). ApoSAA and at least 17 other unrelated normally nonfibrillar proteins are known precursors of amyloid (18Kazatchkine M.D. Husby G. Araki S. Benditt E.P. Benson M.D. Cohen A.S. Frangione B. Glenner G.G. Natvig J.B. Westermark P. Bull. W.H.O. 1993; 71: 105-108PubMed Google Scholar). Each is associated with a specific disease such as Alzheimer's disease, chronic hemodialysis, adult-onset diabetes, rheumatoid arthritis, and certain malignancies. Regardless of the underlying amyloid fibril protein/peptide or associated disease, isolated amyloids fibrils are composed of two or more 3-nm filaments twisted around each other forming nonbranching fibrils, 7–10 nm in diameter, with a crossed β-pleated sheet conformation. They stain with Congo Red, and when stained and viewed under polarized light they exhibit a red/green birefringence, a property diagnostic for amyloid. It has been proposed that the deposition of amyloid requires the formation of a nidus or protofilament around which amyloid fibrillogenesis takes place, and the glycosaminoglycan (GAG) heparan sulfate (HS) plays an important role in this pathological process (17Magnus J.H. Stenstad T. Amyloid Int. J. Exp. Clin. Invest. 1997; 4: 121-134Crossref Scopus (30) Google Scholar,19Kisilevsky R. Fraser P. Bock G.R. Goode J.A. The Nature and Origin of Amyloid Fibrils. John Wiley & Sons Ltd., Chichester, UK1996: 58-67Google Scholar, 20Kisilevsky R. Fraser P. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 361-404Crossref PubMed Scopus (84) Google Scholar). GAGs are sulfated heteropolysaccharides that have been known to be associated with amyloid for over 30 years (21Bitter T. Muir H. J. Clin. Invest. 1966; 45: 963-975Crossref PubMed Scopus (38) Google Scholar, 22Dalferes E.R. Radhakrishnamurthy B. Berenson G.S. Arch. Biochem. Biophys. 1967; 118: 284-291Crossref PubMed Scopus (27) Google Scholar) but attracted little attention until 1987, when Snow and co-workers (23Snow A.D. Kisilevsky R. Stephens C. Anastassiades T. Lab. Invest. 1987; 56: 665-667PubMed Google Scholar) showed that the GAG component of mouse AA amyloid was deposited coincidentally with the amyloid protein and that the GAG was part of the HS proteoglycan, perlecan (24Snow A.D. Kisilevsky R. Lab. Invest. 1988; 57: 687-698Google Scholar, 25Snow A.D. Bramson R. Mar H. Wight T.N. Kisilevsky R. J. Histochem. Cytochem. 1991; 39: 1321-1330Crossref PubMed Scopus (77) Google Scholar). By experimentally varying the induction speed of murine AA-type amyloidosis, it was possible to show that HS was both temporally and spatially deposited with the AA peptide (24Snow A.D. Kisilevsky R. Lab. Invest. 1988; 57: 687-698Google Scholar, 25Snow A.D. Bramson R. Mar H. Wight T.N. Kisilevsky R. J. Histochem. Cytochem. 1991; 39: 1321-1330Crossref PubMed Scopus (77) Google Scholar, 26Ailles L. Kisilevsky R. Young I.D. Lab. Invest. 1993; 69: 443-448PubMed Google Scholar) and that splenic perlecan mRNA was increased prior to the histological detection of AA amyloid (26Ailles L. Kisilevsky R. Young I.D. Lab. Invest. 1993; 69: 443-448PubMed Google Scholar). In vitro experiments have also shown that of a number of different GAGs examined, only HS could increase the β-sheet content (the characteristic conformation of amyloid) for mouse apoSAA2 but not for the nonamyloidogenic isoforms apoSAA1 and apoSAACE/J (27McCubbin W.D. Kay C.M. Narindrasorasak S. Kisilevsky R. Biochem. J. 1988; 256: 775-783Crossref PubMed Scopus (149) Google Scholar, 28de Beer M.C. de Beer F.C. McCubbin W.D. Kay C.M. Kindy M.S. J. Biol. Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar). HS has in fact been found in all amyloid deposits that have been investigated (19Kisilevsky R. Fraser P. Bock G.R. Goode J.A. The Nature and Origin of Amyloid Fibrils. John Wiley & Sons Ltd., Chichester, UK1996: 58-67Google Scholar). High affinity binding between perlecan and three Alzheimer's amyloid (Aβ) precursors, βPP-695, βPP-751, and βPP-770, could be inhibited with dextran sulfate and heparin but not chondroitin sulfate or dermatan sulfate (29Narindrasorasak S. Lowery D. Gonzalez-DeWhitt P. Poorman R.A. Greenberg B. Kisilevsky R. J. Biol. Chem. 1991; 266: 12878-12883Abstract Full Text PDF PubMed Google Scholar). HS has also been found to enhance Aβ fibrillogenesis (30Fraser P.E. Nguyen J.T. Chin D.T. Kirschner D.A. J. Neurochem. 1992; 59: 1531-1540Crossref PubMed Scopus (207) Google Scholar), and analogs of HS (aliphatic polysulfonates) were recently reported to block HS-induced Aβ fibrillogenesis, in vitro, and interfere with in vivo AA-type amyloid accumulation in mice (31Kisilevsky R. Lemieux L.J. Fraser P.E. Kong X. Hultin P.G. Szarek W.A. Nat. Med. 1995; 1: 143-147Crossref PubMed Scopus (338) Google Scholar, 32Inoue S. Hultin P.G. Szarek W.A. Kisilevsky R. Lab. Invest. 1996; 74: 1081-1090PubMed Google Scholar). Congo Red (CR), a disulfonated acidic dye, which has long been used as a stain for amyloid (33Puchtler H. Waldrop F.S. Meloan S.N. Histochemistry. 1983; 77: 431-445Crossref PubMed Scopus (27) Google Scholar), can inhibit AA amyloid in vivo (34Kagan D.Z. Rozinova V.N. Probl. Tuberk. 1974; 40: 72-74Google Scholar). CR and a number of sulfated glycans have also been shown to prevent the accumulation of the protease-resistant prion protein (amyloid form) (35Caughley B. Raymond G.J. J. Virol. 1993; 67: 643-650Crossref PubMed Google Scholar, 36Caughley B. Race R.E. J. Neurochem. 1992; 59: 768-771Crossref PubMed Scopus (218) Google Scholar, 37Priola S.A. Caughley B. Mol. Neurobiol. 1994; 8: 113-120Crossref PubMed Scopus (57) Google Scholar). HS and closely related heparin are negatively charged polymers composed of disaccharide repeats that contain carboxyl and sulfate groups (38Kjellén L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1678) Google Scholar,39Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (396) Google Scholar). Many proteins bind these GAGs through electrostatic interactions, and it has been demonstrated by substitution and chemical modification experiments that the protein binding is dependent on their positively charged (basic) residues (40Barkalow F.J.B. Schwarzbauer J.E. J. Biol. Chem. 1991; 266: 7812-7818Abstract Full Text PDF PubMed Google Scholar, 41Handin R.I. Cohen H.J. J. Biol. Chem. 1976; 251: 4273-4282Abstract Full Text PDF PubMed Google Scholar, 42Liu C.-S. Chang J.Y. J. Biol. Chem. 1987; 262: 17356-17361Abstract Full Text PDF PubMed Google Scholar). Because a wide range of proteins bind specifically to heparin, it was expected that a common structural motif would be found. Cardin and Weintraub in 1989 (43Cardin A. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar) examined a series of heparin-binding sequences and found that the basic residues tended to be arranged on one side of an α-helix with the patternXBBBXXBX (B, basic residue; X,nonbasic residues). A second pattern,XBBXBX, was proposed to align the basic residues on one side of a β-strand. Later, a third consensus sequence was published, XBBXXBBBXXBBX (44Sobel M. Soler D.F. Kermode J.C. Harris R.B. J. Biol. Chem. 1992; 267: 8857-8862Abstract Full Text PDF PubMed Google Scholar). However, there are many examples of heparin/HS-binding sequences lacking these consensus sequences, and recently Margalit et al. (45Margalit H. Fischer N. Ben-Sasson S.A. J. Biol. Chem. 1993; 268: 19228-19231Abstract Full Text PDF PubMed Google Scholar) reported that a common feature of heparin-binding sequences was that the outer basic residues were always 20 Å apart. The co-localization of HS with amyloids, the induction of perlecan expression prior to the appearance of AA amyloid, its ability to promote conformational change in native amyloid precursors and amyloid fibrillogenesis in vitro, and the amyloid-blocking effects of HS analogs and other sulfonates are all consistent with the working hypothesis that HS-amyloid precursor associations take place in situ and are a critical early step leading to protofilament formation and/or amyloid fibrillogenesis. In an effort to understand the underlying mechanism of HS-dependent conversion of apoSAA into AA amyloid fibrils, we undertook this study to characterize the GAG binding activity of apoSAA. This was done by affinity chromatography with defined apoSAA peptides using columns to which different GAGs were covalently attached. The GAG-binding site on apoSAA was initially mapped testing the binding activities of apoSAA CNBr fragments. This was followed with an assessment of the binding activities of a series of synthetic peptides corresponding to the smallest heparin/HS-binding CNBr fragment, containing specific residue substitutions, or a deletion. This approach allowed us not only to identify the heparin/HS-binding peptide sequence but also to rank the relative importance of the individual basic residues within the binding site. The demonstration of heparin/HS binding activity for apoSAA is consistent with some of the functions proposed for apoSAA. Characterization of the apoSAA-binding site also advances our understanding of amyloidogenesis and may assist in the design of therapeutic compounds. Plasma apoSAA concentrations were experimentally elevated in CD1 mice (Charles Rivers, Montreal, Quebec, Canada) by a subcutaneous injection of 0.5 ml of 2% (w/v) AgNO3 (46Axelrad M.A. Kisilevsky R. Willmer J. Chen S.J. Skinner M. Lab. Invest. 1982; 47: 139-146PubMed Google Scholar) which resulted in a sterile abscess. After 18–20 h, mice were sacrificed by CO2narcosis and exsanguinated by cardiac puncture preventing clotting with a small amount (50 μl) of 7% EDTA. High density lipoprotein containing apoSAA (HDL-apoSAA) was isolated from plasma by density flotation (47Havel R.J. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar). The density of the plasma was adjusted to 1.25 g/ml with NaBr and centrifuged at 250,000 × g for 24 h at 10 °C. The top layer was aspirated, pooled, and LDL and HDL-apoSAA were separated by gel filtration on a Sephacryl S-300 column (1.5 × 45 cm) washed with 50 mm Tris, 150 mm NaCl, pH 7.5, at 20 ml/h. Normal HDL and LDL were also purified from uninflamed mice by this procedure. ApoA-I and apoSAAs were purified from total lipoprotein by gel filtration as described previously (48Ancsin J.B. Kisilevsky R. J. Biol. Chem. 1997; 272: 406-413Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The lipoprotein preparations (3–5 ml) were dialyzed against 10% formic acid, pH 2.0 (2 liters), for 18 h at 4 °C and then applied to a Sephacryl-S-100HR column (2.5 × 110 cm) and eluted in the same buffer at 25 ml/h. The sample separated into two peaks which were collected, frozen in liquid N2, lyophilized, and stored at −20 °C. The protein residue from the first peak containing apoA-I was delipidated with diethyl ether (49Osborne Jr., J.C. Methods Enzymol. 1986; 128: 213-222Crossref PubMed Scopus (103) Google Scholar). The dried residue was resuspended in 4m urea, 10% formic acid, and the apoA-I was purified by gel filtration on the Sephacryl S-100HR column. Individual apoSAA isoforms were purified from the second gel filtration peak by reversed phase-high performance liquid chromatography (RP-HPLC) using a Waters (Millipore) HPLC system with a model 680 automated gradient controller, model 501 pump units, and a series 440 absorbance detector connected to a Waters 740 data module integrator. The lyophilized apoSAA powder was solubilized in 20% formic acid and then injected onto a semi-preparative C18 Vydac column (1 × 25 cm), eluted at 3 ml/min with 0.1% trifluoroacetic acid, 10% acetonitrile for 5 min, and then developed with an acetonitrile concentration gradient, increasing 2.5%/min for 10 min followed by 1.0%/min for 20 min, and finally 4.3%/min for 10 min bringing the elution buffer to 98% acetonitrile by 45 min. The eluant was monitored continuously at 214 nm, and absorbance was plotted against retention time. Peaks were collected manually and dried by vacuum centrifugation. The dried residue was stored at −20 °C. Apolipoproteins were identified by their mobility on SDS-urea-polyacrylamide gel electrophoresis (50Goldsmith M.R. Rattner E.C. Koehler M.D. Balikov S.R. Bock S.C. Anal. Biochem. 1979; 99: 33-40Crossref PubMed Scopus (38) Google Scholar). ApoSAA1 and apoSAA2 were cleaved at Met-X peptide bonds with cyanogen bromide (CNBr). Protein was dissolved in 70% formic acid at 1 mg/ml, to which 5.5 mg/ml CNBr was added (250 m excess over Met) plus freel-Trp (5 m excess over Met) to protect Trp residues. The reaction was carried out under nitrogen, at room temperature overnight, and then the solvent was evaporated by vacuum centrifugation. The reaction was evaluated, and peptides were purified by RP-HPLC. A semi-preparative C-18 Vydac column was equilibrated with 10% acetonitrile, 0.1% trifluoroacetic acid. Peptides were dissolved in 40% formic acid, filtered through a 0.2-μm filter, or centrifuged at 10,000 × g, and loaded onto the column, washed for 5 min at 3 ml/min, and then developed with a 1.5%/min acetonitrile linear concentration gradient for 30 min, followed by 4.3%/min acetonitrile for 10 min, bringing the elution buffer to 98% acetonitrile by 45 min. The separated peptides were identified by amino-terminal sequencing and molecular weight determination by mass spectroscopy carried out at the Alberta Peptide Institute (Edmonton, Alberta, Canada). Also, a series of apoSAA peptides corresponding to 1) mouse apoSAA2, residues 77–103 (m27-mer), with specific basic residues substituted with Ala, 2) mouse apoSAA2, residues 77–96 (a 20-mer missing Lys-102), and 3) human apoSAA2, residues 78–104 (h27-mer), were synthesized by Multiple Peptide Systems (San Diego, CA). Heparin/Affi-Gel media were purchased from Bio-Rad. Heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronan were all purchased from Sigma and coupled to either to Sepharose 4B (Amersham Pharmacia Biotech) based on the method of Smithet al. (51Smith P.K. Mallia A.K. Hermanson G.T. Anal. Biochem. 1980; 109: 466-473Crossref PubMed Scopus (314) Google Scholar) or to Affi-Gel 102 (Bio-Rad) as per manufacturer's instructions. Sepharose 4B was washed with 20 bed volumes of water, resuspended in 1 volume of water, and transferred to a beaker with a stir bar on ice. GAGs were dissolved in water at 2 mg/ml and were also placed on ice. The two solutions were mixed, and while stirring the pH was adjusted to pH 11 with NaOH (5n). Cyanogen bromide, 1 g/ml inN,N-dimethylformamide, was added dropwise to a final concentration of 31.3 mg/ml. The pH was maintained at about 11 by adding NaOH for 15 min and then left stirring for 18 h at room temperature. The gel was then washed with 20 bed volumes of water followed by 1 m ethanolamine, pH 9.0, to block excess reactive groups. After further washing with 10 bed volumes of (i) water, (ii) 0.1 m sodium acetate, pH 4.0, and (iii) 0.1m NaHCO3, pH 8.3, the column gel was equilibrated in 20 mm Tris-HCl, pH 7.5. Affi-Gel 102 (4 ml) was washed with 20 bed volumes of 50 mm acetate, pH 6.0, and then 8 mg of GAG in 4 ml of the same buffer was mixed with the gel. The coupling reaction was initiated by the addition of 32 mg of 1-ethyl-3–3-dimethylaminopropyl carbodiimide, adjusting the pH to 5 with 1 n HCl and allowing the reaction to proceed for 3 h. Heparin and HS-Sepharose 4B were also treated with carbodiimide that forms a stable adduct with GAG carboxyl side groups (52Gitel S.N. Medina V.M. Wessler S. Blood. 1985; 65: 902-911Crossref PubMed Google Scholar). The amount of GAG linked to columns ranged from 0.5 to 0.75 mg/ml gel as determined colorimetrically by the toluidine blue method (51Smith P.K. Mallia A.K. Hermanson G.T. Anal. Biochem. 1980; 109: 466-473Crossref PubMed Scopus (314) Google Scholar). Taurine (2-aminoethanesulfonate) and Congo Red (CR) dye were also coupled to Sepharose 4B. Taurine at 30 mg/ml and CR at 5 mg/ml were reacted with the CNBr-activated matrix. Once the reaction was complete the CR-Sepharose 4B was washed with 10% ethanol followed by 2m guanidine HCl, pH 7.5, to remove excess CR prior to equilibration with 20 mm Tris-HCl, pH 7.5. Based on the amount of CR that was washed from the column (absorbance at 480 nm), 3.8 mg of CR was coupled to the column. By including [3H]taurine (15 μCi) with the cold taurine, the amount of taurine coupled to Sepharose 4B was estimated at 12.4 mg/ml by scintillography. The CR and taurine columns contained approximately equimolar amounts of sulfate groups. ApoSAA CNBr cleavage products were resuspended in 20 mm Tris-HCl, pH 7.5, and loaded onto a 6-ml heparin-Affi-Gel column equilibrated with the same buffer at 0.5 ml/min. The column was then washed with 4 bed volumes of buffer and then developed with a 0–1 m NaCl concentration gradient. Fractions (0.6 ml) were collected and their absorbance measured at 214 nm. Heparin/Affi-Gel and other GAG-charged matrices were also packed into a 3-ml stainless steel column and equilibrated with 20 mm Tris-HCl, pH 7.5, at 0.5 ml/min using a Waters HPLC system. Samples (20–80 μg in 150–200 μl) were injected onto the column, washed with 3 bed volumes (18 min) of the same buffer, and then developed with a 0–1.0 m NaCl linear gradient for 10 bed volumes (60 min). The eluate was monitored continuously at 214 nm, and the absorbance was plotted against the retention time (RT). Generally, unbound peptides/proteins eluted 6.5–7.0 min after loading, and based on the RTs for the bound peptides/proteins, the NaCl concentration at which desorption took place could be calculated as follows: desorption [NaCl] = (RT − 6.5 min − 18 min)/60 min. The acute-phase serum amyloid A isoforms, apoSAA1 and apoSAA2, of mouse are 91% identical in amino acid sequence and have a single heparin-binding consensus sequence (XBBXBX, X, non-basic residue; B, basic residue) (43Cardin A. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar) located between residues 82 and 87 near the carboxyl terminus (Fig. 1A). A second potential GAG-binding sequence, rich in basic residues, is located between residues 18 and 46. Unfortunately, the direct testing of heparin binding activity of apoSAA was hampered by the insolubility of delipidated apoSAA under physiological buffer conditions in the absence of chaotropic agents (urea, SDS, and CHAPS). But two Met residues at positions 16 and 23 facilitated the removal of the amino-terminal amphipathic lipid binding domain (residues 1–24) (53Segrest J.P. Pownall H.J. Jackson R.L. Glenner G.C. Pollock P.S. Biochemistry. 1976; 15: 3187-3191Crossref PubMed Scopus (65) Google Scholar) by CNBr cleavage (Fig. 1A). For apoSAA1 this reaction generated an insoluble 16-mer (apoSAA1–16), and two soluble fragments, a 7-mer (apoSAA117–23) and an 80-mer (apoSAA124–103) which constituted about 78% of the native protein. For apoSAA2 a substitution of Ile with Met at residue 76 introduces an additional cleavage site allowing the 80-mer to be cleaved into a 53-mer (apoSAA224–76) and a 27-mer (apoSAA277–103) (Fig. 1A). The GAG-binding consensus sequence was predicted to lie within the 27-mer. Both apoSAA1 and apoSAA2 were purified (Fig. 1B), and cleavage of the individual isoforms with CNBr yielded the expected fragments, with a minimum of secondary by-products (Fig. 1, C and D). The identity of the CNBr-generated peptides was determined by amino-terminal sequencing and molecular weight analysis by mass spectrometry (data not shown). Preliminary chromatographic analysis of the apoSAA1 and apoSAA2 CNBr cleavage products on a heparin/Affi-Gel column (6 ml) revealed that both preparations contained heparin binding activity (Fig. 2). The bound peptides were eluted from the column by increasing the NaCl concentration, suggesting that the association was primarily electrostatic in nature. The interactions involved only heparin since binding was not detected with the uncharged agarose Affi-Gel matrix alone (data not shown). Amino-terminal sequencing of the heparin-bound peptides repurified by RP-HPLC identified the peptides as apoSAA124–103 (m80-mer) and apoSAA277–103 (m27-mer). The second potential GAG binding region contained on the m53-mer did not to have heparin binding activity. Further analysis of GAG binding activities was performed on matrices produced by covalently coupling different GAGs to Affi-Gel or Sepharose 4B and analyzed on a Waters HPLC apparatus. The two different coupling reactions linking the GAGs through their accessible carboxyl (Affi-Gel) or hydroxyl/amino groups (CNBr-Sepharose 4B) al
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