Abstract
Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.
Similar content being viewed by others
References
Almanza M, Vega N, Pérez G (2004) Isolating and characterizing a lectin from Galactia lindenii seeds that recognises blood group H determinants. Arch Biochem Biophys 429:180–190
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Arango R, Rozenblatt S, Sharon N (1990) Cloning and sequence analysis of the Erythrina corallodendron lectin cDNA. FEBS Lett 264:109–111
Bourne Y, Ayouba A, Rouge P, Cambillau C (1994) Interaction of a legume lectin with two components of the bacterial cell wall; a crystallographic study. J Biol Chem 269:9429–9435
Brinda KV, Mitra N, Surolia A, Vishveshwara S (2004) Determinants of quaternary association in legume lectins. Protein Sci 13:1735–1749
Campana PT, Moraes DI, Moteiro-Moreira ACO, Beltramini LM (2002) Unfolding and refolding studies of frutalin, a tetrameric d-galactose binding lectin. Eur J Biochem 269:753–758
Carrington DM, Auffret A, Hanke DE (1985) Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature 313:64–67
Chatterjee A, Mandal DK (2003) Denaturant-induced equilibrium unfolding of concanavalin A is expressed by a three-state mechanism and provides an estimate of its protein stability. Biochim Biophys Acta 1648:174–183
Chatterjee A, Mandal DK (2005) Quaternary association and reactivation of dimeric concanavalin A. Inter J Biol Macromol 35:103–109
Cominetti MR, Terruggi CH, Ramos OH, Fox JW, Mariano-Oliveira A, de Freitas MS, Figueiredo CC, Morandi V, Selistre-De-Araujo HS (2004) Alternagin-C, a disintegrin-like protein, induces vascular endothelial cell growth factor (VEGF) expression and endothelial cell proliferation in vitro. J Biol Chem 279:18247–18255
Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, pp 571–607
Gatehouse AMR, Powell KS, Van Damme EJM, Peumans WJ (1995) Insecticidal properties of plant lectins their potential in plant protection. In: Pustzai A, Bardocz S (eds) Lectins: biomedical perspectives. Taylor and Francis, London, pp 35–58
Grangeiro TB, Schriefer A, Calvete JJ, Raida M, Urbanke C, Barral-Netto M, Cavada BS (1997) Molecular cloning and characterization of ConBr, the lectin of Canavalia brasiliensis seeds. Eur J Biochem 248:43–48
Johnson CR, Morin PE, Arrowsmith CH, Freire E (1995) Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor. Biochem 34:5309–5316
Konarev PV, Petoukhov MV, Svergun DI (2001) MASSHA–a graphics system for rigid-bodymodelling of macromolecular complexes against solution scattering data. J Appl Cryst 34:527–532
Kozin MB, Svergun DI (2001) Automatedmatching of high- and low-resolution structural models. J Appl Cryst 34:33–41
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Plenum Publishers, New York
Larkin MA et al (2007) ClustalW and ClustalX version 2. Bioinformatics 23:2947–2948
Loris R, Hamelryck T, Bouckaert J, Wyns L (1998) Legume lectin structure. Biochim Biophys Acta 1383:9–36
Mallam AL, Jackson SE (2005) Folding studies on a knotted protein. J Mol Biol 346:1409–1421
Moreno FB, de Oliveira TM, Martil DE, Vicoti MM, Bezerra GA, Abrego JR, Cavada BS, Filgueira AJ (2008) Identification of a new quaternary association for legume lectins. J Struct Biol 161:133–143
Petoukhov MV, Svergun DI (2003) New methods for domain structure determination of proteins from solution scattering data. J Appl Cryst 36:540–544
Prabu MM, Sankaranarayanan R, Puri KD, Sharma V, Surolia A, Vijayan M, Suguna K (1998) Carbohydrate specificity and quaternary association in basic winged bean lectin: X-ray analysis of the lectin at 2.5 Å resolution. J Mol Biol 276:787–796
Rabijns A, Verboven C, Rougé P, Barre A, Van Damme EJM, Peumans WJ, De Ranter CJ (2001) Structure of a legume lectin from the bark of Robinia pseudoacacia and its complex with N-Acetylgalactosamine. Proteins Structure Funct Bioinform 44:470–478
Rüdiger H, Gabius HJ (2001) Plant lectins: occurrence, biochemistry, functions and applications. Glycoconjugate J 18:589–613
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Santos-de-Oliveira R, Dias-Baruffi M, Thomaz SM, Beltramini LM, Roque-Barreira MC (1994) A neutrophil migration-inducing lectin from Artocarpus integrifólia. The J Immunol 153:1798–1807
Sharon N (2007) Lectins: carbohydrate-specific reagents and biological recognition molecules. J Biol Chem 282:2753–2764
Sharon N, Lis H (1990) Legume lectins—a large family of homologous proteins. FASEB J 4:3198–3208
Sharon N, Lis H (2004) History of lectins: from hemagglutinins to biological recognition molecules. Glycobiol 14:53–62
Sheng S, Carey J, Seftor EA, Dias L, Hendrix MJ, Sager R (1996) Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells. Proc Natl Acad Sci USA 93:11669–11674
Sinha S, Surolia A (2005) Oligomerization endows enormous stability to soybean agglutinin: a comparison of the stability of monomer and tetramer of soybean agglutinin. Biophys J 88:4243–4251
Sinha S, Mitra N, Kumar G, Bajaj K, Surolia A (2005) Unfolding studies on soybean agglutinin and concanavalin a tetramers: a comparative account. Biophys J 88:1300–1310
Sreerama N, Venyaminov SY, Woody RW (2001) Analysis of protein CD spectra with a reference protein set based on tertiary structure class. Anal Biochem 299:271–274
Srinivas VR, Reddy GB, Ahmad N, Swaminathan CP, Mitra N, Surolia A (2001) Legume lectin family, the “natural mutants of the quaternary state”, provide insights into the relationship between protein stability and oligomerization. Biochim Biophys Acta 1527:102–111
Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503
Svergun DI, Semenyuk AV, Feigin LA (1988) Small angle- scattering-data treatment by the regularization method. Acta Crystallogr A 44:244–250
Svergun D, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Cryst 28:768–773
Svergun DI, Petoukhov MV, Koch MH (2001) Determination of domain structure of proteins from X-ray solution scattering. Biophys J 80:2946–2953
Takada Y, Helmer ME (1989) The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain. J Cell Biol 109:397–407
Van Damme EJ, Barre A, Smeets K, Torrekens S, Van Leuven F, Rouge P, Peumans WJ (1995) The bark of Robinia pseudoacacia contains a complex mixture of lectins. Characterization of the proteins and the cDNA clones. Plant Physiol 107:833–843
Van Damme EJM, Peumans WJ, Barre A, Rougé P (1998) Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles. Crit Rev Plant Sci 17:575–692
Varela ED, Lima JPMS, Galdino AS, Pinto LS, Bezerra WM, Nunes EP, Alves MAO, Grangeiro TB (2004) Relationships in subtribe diocleinae (Leguminosae; Papilionoideae) inferred from internal transcribed spacer sequences from nuclear ribosomal DNA. Phytochem 65:59–69
Yoshida K, Tazaki K (1999) Expression patterns of the genes that encode lectin or lectin-related polypeptides in Robinia pseudoacacia. Aust J Plant Physiol 26:495–502
Acknowledgments
The authors are very grateful to Dr. Lúcia Rossi for botanical identification work carried out at the Botanical Institute, São Paulo, Brazil.
Author information
Authors and Affiliations
Corresponding author
Additional information
F. A. H. Batista and L. S. Goto contributed equally to this work.
Rights and permissions
About this article
Cite this article
Batista, F.A.H., Goto, L.S., Garcia, W. et al. Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis. Eur Biophys J 39, 1193–1205 (2010). https://doi.org/10.1007/s00249-009-0571-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-009-0571-5