A Peptide Found by Phage Display Discriminates a Specific Structure of a Trisaccharide in Heparin
2011; Elsevier BV; Volume: 286; Issue: 14 Linguagem: Inglês
10.1074/jbc.m110.172155
ISSN1083-351X
AutoresTomio Yabe, Ritsuko Hosoda‐Yabe, Yoshihiro Kanamaru, Makoto Kiso,
Tópico(s)Platelet Disorders and Treatments
ResumoA number of recent studies have shown that heparan sulfate can control several important biological events on the cell surface through changes in sulfation pattern. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult. Heparin, which is primarily produced by mast cells, is closely approximated by the structural analog heparan sulfate. Screening of heparin-associating peptides using phage display and antithrombin-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. The peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and heparan sulfate but does not associate with other glycosaminoglycans. HappY recognizes three consecutive monosaccharide residues in heparin through its three arginine residues. HappY should be a useful probe to detect heparin and heparan sulfate in studies of glycobiology. A number of recent studies have shown that heparan sulfate can control several important biological events on the cell surface through changes in sulfation pattern. The in vivo modification of sugar chains with sulfates, however, is complicated, and the discrimination of different sulfation patterns is difficult. Heparin, which is primarily produced by mast cells, is closely approximated by the structural analog heparan sulfate. Screening of heparin-associating peptides using phage display and antithrombin-bound affinity chromatography identified a peptide, heparin-associating peptide Y (HappY), that acts as a target of immobilized heparin. The peptide consists of 12 amino acid residues with characteristic three arginines and exclusively binds to heparin and heparan sulfate but does not associate with other glycosaminoglycans. HappY recognizes three consecutive monosaccharide residues in heparin through its three arginine residues. HappY should be a useful probe to detect heparin and heparan sulfate in studies of glycobiology. IntroductionHeparin, which is comprised of heterogeneous mixtures of complex, linear, sulfated polysaccharides consisting of one to four linked uronic acid and glucosamine residues, is well known for its potent anticoagulant activity (1Linhardt R.J. J. Med. Chem. 2003; 46: 2551-2564Crossref PubMed Scopus (429) Google Scholar, 2Casu B. Lindahl U. Adv. Carbohydr. Chem. Biochem. 2001; 57: 159-206Crossref PubMed Scopus (347) Google Scholar, 3Machovich R. Biochim. Biophys. Acta. 1975; 412: 13-17Crossref PubMed Scopus (62) Google Scholar). The antithrombin-binding (ATB) 2The abbreviations used are: ATB, antithrombin-binding; AT, antithrombin; HS, heparan sulfate; LMWH, low-molecular-weight heparin; SPR, surface plasmon resonance; FC, flow cell; ATU, antithrombin unbound. domain mainly exerts its activity by binding to antithrombin (AT), a 55-kDa single-chain plasma glycoprotein (4Damus P.S. Hicks M. Rosenberg R.D. Nature. 1973; 246: 355-357Crossref PubMed Scopus (417) Google Scholar, 5Rosenberg R.D. Damus P.S. J. Biol. Chem. 1973; 248: 6490-6505Abstract Full Text PDF PubMed Google Scholar), and has a unique pentasaccharide structure (6Oosta G.M. Gardner W.T. Beeler D.L. Rosenberg R.D. Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 829-833Crossref PubMed Scopus (148) Google Scholar). This pentasaccharide sequence is randomly distributed along the heparin chains and is present in only about one-third of the heparins currently used in the treatment of thrombosis (7Lam L.H. Silbert J.E. Rosenberg R.D. Biochem. Biophys. Res. Commun. 1976; 69: 570-577Crossref PubMed Scopus (368) Google Scholar). For a review of the prevention of thrombosis, see Ref. 8Middeldorp S. Thromb. Res. 2008; 122: 753-762Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar.Heparan sulfate (HS), which is a structural analog of heparin, is ubiquitously distributed on the cell surface and in the extracellular matrix, whereas heparin is usually sequestered in mast cells. HS plays important roles in a number of biological phenomena such as blood coagulation, viral infection, tumor metastasis, and various developmental processes (9Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar). HS and heparin are composed of the same disaccharide units, although the proportions of sulfated monosaccharide residues vary. HS is less sulfated and more heterogeneous than heparin. The biosynthetic process of HS generates enormous structural diversity and leads to various physiological functions through different affinities with a variety of proteins such as growth factors, enzymes, and extracellular matrix components. Numerous studies of the critical role of HS and heparin in biological events have stimulated interest in elucidating the detailed structure of the polysaccharides. HS and heparin, however, are structurally heterogeneous. Further, it is difficult to raise useful antibodies against HS or heparin because these polysaccharides are ubiquitous on the cell surfaces of all animals normally used for antibody production, such as chickens, mice, and rabbits. To gain insights into the functions of HS and/or heparin, it is necessary to obtain specific probes against each sulfation pattern of HS or heparin.Thus, we decided to use phage display to obtain ligands that specifically target HS and/or heparin structures. Phage display has become a powerful method for identifying polypeptides since it was described by Smith (10Smith G.P. Science. 1985; 228: 1315-1317Crossref PubMed Scopus (3040) Google Scholar). In this method, a peptide is displayed on the N-terminal domain of the pIII coat protein of filamentous phage M13. The N-terminal domain of pIII is necessary for phage infectivity and is present in five copies at the end tip of the virion (11Deng L.W. Malik P. Perham R.N. Virology. 1999; 253: 271-277Crossref PubMed Scopus (46) Google Scholar). To ensure monovalent display of objective genes, phagemid vectors to express peptides on pIII under the control of a weak promoter were used in this study (12Bass S. Greene R. Wells J.A. Proteins. 1990; 8: 309-314Crossref PubMed Scopus (320) Google Scholar, 13Lowman H.B. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 401-424Crossref PubMed Scopus (138) Google Scholar). This monovalent display allows selection based purely on affinity to the tightest binding variants from a cDNA library.In this study, we selected heparin because of its characterized structure for further elucidating the structure-function relationship between polysaccharides and proteins. We report the identification of a novel peptide through phage display and its characteristics for discriminating heparin in a structure-specific manner.DISCUSSIONWe describe a novel peptide, HappY, which binds to a specific structure of trisaccharide in heparin isolated via phage display. Our computer modeling showed that the peptide binds to ATB heparin through three arginine residues. In particular, the N-terminal arginine residue of the peptide is critical for binding to ATB heparin because the biotinylation of the N-terminal arginine residue, which was mainly labeled at the α-amino group, abolished association with the polysaccharide completely (Fig. 2C). On the basis of a binding experiment for the binding domain of ATB heparin to HappY using fractionated oligosaccharides (Fig. 4B) and for the two LMWH to HappY (Fig. 5B), we concluded that a trisaccharide in heparin was recognized by the peptide as the minimal region. For the neutralization of the anticoagulant effect of heparin by the HappY peptide, the peptide may bind to the unique pentasaccharide region of heparin required for specific binding to AT-III, or the region of heparin that is recognized by the heparin-binding site of thrombin, also known as TABE2 (thrombin anion binding exosite 2) (21Sheehan J.P. Sadler J.E. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 5518-5522Crossref PubMed Scopus (181) Google Scholar). To distinguish the two possibilities for binding, the binding between LMWH and HappY was investigated (Fig. 5B). LMWH has less of an anticoagulant effect on thrombin compared with heparin but maintains the same effect on factor Xa, which means that LMWH contains the unique pentasaccharide region (22Hirsh J. Levine M.N. Blood. 1992; 79: 1-17Crossref PubMed Google Scholar). Our results showed that LMWH competitively bound to HappY, suggesting that the target of HappY is the pentasaccharide region of heparin. In addition, the fact that LMWH5000 competitively bound to HappY more than LMWH3000 (Fig. 5B) would indicate that the recognition domain in heparin by HappY should be consecutive monosaccharide residues. Furthermore, the fact that HappY exclusively bound to heparin or HS (Fig. 3, B and C), competitively bound to ATB heparin unlike ATU heparin (Fig. 5A), and neutralized the anticoagulant effect of heparin (Fig. 6) also indicates that it recognizes the trisaccharide, including an iduronic acid residue, that exists in both heparin and HS. Therefore, these results strongly support the hypothesis that the trisaccharide GlcN,3,6-SO3α1–4IdoA,2-SO3α1–4GlcN,6-SO3 in heparin and HS is the target domain for HappY.Cardin and Weintraub (23Cardin A.D. Weintraub H.J.R. Arterioscler. Thromb. Vasc. Biol. 1989; 9: 21-32Crossref Google Scholar) reported potential heparin-binding sites based on the sequence organizations of basic and nonbasic residues from 49 regions in 21 proteins. Consensus sequences for heparin recognition were determined as X-B-B-X-B-X and X-B-B-B-X-X-B-X, where B is the probability of a basic residue and X is a hydropathic residue. The sequence of the heparin-binding peptide obtained in this study was RTRGSTR, resulting in B-X-B-X-X-X-B. This sequence does not match the reported consensus sequences, suggesting that HappY recognizes unique heparin and HS molecules, including the trisaccharide.Verrecchio et al. (24Verrecchio A. Germann M.W. Schick B.P. Kung B. Twardowski T. San Antonio J.D. J. Biol. Chem. 2000; 275: 7701-7707Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) reported that heparin-binding peptides were designed based on consensus sequences, (AKKARA)n and (ARKKAAKA)n (n = 1–6), and then higher Mr peptides (2000–3500) exhibited significant affinities (KD ≈ 50–150 nm), which increased with peptide Mr. Meanwhile, HappY is a low Mr peptide (Mr = 1423) but showed significant affinities. Furthermore, Tashiro et al. (25Tashiro K. Monji A. Yoshida I. Hayashi Y. Matsuda K. Tashiro N. Mitsuyama Y. Biochem. J. 1999; 340: 119-126Crossref PubMed Scopus (35) Google Scholar) also reported that IKLLI-containing peptides were found to mediate heparin binding. Unlabeled heparin and HS inhibited the peptide-mediated [3H]heparin binding competitively in a dose-dependent manner. The IC50 for inhibition of heparin-peptide binding was observed with 9 μm heparin/HS, and chondroitin sulfate did not inhibit it. In addition, Schuksz et al. (26Schuksz M. Fuster M.M. Brown J.R. Crawford B.E. Ditto D.P. Lawrence R. Glass C.A. Wang L. Tor Y. Esko J.D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13075-13080Crossref PubMed Scopus (129) Google Scholar) reported that the small molecule antagonist of heparan sulfate, surfen, bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. Surfen also inhibited FGF-2-stimulated sprouting by endothelial cells with an IC50 of ≈ 5 μm. On the other hand, HappY-mediated ATB heparin binding was inhibited by 196 μg/ml of heparin in almost 50% inhibition (Fig. 3A). This concentration corresponds approximately to 16 μm (average mass of heparin, 12 kDa). HappY also showed an approximately 50% inhibition of neurite outgrowth induced by FGF-2 in PC-12 cells with ≈ 1 μm (Fig. 7C). Taken together, our data demonstrate that HappY has a high potency comparable with the known materials.As shown in Fig. 1C, three arginine residues in HappY seem to lie in a plane and face the sulfate groups of heparin when interacting with the AT-binding domain. Both the specific heparin pentasaccharide sequence and the AT polypeptide sequence that participate in the interaction were reported previously (27Grootenhuis P.D.J. van Boeckel C.A.A. J. Am. Chem. Soc. 1991; 113: 2743-2747Crossref Scopus (87) Google Scholar, 28Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar), and docking of the pentasaccharide between two peptide segments of AT has been demonstrated. This binding interaction is electrostatic, arising from the negatively charged sulfate and carboxyl groups of the pentasaccharide and the positively charged lysine and arginine side chains of the protein. It has been revealed that Arg-47, Lys-114, Lys-125, and Arg-129 are the most important residues in the heparin-binding site of AT, using a chemically modified, naturally occurring mutant and recombinant AT (29Schedin-Weiss S. Desai U.R. Bock S.C. Gettins P.G. Olson S.T. Björk I. Biochemistry. 2002; 41: 4779-4788Crossref PubMed Scopus (43) Google Scholar, 30Arocas V. Bock S.C. Raja S. Olson S.T. Bjork I. J. Biol. Chem. 2001; 276: 43809-43817Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 31Desai U. Swanson R. Bock S.C. Bjork I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 32Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar). In addition, the Ala-124-Arg145 sequence, found in the D helix of AT, is rich in basic amino acids, including lysines (125, 133, and 136) and arginines (129 and 132) that face the heparin pentasaccharide (27Grootenhuis P.D.J. van Boeckel C.A.A. J. Am. Chem. Soc. 1991; 113: 2743-2747Crossref Scopus (87) Google Scholar, 28Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). Furthermore, Onoue et al. (33Onoue S. Nemoto Y. Harada S. Yajima T. Kashimoto K. Life Sci. 2003; 73: 2793-2806Crossref PubMed Scopus (8) Google Scholar) reported that human AT-III-derived heparin-binding peptide is a novel heparin antagonist in which lysines are substituted with arginines. We have deduced that HappY interacts with heparin through an electrostatic association between the positively charged arginines and negatively charged saccharides. Therefore, HappY associates with its target molecule in a structure-dependent manner, resulting in specific binding to heparin and/or HS.AT associates with a specific pentasaccharide structure that has 3-O-sulfated glucosamine modified by HS 3-O-sulfotransferase 1 (17Liu J. Shworak N.W. Fritze L.M. Edelberg J.M. Rosenberg R.D. J. Biol. Chem. 1996; 271: 27072-27082Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In this study, HappY associated with the trisaccharide structure, including the 3-O-sulfated glucosamine residue. 3-O-sulfation of d-glucosamine residues is the rarest modification in HS/heparin biosynthesis and is a key regulator for generating HS-unique sequences that are believed to define specific protein interaction patterns. Thus, the activities of several effectors associated with HS are influenced by selective binding to 3-O-sulfated HS motifs. Other than AT, the envelope glycoprotein D of the herpes simplex virus type 1 has also been found to bind to the 3-O-sulfated HS on target cells (34Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar, 35McKeehan W.L. Wu X. Kan M. J. Biol. Chem. 1999; 274: 21511-21514Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Furthermore, functions that are accelerated by 3-O-sulfated HS have recently been reported, such as binding to fibroblast growth factor 7 (34Shukla D. Liu J. Blaiklock P. Shworak N.W. Bai X. Esko J.D. Cohen G.H. Eisenberg R.J. Rosenberg R.D. Spear P.G. Cell. 1999; 99: 13-22Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar) and to a receptor for fibroblast growth factors (36Ye S. Luo Y. Lu W. Jones R.B. Linhardt R.J. Capila I. Toida T. Kan M. Pelletier H. McKeehan W.L. Biochemistry. 2001; 40: 14429-14439Crossref PubMed Scopus (127) Google Scholar), the predominant restriction of daytime pineal glands (37Kuberan B. Lech M. Borjigin J. Rosenberg R.D. J. Biol. Chem. 2004; 279: 5053-5054Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), regulation of Notch signaling (38Kamimura K. Rhodes J.M. Ueda R. McNeely M. Shukla D. Kimata K. Spear P.G. Shworak N.W. Nakato H. J. Cell Biol. 2004; 166: 1069-1079Crossref PubMed Scopus (73) Google Scholar), and interaction with cyclophilin B binding to responsive cells (39Vanpouille C. Deligny A. Delehedde M. Denys A. Melchior A. Liénard X. Lyon M. Mazurier J. Fernig D.G. Allain F. J. Biol. Chem. 2007; 282: 24416-24429Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This modification of HS is very difficult to detect in vivo because it is the rarest modification for HS biosynthesis. HappY has the potential to detect this functional moiety of HS in the near future.Heparin has also shown strong anti-inflammatory effects apart from its anticoagulant activity, including the inhibition of complement activation (40Teixeira M.M. Rossi A.G. Hellewell P.G. J. Leukocyte Biol. 1996; 59: 389-396Crossref PubMed Scopus (25) Google Scholar, 41Weiler J.M. Edens R.E. Linhardt R.J. Kapelanski D.P. J. Immunol. 1992; 148: 3210-3215PubMed Google Scholar) and inhibition of neutrophil-derived elastase (42Brown R.A. Lever R. Jones N.A. Page C.P. Br. J. Pharmacol. 2003; 139: 845-853Crossref PubMed Scopus (63) Google Scholar). Although heparin has great potential as an anti-inflammatory agent, its clinical use is impaired by its strong anticoagulant activity and hemorrhagic complications. To overcome this problem, some chemical modifications from mammalian heparin have been developed (43Gao Y. Li N. Fei R. Chen Z. Zheng S. Zeng X. Mol. Cells. 2005; 19: 350-355PubMed Google Scholar, 44Wan J.G. Mu J.S. Zhu H.S. Geng J.G. Inflamm. Res. 2002; 51: 435-443Crossref PubMed Scopus (15) Google Scholar), and a heparin-like compound from shrimp was reported to be a better alternative than mammalian heparin as a possible anti-inflammatory drug (45Brito A.S. Arimatéia D.S. Souza L.R. Lima M.A. Santos V.O. Medeiros V.P. Ferreira P.A. Silva R.A. Ferreira C.V. Justo G.Z. Leite E.L. Andrade G.P. Oliveira F.W. Nader H.B. Chavante S.F. Bioorg. Med. Chem. 2008; 16: 9588-9595Crossref PubMed Scopus (51) Google Scholar). The fact that HappY neutralizes the anticoagulant effect of heparin (Fig. 6) makes it potentially useful for facilitating the anti-inflammatory effects of heparin without the anticoagulant effects. HappY may contribute to the development of treatments for chronic and acute inflammatory diseases.In PC-12 cells, agrin, HS proteoglycan, increases the efficacy of FGF-2 stimulation of neurite outgrowth mediated by the FGF receptor (46Kim M.J. Cotman S.L. Halfter W. Cole G.J. J. Neurobiol. 2003; 55: 261-277Crossref PubMed Scopus (63) Google Scholar). The possible mechanisms by which agrin may modulate neurite outgrowth had been investigated, analyzing ERK phosphorylation and c-fos phosphorylation. HappY inhibited the neurite outgrowth in PC-12 cells by FGF-2 (Fig. 7). This result suggests that HappY binds to HS attached at the agrin and affects neurite outgrowth.A major advantage of the phage display system is that amino acid sequences of exogenous peptides expressed on the phage envelope are easily available after screening. Because almost no glycosaminoglycans are immunoreactive and there are few antibodies against them, this advantage is extremely useful for generating probes that bind to glycosaminoglycans. Thus, phage display technology is increasingly important for studying the functions of glycosaminoglycans (47Bruinsma I.B. te Riet L. Gevers T. ten Dam G.B. van Kuppevelt T.H. David G. Küsters B. de Waal R.M. Verbeek M.M. Acta Neuropathol. 2010; 119: 211-220Crossref PubMed Scopus (48) Google Scholar, 48Timmer N.M. Schirris T.J. Bruinsma I.B. Otte-Höller I. van Kuppevelt T.H. de Waal R.M. Verbeek M.M. Neurosci. Res. 2010; 66: 380-389Crossref PubMed Scopus (23) Google Scholar, 49Ten Dam G.B. Yamada S. Kobayashi F. Purushothaman A. van de Westerlo E.M. Bulten J. Malmström A. Sugahara K. Massuger L.F. van Kuppevelt T.H. Histochem. Cell Biol. 2009; 132: 117-127Crossref PubMed Scopus (28) Google Scholar, 50Thompson S.M. Fernig D.G. Jesudason E.C. Losty P.D. van de Westerlo E.M.A. van Kuppevelt T.H. Turnbull J.E. J. Biol. Chem. 2009; 284: 35621-35631Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 51Li F. Ten Dam G.B. Murugan S. Yamada S. Hashiguchi T. Mizumoto S. Oguri K. Okayama M. van Kuppevelt T.H. Sugahara K. J. Biol. Chem. 2008; 283: 34294-34304Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 52Purushothaman A. Fukuda J. Mizumoto S. ten Dam G.B. van Kuppevelt T.H. Kitagawa H. Mikami T. Sugahara K. J. Biol. Chem. 2007; 282: 19442-19452Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The generation of type-specific anti-HS antibodies using phage display technology was initially applied by van Kuppevelt et al. (53van Kuppevelt T.H. Dennissen M.A. van Venrooij W.J. Hoet R.M. Veerkamp J.H. J. Biol. Chem. 1998; 273: 12960-12966Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) for investigating HS heterogeneity in the kidney. In their study, three different phage clones expressing anti-HS single-chain variable fragment antibodies were obtained by a human semisynthetic phage library that contained 50 different VH genes with synthetic random complementarity-determining region 3 segments. In contrast, we used a mouse brain cDNA library instead of single-chain variable fragment antibodies in our phage display method. Thus, peptides of various sizes that bound to heparin were expected through our method. Recently, some physiological functions regulated by interactions between proteins and glycosaminoglycans have been reported (for reviews, see Refs. 54Laremore T.N. Zhang F. Dordick J.S. Liu J. Linhardt R.J. Curr. Opin. Chem. Biol. 2009; 13: 633-640Crossref PubMed Scopus (96) Google Scholar, 55Bishop J.R. Schuksz M. Esko J.D. Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1240) Google Scholar, 56Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar), and phage display will also be useful for finding novel proteins with these features. Furthermore, if peptides that discriminate the different structures of heparin from HappY or the structures of other glycosaminoglycans are generated through this technique, they should facilitate clinical and basic studies of glycosaminoglycans. In the near future, such peptide tools will make a contribution to studying diseases that are caused by alterations of the structure of glycosaminoglycans in tissues and will potentially be used in diagnosing such diseases.In conclusion, the HappY peptide, which discriminates the specific structure of the trisaccharide GlcN,3,6-SO3α1–4IdoA,2-SO3α1–4GlcN,6-SO3 in heparin and HS, can be used as a probe to detect the structure of heparin and/or HS. Further studies on synthetic trisaccharides of similar structure will be needed to discover the structural specificity with which HappY binds to heparin and/or HS. IntroductionHeparin, which is comprised of heterogeneous mixtures of complex, linear, sulfated polysaccharides consisting of one to four linked uronic acid and glucosamine residues, is well known for its potent anticoagulant activity (1Linhardt R.J. J. Med. Chem. 2003; 46: 2551-2564Crossref PubMed Scopus (429) Google Scholar, 2Casu B. Lindahl U. Adv. Carbohydr. Chem. Biochem. 2001; 57: 159-206Crossref PubMed Scopus (347) Google Scholar, 3Machovich R. Biochim. Biophys. Acta. 1975; 412: 13-17Crossref PubMed Scopus (62) Google Scholar). The antithrombin-binding (ATB) 2The abbreviations used are: ATB, antithrombin-binding; AT, antithrombin; HS, heparan sulfate; LMWH, low-molecular-weight heparin; SPR, surface plasmon resonance; FC, flow cell; ATU, antithrombin unbound. domain mainly exerts its activity by binding to antithrombin (AT), a 55-kDa single-chain plasma glycoprotein (4Damus P.S. Hicks M. Rosenberg R.D. Nature. 1973; 246: 355-357Crossref PubMed Scopus (417) Google Scholar, 5Rosenberg R.D. Damus P.S. J. Biol. Chem. 1973; 248: 6490-6505Abstract Full Text PDF PubMed Google Scholar), and has a unique pentasaccharide structure (6Oosta G.M. Gardner W.T. Beeler D.L. Rosenberg R.D. Proc. Natl. Acad. Sci. U.S.A. 1981; 78: 829-833Crossref PubMed Scopus (148) Google Scholar). This pentasaccharide sequence is randomly distributed along the heparin chains and is present in only about one-third of the heparins currently used in the treatment of thrombosis (7Lam L.H. Silbert J.E. Rosenberg R.D. Biochem. Biophys. Res. Commun. 1976; 69: 570-577Crossref PubMed Scopus (368) Google Scholar). For a review of the prevention of thrombosis, see Ref. 8Middeldorp S. Thromb. Res. 2008; 122: 753-762Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar.Heparan sulfate (HS), which is a structural analog of heparin, is ubiquitously distributed on the cell surface and in the extracellular matrix, whereas heparin is usually sequestered in mast cells. HS plays important roles in a number of biological phenomena such as blood coagulation, viral infection, tumor metastasis, and various developmental processes (9Bernfield M. Götte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar). HS and heparin are composed of the same disaccharide units, although the proportions of sulfated monosaccharide residues vary. HS is less sulfated and more heterogeneous than heparin. The biosynthetic process of HS generates enormous structural diversity and leads to various physiological functions through different affinities with a variety of proteins such as growth factors, enzymes, and extracellular matrix components. Numerous studies of the critical role of HS and heparin in biological events have stimulated interest in elucidating the detailed structure of the polysaccharides. HS and heparin, however, are structurally heterogeneous. Further, it is difficult to raise useful antibodies against HS or heparin because these polysaccharides are ubiquitous on the cell surfaces of all animals normally used for antibody production, such as chickens, mice, and rabbits. To gain insights into the functions of HS and/or heparin, it is necessary to obtain specific probes against each sulfation pattern of HS or heparin.Thus, we decided to use phage display to obtain ligands that specifically target HS and/or heparin structures. Phage display has become a powerful method for identifying polypeptides since it was described by Smith (10Smith G.P. Science. 1985; 228: 1315-1317Crossref PubMed Scopus (3040) Google Scholar). In this method, a peptide is displayed on the N-terminal domain of the pIII coat protein of filamentous phage M13. The N-terminal domain of pIII is necessary for phage infectivity and is present in five copies at the end tip of the virion (11Deng L.W. Malik P. Perham R.N. Virology. 1999; 253: 271-277Crossref PubMed Scopus (46) Google Scholar). To ensure monovalent display of objective genes, phagemid vectors to express peptides on pIII under the control of a weak promoter were used in this study (12Bass S. Greene R. Wells J.A. Proteins. 1990; 8: 309-314Crossref PubMed Scopus (320) Google Scholar, 13Lowman H.B. Annu. Rev. Biophys. Biomol. Struct. 1997; 26: 401-424Crossref PubMed Scopus (138) Google Scholar). This monovalent display allows selection based purely on affinity to the tightest binding variants from a cDNA library.In this study, we selected heparin because of its characterized structure for further elucidating the structure-function relationship between polysaccharides and proteins. We report the identification of a novel peptide through phage display and its characteristics for discriminating heparin in a structure-specific manner.
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