An Unusual Carbohydrate Binding Site Revealed by the Structures of Two Maackia amurensis Lectins Complexed with Sialic Acid-containing Oligosaccharides
2000; Elsevier BV; Volume: 275; Issue: 23 Linguagem: Inglês
10.1074/jbc.m000560200
ISSN1083-351X
AutoresAnne Imberty, Catherine Gautier, Julien Lescar, Serge Pérez, Lode Wyns, Remy Loris,
Tópico(s)Galectins and Cancer Biology
ResumoSeeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (αNeuAc(2–3)βGal(1–4)βGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (αNeuAc(2–3)βGal(1–3)[αNeuAc(2–6)]αGalNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-Å resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensishemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations. Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (αNeuAc(2–3)βGal(1–4)βGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (αNeuAc(2–3)βGal(1–3)[αNeuAc(2–6)]αGalNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-Å resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensishemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations. M. amurensis leukoagglutinin M. amurensishemagglutinin sialyl-N-acetyllactosamine Galβ1,3GalNAcβ1,4(NeuAcα2,3)Galβ1,4Glcβ1, 4Glc-ceramide Lectins are oligomeric proteins that specifically recognize carbohydrates (for a review, see Ref. 1.Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar). Widespread in all living organisms, from microbes and invertebrates to plants and vertebrates, they are key players in many recognition events involved in fertilization, embryogenesis, inflammation, metastasis, and host-parasite recognition. In the plant kingdom, lectins have been demonstrated to play a role in establishing symbiosis with bacteria and mushrooms as well as in defense against pathogens or predators (2.Etzler M.E. Annu. Rev. Plant Physiol. 1985; 36: 209-234Crossref Google Scholar, 3.Chrispeels M.J. Raikhel N.V. Plant Cell. 1991; 3: 1-9Crossref PubMed Scopus (547) Google Scholar, 4.Van Damme E.J.M. Peumans W.J. Barre A. Rougé P. Crit. Rev. Plant Sci. 1998; 17: 575-692Crossref Scopus (566) Google Scholar). In addition, their fine specificity makes plant lectins excellent tools for the identification and purification of complex carbohydrates, hence their wide use in biochemical and medical research (5.Lis H. Sharon N. Annu. Rev. Biochem. 1986; 1986: 35-67Crossref Scopus (618) Google Scholar, 6.Van Damme E.J.M. Peumans W.J. Pusztai A. Bardocz S. Handbook of Plant Lectins: Properties and Biomedical Applications. John Wiley & Sons, New York1997Google Scholar).Lectins from the seeds, stem, and bark of legumes represent the largest and most thoroughly studied family of lectins (7.Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (468) Google Scholar). They consist of two or four identical or almost identical subunits, each containing one carbohydrate binding site and two metal binding sites for Ca2+ and for Mn2+. At present, more than 80 crystal structures of legume lectins have been determined from 15 different plants (see Protein Data Bank and 3D Lectin Data Base). Most of these structures have been obtained as a complex with a bound sugar. Although no lectins specific for fucose or GlcNAc have been crystallized, much information is available for two classes of legume lectins: (i) the Man/Glc specificity group such as concanavalin A and pea lectin and (ii) the Gal/GalNAc specificity group that includes peanut lectin and soybean agglutinin. As for the complex specificity group, the only co-crystals reported so far are those ofGriffonia simplicifolia isolectin IV complexed with two histo-blood group oligosaccharides (8.Delbaere L.T. Vandonselaar M. Prasad L. Quail J.W. Wilson K.S. Dauter Z. J. Mol. Biol. 1993; 230: 950-965Crossref PubMed Scopus (151) Google Scholar).The presence of a compound with hemagglutinating activity in the seeds of Maackia amurensis was described in the early 1960s (9.Boyd W.C. Waszczenko-Zacharczenko E. Goldwasser S.M. Transfusion. 1961; 1: 374-382Crossref PubMed Scopus (34) Google Scholar). Two isolectins were purified and designated hemagglutinin (MAH)1 and leukoagglutinin (MAL), reflecting their specific agglutination activity toward different blood cell types (10.Kawaguchi T. Matsumoto I. Osawa T. J. Biol. Chem. 1974; 249: 2786-2792Abstract Full Text PDF PubMed Google Scholar). The first carbohydrate binding specificity studies, conducted using competitive binding assays to human erythrocytes, indicated that MAH preferentially binds toO-linked carbohydrate chains, whereas MAL bindsN-linked glycans (10.Kawaguchi T. Matsumoto I. Osawa T. J. Biol. Chem. 1974; 249: 2786-2792Abstract Full Text PDF PubMed Google Scholar, 11.Kawaguchi T. Osawa T. Biochemistry. 1976; 15: 4581-4586Crossref PubMed Scopus (38) Google Scholar). Later, MAL was shown to bind strongly to carbohydrate chains containing sialic acid and particularly to the αNeuAc(2–3)βGal(1–4)βGlcNAc/Glc sequence (12.Wang W.-C. Cummings R. J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar, 13.Knibbs R.N. Goldstein I.J. Ratcliffe R.M. Shibuya N. J. Biol. Chem. 1991; 266: 83-88Abstract Full Text PDF PubMed Google Scholar). Carbohydrate specificity of MAH is slightly different since its highest affinity is directed toward the αNeuAc(2–3)βGal(1–3)[αNeuAc(2–6)]αGalNAc tetrasaccharide (14.Konami Y. Yamamoto K. Osawa T. Irimura T. FEBS Lett. 1994; 342: 334-338Crossref PubMed Scopus (78) Google Scholar).Amino acid sequences of MAL (15.Yamamoto K. Konami Y. Irimura T. J. Biochem. (Tokyo). 1997; 121: 756-761Crossref PubMed Scopus (49) Google Scholar) and MAH (16.Konami Y. Ishida C. Yamamoto K. Osawa T. Irimura T. J. Biochem. 1994; 115: 767-777Crossref PubMed Scopus (17) Google Scholar, 17.Yamamoto K. Ishida C. Saito M. Konami Y. Osawa T. Irimura T. Glycoconj. J. 1994; 11: 572-575Crossref PubMed Scopus (14) Google Scholar) share 86% identity but are rather different from other legume lectins with an average sequence identity of 40%. Indeed, these two lectins have peculiarities in their binding sites. In all legume lectins, a triad of conserved Asp, Gly, and Asn residues located at the bottom of the binding site, establish hydrogen bonds with the carbohydrate. For both MAL and MAH, the Asp residue is conserved but Gly is replaced by Lys and Asn by Asp. Mutagenesis studies conducted on MAH demonstrated that the ligand binding is abolished by Lys105 → Gly or Asp135 → Asn mutations (15.Yamamoto K. Konami Y. Irimura T. J. Biochem. (Tokyo). 1997; 121: 756-761Crossref PubMed Scopus (49) Google Scholar).We report here the structures of these two legume lectins in order to understand their fine specificity toward sialylated glycans. The crystal structure of the MAL/sialyllactose determined at 2.75-Å resolution reveals unexpected details of this legume lectin binding site, with a new mode of binding for the central residue in the monosaccharide binding site, and extensive contacts with the three carbohydrate residues. This crystal structure served as a template for modeling the MAH protein and its interaction with a disialylated tetrasaccharide. A secondary binding site is predicted to accommodate the second sialic acid residue. Lectins are oligomeric proteins that specifically recognize carbohydrates (for a review, see Ref. 1.Lis H. Sharon N. Chem. Rev. 1998; 98: 637-674Crossref PubMed Scopus (1626) Google Scholar). Widespread in all living organisms, from microbes and invertebrates to plants and vertebrates, they are key players in many recognition events involved in fertilization, embryogenesis, inflammation, metastasis, and host-parasite recognition. In the plant kingdom, lectins have been demonstrated to play a role in establishing symbiosis with bacteria and mushrooms as well as in defense against pathogens or predators (2.Etzler M.E. Annu. Rev. Plant Physiol. 1985; 36: 209-234Crossref Google Scholar, 3.Chrispeels M.J. Raikhel N.V. Plant Cell. 1991; 3: 1-9Crossref PubMed Scopus (547) Google Scholar, 4.Van Damme E.J.M. Peumans W.J. Barre A. Rougé P. Crit. Rev. Plant Sci. 1998; 17: 575-692Crossref Scopus (566) Google Scholar). In addition, their fine specificity makes plant lectins excellent tools for the identification and purification of complex carbohydrates, hence their wide use in biochemical and medical research (5.Lis H. Sharon N. Annu. Rev. Biochem. 1986; 1986: 35-67Crossref Scopus (618) Google Scholar, 6.Van Damme E.J.M. Peumans W.J. Pusztai A. Bardocz S. Handbook of Plant Lectins: Properties and Biomedical Applications. John Wiley & Sons, New York1997Google Scholar). Lectins from the seeds, stem, and bark of legumes represent the largest and most thoroughly studied family of lectins (7.Loris R. Hamelryck T. Bouckaert J. Wyns L. Biochim. Biophys. Acta. 1998; 1383: 9-36Crossref PubMed Scopus (468) Google Scholar). They consist of two or four identical or almost identical subunits, each containing one carbohydrate binding site and two metal binding sites for Ca2+ and for Mn2+. At present, more than 80 crystal structures of legume lectins have been determined from 15 different plants (see Protein Data Bank and 3D Lectin Data Base). Most of these structures have been obtained as a complex with a bound sugar. Although no lectins specific for fucose or GlcNAc have been crystallized, much information is available for two classes of legume lectins: (i) the Man/Glc specificity group such as concanavalin A and pea lectin and (ii) the Gal/GalNAc specificity group that includes peanut lectin and soybean agglutinin. As for the complex specificity group, the only co-crystals reported so far are those ofGriffonia simplicifolia isolectin IV complexed with two histo-blood group oligosaccharides (8.Delbaere L.T. Vandonselaar M. Prasad L. Quail J.W. Wilson K.S. Dauter Z. J. Mol. Biol. 1993; 230: 950-965Crossref PubMed Scopus (151) Google Scholar). The presence of a compound with hemagglutinating activity in the seeds of Maackia amurensis was described in the early 1960s (9.Boyd W.C. Waszczenko-Zacharczenko E. Goldwasser S.M. Transfusion. 1961; 1: 374-382Crossref PubMed Scopus (34) Google Scholar). Two isolectins were purified and designated hemagglutinin (MAH)1 and leukoagglutinin (MAL), reflecting their specific agglutination activity toward different blood cell types (10.Kawaguchi T. Matsumoto I. Osawa T. J. Biol. Chem. 1974; 249: 2786-2792Abstract Full Text PDF PubMed Google Scholar). The first carbohydrate binding specificity studies, conducted using competitive binding assays to human erythrocytes, indicated that MAH preferentially binds toO-linked carbohydrate chains, whereas MAL bindsN-linked glycans (10.Kawaguchi T. Matsumoto I. Osawa T. J. Biol. Chem. 1974; 249: 2786-2792Abstract Full Text PDF PubMed Google Scholar, 11.Kawaguchi T. Osawa T. Biochemistry. 1976; 15: 4581-4586Crossref PubMed Scopus (38) Google Scholar). Later, MAL was shown to bind strongly to carbohydrate chains containing sialic acid and particularly to the αNeuAc(2–3)βGal(1–4)βGlcNAc/Glc sequence (12.Wang W.-C. Cummings R. J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar, 13.Knibbs R.N. Goldstein I.J. Ratcliffe R.M. Shibuya N. J. Biol. Chem. 1991; 266: 83-88Abstract Full Text PDF PubMed Google Scholar). Carbohydrate specificity of MAH is slightly different since its highest affinity is directed toward the αNeuAc(2–3)βGal(1–3)[αNeuAc(2–6)]αGalNAc tetrasaccharide (14.Konami Y. Yamamoto K. Osawa T. Irimura T. FEBS Lett. 1994; 342: 334-338Crossref PubMed Scopus (78) Google Scholar). Amino acid sequences of MAL (15.Yamamoto K. Konami Y. Irimura T. J. Biochem. (Tokyo). 1997; 121: 756-761Crossref PubMed Scopus (49) Google Scholar) and MAH (16.Konami Y. Ishida C. Yamamoto K. Osawa T. Irimura T. J. Biochem. 1994; 115: 767-777Crossref PubMed Scopus (17) Google Scholar, 17.Yamamoto K. Ishida C. Saito M. Konami Y. Osawa T. Irimura T. Glycoconj. J. 1994; 11: 572-575Crossref PubMed Scopus (14) Google Scholar) share 86% identity but are rather different from other legume lectins with an average sequence identity of 40%. Indeed, these two lectins have peculiarities in their binding sites. In all legume lectins, a triad of conserved Asp, Gly, and Asn residues located at the bottom of the binding site, establish hydrogen bonds with the carbohydrate. For both MAL and MAH, the Asp residue is conserved but Gly is replaced by Lys and Asn by Asp. Mutagenesis studies conducted on MAH demonstrated that the ligand binding is abolished by Lys105 → Gly or Asp135 → Asn mutations (15.Yamamoto K. Konami Y. Irimura T. J. Biochem. (Tokyo). 1997; 121: 756-761Crossref PubMed Scopus (49) Google Scholar). We report here the structures of these two legume lectins in order to understand their fine specificity toward sialylated glycans. The crystal structure of the MAL/sialyllactose determined at 2.75-Å resolution reveals unexpected details of this legume lectin binding site, with a new mode of binding for the central residue in the monosaccharide binding site, and extensive contacts with the three carbohydrate residues. This crystal structure served as a template for modeling the MAH protein and its interaction with a disialylated tetrasaccharide. A secondary binding site is predicted to accommodate the second sialic acid residue. We thank the ESRF (Grenoble, France) for access to synchrotron beamline and particularly Dr. Wilhelm Burmeister and Dr. Eward Mitchell for their help with data collection.
Referência(s)