Characterization of the Heparin Binding Properties of Annexin II Tetramer
1997; Elsevier BV; Volume: 272; Issue: 24 Linguagem: Inglês
10.1074/jbc.272.24.15093
ISSN1083-351X
AutoresGeetha Kassam, Akhil Manro, Carol E. Braat, Peter Louie, Sandra L. Fitzpatrick, David M. Waisman,
Tópico(s)Machine Learning in Bioinformatics
ResumoIn this report, we have characterized the interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt). Analysis of the circular dichroism spectra demonstrated that the Ca2+-dependent binding of AIIt to heparin caused a large decrease in the α-helical content of AIIt from ∼44 to 31%, a small decrease in the β-sheet content from ∼27 to 24%, and an increase in the unordered structure from 20 to 29%. The binding of heparin also decreased the Ca2+ concentration required for a half-maximal conformational change in AIIt from 360 to 84 μm. AIIt bound to heparin with an apparentK d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). The binding of heparin to AIIt was specific as other sulfated polysaccharides did not elicit a conformational change in AIIt. A region of the p36 subunit of AIIt (Phe306–Ser313) was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition. A peptide to this region underwent a conformational change upon heparin binding. Other annexins contained the Cardin-Weintraub consensus sequence, but did not undergo a substantial conformational change upon heparin binding. In this report, we have characterized the interaction of heparin with the Ca2+- and phospholipid-binding protein annexin II tetramer (AIIt). Analysis of the circular dichroism spectra demonstrated that the Ca2+-dependent binding of AIIt to heparin caused a large decrease in the α-helical content of AIIt from ∼44 to 31%, a small decrease in the β-sheet content from ∼27 to 24%, and an increase in the unordered structure from 20 to 29%. The binding of heparin also decreased the Ca2+ concentration required for a half-maximal conformational change in AIIt from 360 to 84 μm. AIIt bound to heparin with an apparentK d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). The binding of heparin to AIIt was specific as other sulfated polysaccharides did not elicit a conformational change in AIIt. A region of the p36 subunit of AIIt (Phe306–Ser313) was found to contain a Cardin-Weintraub consensus sequence for glycosaminoglycan recognition. A peptide to this region underwent a conformational change upon heparin binding. Other annexins contained the Cardin-Weintraub consensus sequence, but did not undergo a substantial conformational change upon heparin binding. The annexins are a family of ∼13 proteins that bind to acidic phospholipids and biological membranes in a Ca2+-dependent manner (see Refs. 1Kaetzel M.A. Dedman J.R. News Physiol. Sci. 1995; 10: 171-176Google Scholar, 2Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1022) Google Scholar, 3Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar for reviews). These proteins are expressed in a wide range of organisms such as slime molds, higher plants, invertebrates, and vertebrates. Studies of the amino acid sequence of the annexins have established the homology of these proteins. All annexins contain four repeats (eight repeats in the case of annexin VI) of ∼70 amino acids that are highly homologous. In contrast, the N terminus of each of the annexins is unique and displays the greatest variation in sequence and length. The crystal structure of several of the annexins has been reported (4Favier-Perron B. Lewit-Bentley A. Russo-Marie F. Biochemistry. 1996; 35: 1740-1744Crossref PubMed Scopus (66) Google Scholar, 5Luecke H. Chang B.T. Mailliard W.S. Schlaepfer D.D. Haigler H.T. Nature. 1995; 378: 512-515Crossref PubMed Scopus (131) Google Scholar, 6Swairjo M.A. Concha N.O. Kaetzel M.A. Dedman J.R. Seaton B.A. Nat. Struct. Biol. 1995; 2: 968-974Crossref PubMed Scopus (278) Google Scholar) and has established that the annexins are composed of two distinct sides. The convex side faces the biological membrane and contains the Ca2+- and phospholipid-binding sites The concave side faces the cytosol and contains the N and C termini.Annexin II (p36) contains three distinct functional regions, the N-terminal region, the C-terminal region, and the core region. The core region of p36 contains the Ca2+- and phospholipid-binding sites, whereas the C-terminal region contains the 14-3-3 homology domain (7Roth D. Morgan A. Burgoyne R.D. FEBS Lett. 1993; 320: 207-210Crossref PubMed Scopus (58) Google Scholar) and the plasminogen-binding domain (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar). The N terminus of annexin II (p36) contains two important regulatory domains, the L and P domains. The L domain consists of the first 14 residues of the N terminus and contains a high affinity binding site for the p11 protein (reviewed in Ref. 9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). The P domain of p36 contains the phosphorylation sites for protein kinase C (Ser25) and pp60 src (Tyr23). The N-terminal L and P domains play regulatory roles; activation of the phosphorylation sites of annexin II tetramer results in an increase in the A 0.5(Ca2+) for chromaffin granule aggregation and F-actin binding, whereas binding of the p11 subunit decreases theA 0.5(Ca2+) for these activities. The heterotetrameric complex (p362·p112) formed by the binding of p11 to p36, referred to as annexin II tetramer (AIIt), 1The abbreviations used are: AIIt, annexin II tetramer; DTT, dithiothreitol.1The abbreviations used are: AIIt, annexin II tetramer; DTT, dithiothreitol. is the predominant form in most cells (reviewed in Ref. 9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar).AIIt has been shown to be present at both the cytosolic and extracellular surfaces of the plasma membrane of many cells (9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). Extracellular AIIt has been proposed to function as a cell adhesion factor (10Tressler R.J. Updyke T.V. Yeatman T. Nicolson G.L. J. Cell. Biochem. 1993; 53: 265-276Crossref PubMed Scopus (94) Google Scholar, 11Tressler R.J. Nicolson G.L. J. Cell. Biochem. 1992; 48: 162-171Crossref PubMed Scopus (22) Google Scholar), a receptor for plasminogen and tissue plasminogen activator (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 12Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar), and a receptor for tenascin-C (13Chung C.Y. Murphy-Ullrich J.E. Erickson H.P. Mol. Biol. Cell. 1996; 7: 883-892Crossref PubMed Scopus (180) Google Scholar, 14Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar).In a previous study (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar), we reported that AIIt bound to a heparin affinity column and that the phosphorylation of AIIt on tyrosine residues blocked the heparin-binding activity of the protein. In this report, we have characterized the interaction of AIIt with heparin. Our results identify AIIt as a specific, high affinity heparin-binding protein. Furthermore, we show that the Ca2+-dependent binding of heparin to AIIt causes a dramatic conformational change in the protein. Last, we show that the p36 subunit of AIIt contains a Cardin-Weintraub glycosaminoglycan recognition site (16Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) and that a peptide to this region of AIIt binds heparin.DISCUSSIONPrevious work from our laboratory established that AIIt is a Ca2+-dependent heparin-binding protein (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). The interaction of AIIt with heparin was also shown to be inhibited by tyrosine phosphorylation of AIIt (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). Since the role that heparin binding plays in the structure or function of AIIt is unknown, the current study was aimed at the characterization of the interaction of heparin with AIIt. Analysis of the CD spectra of AIIt showed that the binding of heparin to AIIt resulted in a profound change in the conformation of AIIt (Fig. 1 and Table I). We also found that in the absence of Ca2+, a small change in the conformation of AIIt occurred upon heparin binding.Animal carbohydrate-binding proteins can be broadly classified into seven major groups. These include the C-type or Ca2+-dependent lectins, the S-type or Gal-binding galectins, P-type mannose 6-phosphate receptors, the I-type lectins, the pentraxins, the hyaluronan-binding proteins, and the heparin-binding proteins (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The C-type lectins bind several carbohydrates including mannose and galactose and require Ca2+ to form a coordination bond with the sugar ligand. The galectins bind only β-galactoside, whereas the P-type proteins bind only mannose 6-phosphate. The I-type lectins bind only sialic acid, whereas the pentraxins bind several carbohydrates such as heparin and sialic acid as well as phosphorylcholine. The hyaluronan-binding proteins bind only hyaluronan. The heparin-binding proteins generally demonstrate Ca2+-independent binding of both heparin and heparan sulfate. Since AIIt binds heparin (Table II) but not phosphorylcholine (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar), AIIt is most likely a member of the heparin-binding family of proteins. However, AIIt appears to be unique among heparin-binding protein members in that the binding of AIIt to heparin is stimulated by Ca2+. Furthermore, AIIt appears to be a unique member of the heparin-binding proteins because AIIt can discriminate between heparin and heparan sulfate ligands.Several consensus sequences have been identified among members of the heparin-binding family of proteins. For example, the heparin-binding sequence of the C-terminal region of fibronectin has been identified asWQPPRARI (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). In contrast, a region of thrombospondin containing the sequence WSPW has been identified as the heparin-binding region of the protein (28Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar, 29Guo N. Krutzsch H.C. Negre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar). Analysis of several heparin-binding proteins has suggested the potential existence of two consensus sequences referred to as Cardin-Weintraub heparin-binding consensus sequences (16Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar, 30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar). Site-directed mutagenesis and binding studies with synthetic or isolated peptides from several of these proteins have confirmed that this consensus region is often involved in binding heparin (30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar, 31Bae J. Desai U.R. Pervin A. Caldwell E.E. Weiler J.M. Linhardt R.J. Biochem. J. 1994; 301: 121-129Crossref PubMed Scopus (51) Google Scholar, 32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 34Barkalow F.J. Schwarzbauer J.E. J. Biol. Chem. 1994; 269: 3957-3962Abstract Full Text PDF PubMed Google Scholar, 36Booth B.A. Boes M. Andress D.L. Dake B.L. Kiefer M.C. Maack C. Linhardt R.J. Bar K. Caldwell E.E. Weiler J. Bar R.S. Growth Regul. 1995; 5: 1-17PubMed Google Scholar). Other studies have shown that the orientation of the Cardin-Weintraub consensus sequence within the protein is critical and may determine if the consensus sequence participates in heparin binding (33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 37Wong P. Hampton B. Szylobryt E. Gallagher A.M. Jaye M. Burgess W.H. J. Biol. Chem. 1995; 270: 25805-25811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As shown in Table III, the p36 subunit of AIIt contains a Cardin-Weintraub heparin-binding consensus sequence. Furthermore, a peptide to this region of the p36 subunit of AIIt (300LKIRSEFKKKYGKSLYY316) undergoes a conformational change upon heparin binding (Fig. 8). These results therefore suggest that residues 300–316 of the p36 subunit of AIIt are involved in heparin binding.Although the monomeric annexins I–VI bind to a heparin affinity column in the presence of Ca2+, a heparin-dependent conformational change was not observed for these proteins (Table IV). The p36 subunit of AIIt can exist as a monomer or as a heterotetramer. Heterotetrameric AIIt is composed of two p36 subunits and two p11 subunits. Considering that the p36 subunit (annexin II) binds to a heparin affinity column and contains the Cardin-Weintraub consensus sequence, it was surprising that the p36 subunit did not undergo a conformational change upon heparin binding. This suggests that the heparin-binding site of the p36 subunit and other monomeric annexins is preformed and does not require the recruitment of residues from other regions of the protein. This is consistent with the observation that carbohydrate-binding proteins undergo few if any changes in conformation upon carbohydrate binding (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The p11 subunit of AIIt does not bind heparin and does not contain any heparin-binding consensus sequences. It is therefore unlikely that the heparin-dependent conformational change in AIIt was due to the coordinated binding of heparin by both the p36 and p11 subunits of AIIt. We cannot, however, rule out the possibility that the binding of the p36 subunit to the p11 subunit induces a conformational change in the p11 subunit that results in exposure of a novel heparin-binding domain. The simplest explanation for the large conformational change in AIIt upon heparin binding is that the orientation of the p36 subunits in AIIt is not optimal for heparin binding. Therefore, the binding of heparin to AIIt results in the realignment of the p36 subunits.Of particular interest was our observation that the Ca2+-dependent conformational change in AIIt was induced by heparin, but not by other negatively charged glycosaminoglycans such as heparan sulfate, chondroitin sulfate, and dextran sulfate. Heparan sulfates are structurally related glycosaminoglycans that are found on cell surfaces and in the extracellular matrix, where they form the chains of heparan sulfate proteoglycans and bear only short stretches of dense sulfation. In contrast, heparin is the glycosaminoglycan that is secreted by mast cells and other hematopoietic cells and therefore may serve as a signaling molecule (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar, 39Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). To date, a heparin-binding protein capable of distinguishing between heparin and heparan sulfate has not been described. Recently, annexin IV was shown to bind heparin, but the binding of heparin to this protein was inhibited by a variety of carbohydrates including glucose, N-acetylneuraminic acid, heparan sulfate, and chondroitin sulfate (40Kojima K. Yamamoto K. Irimura T. Osawa T. Ogawa H. Matsumoto I. J. Biol. Chem. 1996; 271: 7679-7685Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In contrast, we have found that heparan sulfate or other glycosaminoglycans do not induce a conformational change in AIIt (Table II). Furthermore, high concentrations of heparan sulfate (50 μm) do not inhibit the conformational change in AIIt elicited by 0.5 μmheparin (Table II), therefore suggesting that heparan sulfate does not bind to AIIt. However, considering the heterogeneity of the cell-surface heparan sulfate proteoglycan (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar), it is possible that AIIt may interact with other heparan sulfate proteoglycans.We also observed that AIIt formed a large complex with heparin and that this complex was pelleted by centrifugation at 400,000 ×g. Analysis of the binding isotherm suggested that AIIt bound heparin with an apparent K d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). This K d for the binding of heparin to AIIt is slightly lower than theK d reported for the binding of heparin to heparinase (60 nm), acidic fibroblast growth factor (50–140 nm), or fibronectin (34 nm), but higher than that reported for the binding of heparin to basic fibroblast growth factor (2.2 nm) or antithrombin III (11 nm) (32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 41Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Crossref PubMed Scopus (189) Google Scholar, 42Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). AIIt does not bind to disaccharides of heparin, but does bind to 3-kDa heparin that contains ∼10 monosaccharides (Table II). The binding of ∼11 molecules of AIIt to a single 17-kDa heparin strand that contains ∼50 monosaccharide units (Fig. 5) suggests that AIIt requires ∼4–5 monosaccharide units for binding.The physiological significance of the binding of heparin to AIIt is unclear. Heparin has been shown to interact with enzymes of the clotting and fibrinolysis systems (24Agnelli, G. (1996) Hemostasis , 26, Suppl. 2, 2–9.Google Scholar), protect proteins from inactivation, play an essential role in the interaction of growth factors with their receptors, directly activate growth factor receptors, and serve as an essential cofactor in cell-cell recognition and cell-matrix adhesion processes (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar, 35Persson B. Bengtsson-Olivecrona G. Enerback S. Olivecrona T. Jornvall H. Eur. J. Biochem. 1989; 179: 39-45Crossref PubMed Scopus (96) Google Scholar, 43Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (472) Google Scholar, 44Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 45Gleizes P.E. Noaillac-Depeyre J. Amalric F. Gas N. Eur. J. Cell Biol. 1995; 66: 47-59PubMed Google Scholar, 46Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Hook M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar, 47Gao G. Goldfarb M. EMBO J. 1995; 14: 2183-2190Crossref PubMed Scopus (94) Google Scholar). AIIt is the major cellular receptor for tenascin-C and plasminogen (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 14Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar). It is therefore possible that heparin might be involved in the regulation of the interaction of AIIt with these ligands. The annexins are a family of ∼13 proteins that bind to acidic phospholipids and biological membranes in a Ca2+-dependent manner (see Refs. 1Kaetzel M.A. Dedman J.R. News Physiol. Sci. 1995; 10: 171-176Google Scholar, 2Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1022) Google Scholar, 3Swairjo M.A. Seaton B.A. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 193-213Crossref PubMed Scopus (188) Google Scholar for reviews). These proteins are expressed in a wide range of organisms such as slime molds, higher plants, invertebrates, and vertebrates. Studies of the amino acid sequence of the annexins have established the homology of these proteins. All annexins contain four repeats (eight repeats in the case of annexin VI) of ∼70 amino acids that are highly homologous. In contrast, the N terminus of each of the annexins is unique and displays the greatest variation in sequence and length. The crystal structure of several of the annexins has been reported (4Favier-Perron B. Lewit-Bentley A. Russo-Marie F. Biochemistry. 1996; 35: 1740-1744Crossref PubMed Scopus (66) Google Scholar, 5Luecke H. Chang B.T. Mailliard W.S. Schlaepfer D.D. Haigler H.T. Nature. 1995; 378: 512-515Crossref PubMed Scopus (131) Google Scholar, 6Swairjo M.A. Concha N.O. Kaetzel M.A. Dedman J.R. Seaton B.A. Nat. Struct. Biol. 1995; 2: 968-974Crossref PubMed Scopus (278) Google Scholar) and has established that the annexins are composed of two distinct sides. The convex side faces the biological membrane and contains the Ca2+- and phospholipid-binding sites The concave side faces the cytosol and contains the N and C termini. Annexin II (p36) contains three distinct functional regions, the N-terminal region, the C-terminal region, and the core region. The core region of p36 contains the Ca2+- and phospholipid-binding sites, whereas the C-terminal region contains the 14-3-3 homology domain (7Roth D. Morgan A. Burgoyne R.D. FEBS Lett. 1993; 320: 207-210Crossref PubMed Scopus (58) Google Scholar) and the plasminogen-binding domain (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar). The N terminus of annexin II (p36) contains two important regulatory domains, the L and P domains. The L domain consists of the first 14 residues of the N terminus and contains a high affinity binding site for the p11 protein (reviewed in Ref. 9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). The P domain of p36 contains the phosphorylation sites for protein kinase C (Ser25) and pp60 src (Tyr23). The N-terminal L and P domains play regulatory roles; activation of the phosphorylation sites of annexin II tetramer results in an increase in the A 0.5(Ca2+) for chromaffin granule aggregation and F-actin binding, whereas binding of the p11 subunit decreases theA 0.5(Ca2+) for these activities. The heterotetrameric complex (p362·p112) formed by the binding of p11 to p36, referred to as annexin II tetramer (AIIt), 1The abbreviations used are: AIIt, annexin II tetramer; DTT, dithiothreitol.1The abbreviations used are: AIIt, annexin II tetramer; DTT, dithiothreitol. is the predominant form in most cells (reviewed in Ref. 9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). AIIt has been shown to be present at both the cytosolic and extracellular surfaces of the plasma membrane of many cells (9Waisman D.M. Mol. Cell. Biochem. 1995; 149/150: 301-322Crossref Scopus (261) Google Scholar). Extracellular AIIt has been proposed to function as a cell adhesion factor (10Tressler R.J. Updyke T.V. Yeatman T. Nicolson G.L. J. Cell. Biochem. 1993; 53: 265-276Crossref PubMed Scopus (94) Google Scholar, 11Tressler R.J. Nicolson G.L. J. Cell. Biochem. 1992; 48: 162-171Crossref PubMed Scopus (22) Google Scholar), a receptor for plasminogen and tissue plasminogen activator (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 12Cesarman G.M. Guevara C.A. Hajjar K.A. J. Biol. Chem. 1994; 269: 21198-21203Abstract Full Text PDF PubMed Google Scholar), and a receptor for tenascin-C (13Chung C.Y. Murphy-Ullrich J.E. Erickson H.P. Mol. Biol. Cell. 1996; 7: 883-892Crossref PubMed Scopus (180) Google Scholar, 14Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar). In a previous study (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar), we reported that AIIt bound to a heparin affinity column and that the phosphorylation of AIIt on tyrosine residues blocked the heparin-binding activity of the protein. In this report, we have characterized the interaction of AIIt with heparin. Our results identify AIIt as a specific, high affinity heparin-binding protein. Furthermore, we show that the Ca2+-dependent binding of heparin to AIIt causes a dramatic conformational change in the protein. Last, we show that the p36 subunit of AIIt contains a Cardin-Weintraub glycosaminoglycan recognition site (16Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) and that a peptide to this region of AIIt binds heparin. DISCUSSIONPrevious work from our laboratory established that AIIt is a Ca2+-dependent heparin-binding protein (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). The interaction of AIIt with heparin was also shown to be inhibited by tyrosine phosphorylation of AIIt (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). Since the role that heparin binding plays in the structure or function of AIIt is unknown, the current study was aimed at the characterization of the interaction of heparin with AIIt. Analysis of the CD spectra of AIIt showed that the binding of heparin to AIIt resulted in a profound change in the conformation of AIIt (Fig. 1 and Table I). We also found that in the absence of Ca2+, a small change in the conformation of AIIt occurred upon heparin binding.Animal carbohydrate-binding proteins can be broadly classified into seven major groups. These include the C-type or Ca2+-dependent lectins, the S-type or Gal-binding galectins, P-type mannose 6-phosphate receptors, the I-type lectins, the pentraxins, the hyaluronan-binding proteins, and the heparin-binding proteins (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The C-type lectins bind several carbohydrates including mannose and galactose and require Ca2+ to form a coordination bond with the sugar ligand. The galectins bind only β-galactoside, whereas the P-type proteins bind only mannose 6-phosphate. The I-type lectins bind only sialic acid, whereas the pentraxins bind several carbohydrates such as heparin and sialic acid as well as phosphorylcholine. The hyaluronan-binding proteins bind only hyaluronan. The heparin-binding proteins generally demonstrate Ca2+-independent binding of both heparin and heparan sulfate. Since AIIt binds heparin (Table II) but not phosphorylcholine (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar), AIIt is most likely a member of the heparin-binding family of proteins. However, AIIt appears to be unique among heparin-binding protein members in that the binding of AIIt to heparin is stimulated by Ca2+. Furthermore, AIIt appears to be a unique member of the heparin-binding proteins because AIIt can discriminate between heparin and heparan sulfate ligands.Several consensus sequences have been identified among members of the heparin-binding family of proteins. For example, the heparin-binding sequence of the C-terminal region of fibronectin has been identified asWQPPRARI (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). In contrast, a region of thrombospondin containing the sequence WSPW has been identified as the heparin-binding region of the protein (28Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar, 29Guo N. Krutzsch H.C. Negre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar). Analysis of several heparin-binding proteins has suggested the potential existence of two consensus sequences referred to as Cardin-Weintraub heparin-binding consensus sequences (16Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar, 30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar). Site-directed mutagenesis and binding studies with synthetic or isolated peptides from several of these proteins have confirmed that this consensus region is often involved in binding heparin (30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar, 31Bae J. Desai U.R. Pervin A. Caldwell E.E. Weiler J.M. Linhardt R.J. Biochem. J. 1994; 301: 121-129Crossref PubMed Scopus (51) Google Scholar, 32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 34Barkalow F.J. Schwarzbauer J.E. J. Biol. Chem. 1994; 269: 3957-3962Abstract Full Text PDF PubMed Google Scholar, 36Booth B.A. Boes M. Andress D.L. Dake B.L. Kiefer M.C. Maack C. Linhardt R.J. Bar K. Caldwell E.E. Weiler J. Bar R.S. Growth Regul. 1995; 5: 1-17PubMed Google Scholar). Other studies have shown that the orientation of the Cardin-Weintraub consensus sequence within the protein is critical and may determine if the consensus sequence participates in heparin binding (33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 37Wong P. Hampton B. Szylobryt E. Gallagher A.M. Jaye M. Burgess W.H. J. Biol. Chem. 1995; 270: 25805-25811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As shown in Table III, the p36 subunit of AIIt contains a Cardin-Weintraub heparin-binding consensus sequence. Furthermore, a peptide to this region of the p36 subunit of AIIt (300LKIRSEFKKKYGKSLYY316) undergoes a conformational change upon heparin binding (Fig. 8). These results therefore suggest that residues 300–316 of the p36 subunit of AIIt are involved in heparin binding.Although the monomeric annexins I–VI bind to a heparin affinity column in the presence of Ca2+, a heparin-dependent conformational change was not observed for these proteins (Table IV). The p36 subunit of AIIt can exist as a monomer or as a heterotetramer. Heterotetrameric AIIt is composed of two p36 subunits and two p11 subunits. Considering that the p36 subunit (annexin II) binds to a heparin affinity column and contains the Cardin-Weintraub consensus sequence, it was surprising that the p36 subunit did not undergo a conformational change upon heparin binding. This suggests that the heparin-binding site of the p36 subunit and other monomeric annexins is preformed and does not require the recruitment of residues from other regions of the protein. This is consistent with the observation that carbohydrate-binding proteins undergo few if any changes in conformation upon carbohydrate binding (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The p11 subunit of AIIt does not bind heparin and does not contain any heparin-binding consensus sequences. It is therefore unlikely that the heparin-dependent conformational change in AIIt was due to the coordinated binding of heparin by both the p36 and p11 subunits of AIIt. We cannot, however, rule out the possibility that the binding of the p36 subunit to the p11 subunit induces a conformational change in the p11 subunit that results in exposure of a novel heparin-binding domain. The simplest explanation for the large conformational change in AIIt upon heparin binding is that the orientation of the p36 subunits in AIIt is not optimal for heparin binding. Therefore, the binding of heparin to AIIt results in the realignment of the p36 subunits.Of particular interest was our observation that the Ca2+-dependent conformational change in AIIt was induced by heparin, but not by other negatively charged glycosaminoglycans such as heparan sulfate, chondroitin sulfate, and dextran sulfate. Heparan sulfates are structurally related glycosaminoglycans that are found on cell surfaces and in the extracellular matrix, where they form the chains of heparan sulfate proteoglycans and bear only short stretches of dense sulfation. In contrast, heparin is the glycosaminoglycan that is secreted by mast cells and other hematopoietic cells and therefore may serve as a signaling molecule (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar, 39Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). To date, a heparin-binding protein capable of distinguishing between heparin and heparan sulfate has not been described. Recently, annexin IV was shown to bind heparin, but the binding of heparin to this protein was inhibited by a variety of carbohydrates including glucose, N-acetylneuraminic acid, heparan sulfate, and chondroitin sulfate (40Kojima K. Yamamoto K. Irimura T. Osawa T. Ogawa H. Matsumoto I. J. Biol. Chem. 1996; 271: 7679-7685Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In contrast, we have found that heparan sulfate or other glycosaminoglycans do not induce a conformational change in AIIt (Table II). Furthermore, high concentrations of heparan sulfate (50 μm) do not inhibit the conformational change in AIIt elicited by 0.5 μmheparin (Table II), therefore suggesting that heparan sulfate does not bind to AIIt. However, considering the heterogeneity of the cell-surface heparan sulfate proteoglycan (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar), it is possible that AIIt may interact with other heparan sulfate proteoglycans.We also observed that AIIt formed a large complex with heparin and that this complex was pelleted by centrifugation at 400,000 ×g. Analysis of the binding isotherm suggested that AIIt bound heparin with an apparent K d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). This K d for the binding of heparin to AIIt is slightly lower than theK d reported for the binding of heparin to heparinase (60 nm), acidic fibroblast growth factor (50–140 nm), or fibronectin (34 nm), but higher than that reported for the binding of heparin to basic fibroblast growth factor (2.2 nm) or antithrombin III (11 nm) (32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 41Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Crossref PubMed Scopus (189) Google Scholar, 42Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). AIIt does not bind to disaccharides of heparin, but does bind to 3-kDa heparin that contains ∼10 monosaccharides (Table II). The binding of ∼11 molecules of AIIt to a single 17-kDa heparin strand that contains ∼50 monosaccharide units (Fig. 5) suggests that AIIt requires ∼4–5 monosaccharide units for binding.The physiological significance of the binding of heparin to AIIt is unclear. Heparin has been shown to interact with enzymes of the clotting and fibrinolysis systems (24Agnelli, G. (1996) Hemostasis , 26, Suppl. 2, 2–9.Google Scholar), protect proteins from inactivation, play an essential role in the interaction of growth factors with their receptors, directly activate growth factor receptors, and serve as an essential cofactor in cell-cell recognition and cell-matrix adhesion processes (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar, 35Persson B. Bengtsson-Olivecrona G. Enerback S. Olivecrona T. Jornvall H. Eur. J. Biochem. 1989; 179: 39-45Crossref PubMed Scopus (96) Google Scholar, 43Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (472) Google Scholar, 44Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 45Gleizes P.E. Noaillac-Depeyre J. Amalric F. Gas N. Eur. J. Cell Biol. 1995; 66: 47-59PubMed Google Scholar, 46Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Hook M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar, 47Gao G. Goldfarb M. EMBO J. 1995; 14: 2183-2190Crossref PubMed Scopus (94) Google Scholar). AIIt is the major cellular receptor for tenascin-C and plasminogen (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 14Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar). It is therefore possible that heparin might be involved in the regulation of the interaction of AIIt with these ligands. Previous work from our laboratory established that AIIt is a Ca2+-dependent heparin-binding protein (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). The interaction of AIIt with heparin was also shown to be inhibited by tyrosine phosphorylation of AIIt (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar). Since the role that heparin binding plays in the structure or function of AIIt is unknown, the current study was aimed at the characterization of the interaction of heparin with AIIt. Analysis of the CD spectra of AIIt showed that the binding of heparin to AIIt resulted in a profound change in the conformation of AIIt (Fig. 1 and Table I). We also found that in the absence of Ca2+, a small change in the conformation of AIIt occurred upon heparin binding. Animal carbohydrate-binding proteins can be broadly classified into seven major groups. These include the C-type or Ca2+-dependent lectins, the S-type or Gal-binding galectins, P-type mannose 6-phosphate receptors, the I-type lectins, the pentraxins, the hyaluronan-binding proteins, and the heparin-binding proteins (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The C-type lectins bind several carbohydrates including mannose and galactose and require Ca2+ to form a coordination bond with the sugar ligand. The galectins bind only β-galactoside, whereas the P-type proteins bind only mannose 6-phosphate. The I-type lectins bind only sialic acid, whereas the pentraxins bind several carbohydrates such as heparin and sialic acid as well as phosphorylcholine. The hyaluronan-binding proteins bind only hyaluronan. The heparin-binding proteins generally demonstrate Ca2+-independent binding of both heparin and heparan sulfate. Since AIIt binds heparin (Table II) but not phosphorylcholine (15Hubaishy I. Jones P.G. Bjorge J. Bellagamba C. Fitzpatrick S. Fujita D.J. Waisman D.M. Biochemistry. 1995; 34: 14527-14534Crossref PubMed Scopus (80) Google Scholar), AIIt is most likely a member of the heparin-binding family of proteins. However, AIIt appears to be unique among heparin-binding protein members in that the binding of AIIt to heparin is stimulated by Ca2+. Furthermore, AIIt appears to be a unique member of the heparin-binding proteins because AIIt can discriminate between heparin and heparan sulfate ligands. Several consensus sequences have been identified among members of the heparin-binding family of proteins. For example, the heparin-binding sequence of the C-terminal region of fibronectin has been identified asWQPPRARI (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar). In contrast, a region of thrombospondin containing the sequence WSPW has been identified as the heparin-binding region of the protein (28Guo N.H. Krutzsch H.C. Negre E. Vogel T. Blake D.A. Roberts D.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3040-3044Crossref PubMed Scopus (141) Google Scholar, 29Guo N. Krutzsch H.C. Negre E. Zabrenetzky V.S. Roberts D.D. J. Biol. Chem. 1992; 267: 19349-19355Abstract Full Text PDF PubMed Google Scholar). Analysis of several heparin-binding proteins has suggested the potential existence of two consensus sequences referred to as Cardin-Weintraub heparin-binding consensus sequences (16Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar, 30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar). Site-directed mutagenesis and binding studies with synthetic or isolated peptides from several of these proteins have confirmed that this consensus region is often involved in binding heparin (30Cardin A.D. Demeter D.A. Weintraub H.J. Jackson R.L. Methods Enzymol. 1991; 203: 556-583Crossref PubMed Scopus (52) Google Scholar, 31Bae J. Desai U.R. Pervin A. Caldwell E.E. Weiler J.M. Linhardt R.J. Biochem. J. 1994; 301: 121-129Crossref PubMed Scopus (51) Google Scholar, 32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 34Barkalow F.J. Schwarzbauer J.E. J. Biol. Chem. 1994; 269: 3957-3962Abstract Full Text PDF PubMed Google Scholar, 36Booth B.A. Boes M. Andress D.L. Dake B.L. Kiefer M.C. Maack C. Linhardt R.J. Bar K. Caldwell E.E. Weiler J. Bar R.S. Growth Regul. 1995; 5: 1-17PubMed Google Scholar). Other studies have shown that the orientation of the Cardin-Weintraub consensus sequence within the protein is critical and may determine if the consensus sequence participates in heparin binding (33Ma Y. Henderson H.E. Liu M.S. Zhang H. Forsythe I.J. Clarke-Lewis I. Hayden M.R. Brunzell J.D. J. Lipid Res. 1994; 35: 2049-2059Abstract Full Text PDF PubMed Google Scholar, 37Wong P. Hampton B. Szylobryt E. Gallagher A.M. Jaye M. Burgess W.H. J. Biol. Chem. 1995; 270: 25805-25811Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As shown in Table III, the p36 subunit of AIIt contains a Cardin-Weintraub heparin-binding consensus sequence. Furthermore, a peptide to this region of the p36 subunit of AIIt (300LKIRSEFKKKYGKSLYY316) undergoes a conformational change upon heparin binding (Fig. 8). These results therefore suggest that residues 300–316 of the p36 subunit of AIIt are involved in heparin binding. Although the monomeric annexins I–VI bind to a heparin affinity column in the presence of Ca2+, a heparin-dependent conformational change was not observed for these proteins (Table IV). The p36 subunit of AIIt can exist as a monomer or as a heterotetramer. Heterotetrameric AIIt is composed of two p36 subunits and two p11 subunits. Considering that the p36 subunit (annexin II) binds to a heparin affinity column and contains the Cardin-Weintraub consensus sequence, it was surprising that the p36 subunit did not undergo a conformational change upon heparin binding. This suggests that the heparin-binding site of the p36 subunit and other monomeric annexins is preformed and does not require the recruitment of residues from other regions of the protein. This is consistent with the observation that carbohydrate-binding proteins undergo few if any changes in conformation upon carbohydrate binding (26Weis W.I. Drickamer K. Annu. Rev. Biochem. 1996; 65: 441-473Crossref PubMed Scopus (995) Google Scholar). The p11 subunit of AIIt does not bind heparin and does not contain any heparin-binding consensus sequences. It is therefore unlikely that the heparin-dependent conformational change in AIIt was due to the coordinated binding of heparin by both the p36 and p11 subunits of AIIt. We cannot, however, rule out the possibility that the binding of the p36 subunit to the p11 subunit induces a conformational change in the p11 subunit that results in exposure of a novel heparin-binding domain. The simplest explanation for the large conformational change in AIIt upon heparin binding is that the orientation of the p36 subunits in AIIt is not optimal for heparin binding. Therefore, the binding of heparin to AIIt results in the realignment of the p36 subunits. Of particular interest was our observation that the Ca2+-dependent conformational change in AIIt was induced by heparin, but not by other negatively charged glycosaminoglycans such as heparan sulfate, chondroitin sulfate, and dextran sulfate. Heparan sulfates are structurally related glycosaminoglycans that are found on cell surfaces and in the extracellular matrix, where they form the chains of heparan sulfate proteoglycans and bear only short stretches of dense sulfation. In contrast, heparin is the glycosaminoglycan that is secreted by mast cells and other hematopoietic cells and therefore may serve as a signaling molecule (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar, 39Matsumoto R. Sali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). To date, a heparin-binding protein capable of distinguishing between heparin and heparan sulfate has not been described. Recently, annexin IV was shown to bind heparin, but the binding of heparin to this protein was inhibited by a variety of carbohydrates including glucose, N-acetylneuraminic acid, heparan sulfate, and chondroitin sulfate (40Kojima K. Yamamoto K. Irimura T. Osawa T. Ogawa H. Matsumoto I. J. Biol. Chem. 1996; 271: 7679-7685Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In contrast, we have found that heparan sulfate or other glycosaminoglycans do not induce a conformational change in AIIt (Table II). Furthermore, high concentrations of heparan sulfate (50 μm) do not inhibit the conformational change in AIIt elicited by 0.5 μmheparin (Table II), therefore suggesting that heparan sulfate does not bind to AIIt. However, considering the heterogeneity of the cell-surface heparan sulfate proteoglycan (38Lindblom A. Bengtsson-Olivecrona G. Fransson L.A. Biochem. J. 1991; 279: 821-829Crossref PubMed Scopus (36) Google Scholar), it is possible that AIIt may interact with other heparan sulfate proteoglycans. We also observed that AIIt formed a large complex with heparin and that this complex was pelleted by centrifugation at 400,000 ×g. Analysis of the binding isotherm suggested that AIIt bound heparin with an apparent K d of 32 ± 6 nm (mean ± S.D., n = 3) and a stoichiometry of 11 ± 0.9 mol of AIIt/mol of heparin (mean ± S.D., n = 3). This K d for the binding of heparin to AIIt is slightly lower than theK d reported for the binding of heparin to heparinase (60 nm), acidic fibroblast growth factor (50–140 nm), or fibronectin (34 nm), but higher than that reported for the binding of heparin to basic fibroblast growth factor (2.2 nm) or antithrombin III (11 nm) (32Sasisekharan R. Venkataraman G. Godavarti R. Ernst S. Cooney C.L. Langer R. J. Biol. Chem. 1996; 271: 3124-3131Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 41Mach H. Volkin D.B. Burke C.J. Middaugh C.R. Linhardt R.J. Fromm J.R. Loganathan D. Mattsson L. Biochemistry. 1993; 32: 5480-5489Crossref PubMed Scopus (189) Google Scholar, 42Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). AIIt does not bind to disaccharides of heparin, but does bind to 3-kDa heparin that contains ∼10 monosaccharides (Table II). The binding of ∼11 molecules of AIIt to a single 17-kDa heparin strand that contains ∼50 monosaccharide units (Fig. 5) suggests that AIIt requires ∼4–5 monosaccharide units for binding. The physiological significance of the binding of heparin to AIIt is unclear. Heparin has been shown to interact with enzymes of the clotting and fibrinolysis systems (24Agnelli, G. (1996) Hemostasis , 26, Suppl. 2, 2–9.Google Scholar), protect proteins from inactivation, play an essential role in the interaction of growth factors with their receptors, directly activate growth factor receptors, and serve as an essential cofactor in cell-cell recognition and cell-matrix adhesion processes (27Woods A. McCarthy J.B. Furcht L.T. Couchman J.R. Mol. Biol. Cell. 1993; 4: 605-613Crossref PubMed Scopus (182) Google Scholar, 35Persson B. Bengtsson-Olivecrona G. Enerback S. Olivecrona T. Jornvall H. Eur. J. Biochem. 1989; 179: 39-45Crossref PubMed Scopus (96) Google Scholar, 43Kan M. Wang F. Xu J. Crabb J.W. Hou J. McKeehan W.L. Science. 1993; 259: 1918-1921Crossref PubMed Scopus (472) Google Scholar, 44Gitay-Goren H. Soker S. Vlodavsky I. Neufeld G. J. Biol. Chem. 1992; 267: 6093-6098Abstract Full Text PDF PubMed Google Scholar, 45Gleizes P.E. Noaillac-Depeyre J. Amalric F. Gas N. Eur. J. Cell Biol. 1995; 66: 47-59PubMed Google Scholar, 46Murphy-Ullrich J.E. Gurusiddappa S. Frazier W.A. Hook M. J. Biol. Chem. 1993; 268: 26784-26789Abstract Full Text PDF PubMed Google Scholar, 47Gao G. Goldfarb M. EMBO J. 1995; 14: 2183-2190Crossref PubMed Scopus (94) Google Scholar). AIIt is the major cellular receptor for tenascin-C and plasminogen (8Hajjar K.A. Jacovina A.T. Chacko J. J. Biol. Chem. 1994; 269: 21191-21197Abstract Full Text PDF PubMed Google Scholar, 14Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar). It is therefore possible that heparin might be involved in the regulation of the interaction of AIIt with these ligands. We thank Dr. Robert W. Woody for the generous gift of the SELCON computer program and Dr. Narasimha Sreerama (Colorado State University) for helpful discussions concerning interpretation of CD data using the SELCON computer program.
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