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In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDLcontaining proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and thatCHDLdomains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Documento: Artículo
Título:RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize rhizobium leguminosarum acidic exopolysaccharides
Autor:Abdian, P.L.; Caramelo, J.J.; Ausmees, N.; Zorreguieta, A.
Filiación:Fundación Instituto Leloir, IIBBA Consejo Nacional de Investigaciones Científicas y Tecnológicas, Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Intendente Güiraldes 2160, C1428EGA Buenos Aires, Argentina
Department of Biology, Lund University, Sölvegatan 35, 22362 Lund, Sweden
Palabras clave:Archaea; Archaeal; Biofilm formation; Biofilm matrix; Cadherins; Calcium binding; Calcium ions; Carbohydrate binding; Cellular interaction; Conserved proteins; Exopolysaccharides; Homo-oligomers; In-silico; Isothermal titration calorimetry; Plant roots; Protein molecules; Rhizobium leguminosarum; Structural similarity; Bacteria; Biofilms; Calcium; Circular dichroism spectroscopy; Glycoproteins; Light scattering; Metabolites; Oligomerization; Oligomers; Polysaccharides; Proteins; cadherin; calcium binding protein; calcium ion; exopolysaccharide; oligomer; protein RapA2; unclassified drug; article; beta sheet; circular dichroism; controlled study; isothermal titration calorimetry; light scattering; nonhuman; priority journal; protein conformation; protein determination; protein domain; protein folding; protein function; protein protein interaction; protein stability; Rhizobium leguminosarum; Amino Acid Sequence; Bacterial Proteins; Cadherins; Calcium; Calcium-Binding Proteins; Calorimetry; Lectins; Molecular Sequence Data; Polysaccharides; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Cell Surface; Recombinant Proteins; Rhizobium leguminosarum; Sequence Homology, Amino Acid; Solvents
Página de inicio:2893
Página de fin:2904
Título revista:Journal of Biological Chemistry
Título revista abreviado:J. Biol. Chem.
CAS:calcium ion, 14127-61-8; Bacterial Proteins; Cadherins; Calcium, 7440-70-2; Calcium-Binding Proteins; Lectins; Polysaccharides; Receptors, Cell Surface; Recombinant Proteins; Solvents; saccharide-binding proteins


  • Downie, J.A., The roles of extracellular proteins, polysaccharides, and signals in the interactions of rhizobia with legume roots (2010) FEMS Microbiol. Rev., 34, pp. 150-170
  • Rinaudi, L.V., Giordano, W., An integrated view of biofilm formation in rhizobia (2010) FEMS Microbiol. Lett., 304, pp. 1-11
  • Russo, D.M., Williams, A., Edwards, A., Posadas, D.M., Finnie, C., Dankert, M., Downie, J.A., Zorreguieta, A., Proteins exported via the PrsD-PrsE type i secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum (2006) J. Bacteriol., 188, pp. 4474-4486
  • Williams, A., Wilkinson, A., Krehenbrink, M., Russo, D.M., Zorreguieta, A., Downie, J.A., Glucomannan-mediated attachment of Rhizobium leguminosarum to pea root hairs is required for competitive nodule infection (2008) J. Bacteriol., 190, pp. 4706-4715
  • Krehenbrink, M., Downie, J.A., Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv viciae (2008) BMC Genomics, 9, p. 55
  • Ausmees, N., Jacobsson, K., Lindberg, M., A unipolarly located, cell surface-associated agglutinin, RapA, belongs to a family of Rhizobiumadhering proteins (Rap) in Rhizobium leguminosarum bv trifolii (2001) Microbiology, 147, pp. 549-559
  • Zorreguieta, A., Finnie, C., Downie, J.A., Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component (2000) J. Bacteriol., 182, pp. 1304-1312
  • Mongiardini, E.J., Ausmees, N., Pérez-Giménez, J., Julia Althabegoiti, M., Ignacio Quelas, J., López-García, S.L., Lodeiro, A.R., The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation (2008) FEMS Microbiol. Ecol., 65, pp. 279-288
  • Cao, L., Yan, X., Borysenko, C.W., Blair, H.C., Wu, C., Yu, L., CHDL. A cadherin-like domain in Proteobacteria and Cyanobacteria (2005) FEMS Microbiol. Lett., 251, pp. 203-209
  • Nollet, F., Kools, P., Van Roy, F., Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members (2000) J. Mol. Biol., 299, pp. 551-572
  • Fraiberg, M., Borovok, I., Bayer, E.A., Weiner, R.M., Lamed, R., Cadherin domains in the polysaccharide-degrading marine bacterium Saccharophagus degradans 2-40 are carbohydrate binding modules (2011) J. Bacteriol., 193, pp. 283-285
  • Fraiberg, M., Borovok, I., Weiner, R.M., Lamed, R., Discovery and characterization of cadherin domains in Saccharophagus degradans 2-40 (2010) J. Bacteriol., 192, pp. 1066-1074
  • Dickens, N.J., Beatson, S., Ponting, C.P., Cadherin-like domains in α-dystroglycan, α/ε- sarcoglycan, and yeast and bacterial proteins (2002) Curr. Biol., 12, pp. R197-R199
  • Young, J.P., Crossman, L.C., Johnston, A.W., Thomson, N.R., Ghazoui, Z.F., Hull, K.H., Wexler, M., Parkhill, J., The genome of Rhizobium leguminosarum has recognizable core and accessory components (2006) Genome Biol, 7, pp. R34
  • Butcher, B.G., Lin, Y.-P., Helmann, J.D., The yydFGHIJ operon of Bacillus subtilis encodes a peptide that induces the LiaRS two-component system (2007) J. Bacteriol., 189, pp. 8616-8625
  • Johnston, A.W., Beringer, J.E., Identification of the Rhizobium strains in pea root nodules using genetic markers (1975) J. Gen. Microbiol., 87, pp. 343-350
  • Downie, J.A., Ma, Q.S., Knight, C.D., Hombrecher, G., Johnston, A.W., Cloning of the symbiotic region of Rhizobium leguminosarum. The nodulation genes are between the nitrogenase genes and a nifA-like gene (1983) EMBO J., 2, pp. 947-952
  • Finnie, C., Hartley, N.M., Findlay, K.C., Downie, J.A., The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation, and exopolysaccharide modification (1997) Mol. Microbiol., 25, pp. 135-146
  • Beringer, J.E., R factor transfer in Rhizobium leguminosarum (1974) J. Gen Microbiol., 84, pp. 188-198
  • Sherwood, M.T., Improved synthetic medium for the growth of Rhizobium (1970) J. Appl Bacteriol., 33, pp. 708-713
  • Leigh, J.A., Signer, E.R., Walker, G.C., Exopolysaccharidedeficient mutants of Rhizobium meliloti that form ineffective nodules (1985) Proc. Natl. Acad. Sci. U.S.A., 82, pp. 6231-6235
  • Miller, K.J., Gore, R.S., Johnson, R., Benesi, A.J., Reinhold, V.N., Cell-associated oligosaccharides of Bradyrhizobium spp (1990) J. Bacteriol., 172, pp. 136-142
  • Zevenhuizen, L.P.T.M., Van Neerven, A.R.W., (1, 2)-β-D Glucan and acidic oligosaccharides produced by Rhizobium meliloti (1983) Carbohydr Res, 118, pp. 127-134
  • Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., Gapped BLAST and PSI-BLAST. A new generation of protein database search programs (1997) Nucleic Acids Res., 25, pp. 3389-3402
  • Edgar, R.C., MUSCLE. Multiple sequence alignment with high accuracy and high throughput (2004) Nucleic Acids Res., 32, pp. 1792-1797
  • Goodstadt, L., Ponting, C.P., CHROMA. Consensus-based colouring of multiple alignments for publication (2001) Bioinformatics, 17, pp. 845-846
  • Ginalski, K., Elofsson, A., Fischer, D., Rychlewski, L., 3D-Jury. A simple approach to improve protein structure predictions (2003) Bioinformatics, 19, pp. 1015-1018
  • Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (1970) Nature, 227, pp. 680-685
  • Loewus, M.W., Briggs, D.R., The number of catalytically active sites present on the chymotrypsin molecule (1952) J. Biol. Chem., 199, pp. 857-864
  • Filisetti-Cozzi, T.M., Carpita, N.C., Measurement of uronic acids without interference from neutral sugars (1991) Anal. Biochem., 197, pp. 157-162
  • Jefferson, K.K., Cerca, N., Methods in molecular biology (2006) Cell- Cell Interactions: Methods and Protocols, pp. 119-126. , Humana Press Inc., Totowa, NJ
  • Sandford, P.A., Pittsley, J.E., Knutson, C.A., Watson, P.R., Cadmus, M.C., Janes, A., Variation in Xanthomonas campestris NRRL B-1459 (1977) Characterization of Xanthan Products of Differing Pyruvic Acid Content: American Chemical Society, pp. 192-210. , Symposium Series No. 45 Sandford P A and Laskin A eds American Chemical Society Washington D C
  • Guex, N., Peitsch, M.C., SWISS-MODEL and the Swiss-Pdb- Viewer. An environment for comparative protein modeling (1997) Electrophoresis, 18, pp. 2714-2723
  • Nagar, B., Overduin, M., Ikura, M., Rini, J.M., Structural basis of calcium-induced E-cadherin rigidification and dimerization (1996) Nature, 380, pp. 360-364
  • Pertz, O., Bozic, D., Koch, A.W., Fauser, C., Brancaccio, A., Engel, J., A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation (1999) EMBO J., 18, pp. 1738-1747
  • Robertsen, B.K., Aman, P., Darvill, A.G., McNeil, M., Albersheim, P., Host-symbiont interactions. V. The structure of acidic extracellular polysaccharides secreted by Rhizobium leguminosarum and Rhizobium trifolii (1981) Plant Physiol., 67, pp. 389-400
  • Laus, M.C., Logman, T.J., Van Brussel, A.A., Carlson, R.W., Azadi, P., Gao, M.Y., Kijne, J.W., Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra (2004) J. Bacteriol., 186, pp. 6617-6625
  • Chen, C.P., Song, S.C., Gilboa-Garber, N., Chang, K.S., Wu, A.M., Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa (1998) Glycobiology, 8, pp. 7-16
  • McNeil, M., Darvill, J., Darvill, A.G., Albersheim, P., Van Veen, R., Hooykaas, P., Schilperoort, R., Dell, A., The discernible, structural features of the acidic polysaccharides secreted by different Rhizobium species are the same (1986) Carbohydr. Res., 146, pp. 307-326
  • Zeng, X., Andrade, C.A., Oliveira, M.D., Sun, X.L., Carbohydrate- protein interactions and their biosensing applications (2012) Anal Bioanal Chem, 402, pp. 3161-3176
  • Finnie, C., Zorreguieta, A., Hartley, N.M., Downie, J.A., Characterization of Rhizobium leguminosarum exopolysaccharide glycanases that are secreted via a type i exporter and have a novel heptapeptide repeat motif (1998) J. Bacteriol., 180, pp. 1691-1699
  • Abbott, D.W., Boraston, A.B., The structural basis for exopolygalacturonase activity in a family 28 glycoside hydrolase (2007) J. Mol. Biol., 368, pp. 1215-1222
  • Kataeva, I.A., Seidel Iii, R.D., Shah, A., West, L.T., Li, X.L., Ljungdahl, L.G., The fibronectin type 3-like repeat from the Clostridium thermocellum cellobiohydrolase CbhA promotes hydrolysis of cellulose by modifying its surface (2002) Appl. Environ. Microbiol., 68, pp. 4292-4300
  • Dazzo, F.B., Truchet, G.L., Sherwood, J.E., Hrabak, E.M., Gardiol, A.E., Alteration of the trifoliin A-binding capsule of Rhizobium trifolii 0403 by enzymes released from clover roots (1982) Appl. Environ. Microbiol., 44, pp. 478-490
  • Watkins, N.J., Knight, M.R., Trewavas, A.J., Campbell, A.K., Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin (1995) Biochem. J., 306, pp. 865-869
  • Delepelaire, P., Type i secretion in gram-negative bacteria (2004) Biochim. Biophys. Acta, 1694, pp. 149-161
  • Holland, I.B., Schmitt, L., Young, J., Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review) (2005) Mol. Membr. Biol., 22, pp. 29-39
  • Chenal, A., Guijarro, J.I., Raynal, B., Delepierre, M., Ladant, D., RTX calcium binding motifs are intrinsically disordered in the absence of calcium. Implication for protein secretion (2009) J. Biol. Chem., 284, pp. 1781-1789
  • Wolff, N., Sapriel, G., Bodenreider, C., Chaffotte, A., Delepelaire, P., Antifolding activity of the SecB chaperone is essential for secretion of HasA, a quickly folding ABC pathway substrate (2003) J. Biol. Chem., 278, pp. 38247-38253
  • Michiels, J., Xi, C., Verhaert, J., Vanderleyden, J., The functions of Ca2+ in bacteria. A role for EF-hand proteins? (2002) Trends Microbiol., 10, pp. 87-93
  • Orans, J., Johnson, M.D., Coggan, K.A., Sperlazza, J.R., Heiniger, R.W., Wolfgang, M.C., Redinbo, M.R., Crystal structure analysis reveals Pseudomonas PilY1 as an essential calcium-dependent regulator of bacterial surface motility (2010) Proc. Natl. Acad. Sci. U.S.A., 107, pp. 1065-1070
  • Aravind, P., Mishra, A., Suman, S.K., Jobby, M.K., Sankaranarayanan, R., Sharma, Y., The βγ-crystallin superfamily contains a universal motif for binding calcium (2009) Biochemistry, 48, pp. 12180-12190
  • Aravind, P., Suman, S.K., Mishra, A., Sharma, Y., Sankaranarayanan, R., Three-dimensional domain swapping in nitrollin, a single-domain βγ-crystallin from Nitrosospira multiformis, controls protein conformation and stability but not dimerization (2009) J. Mol. Biol., 385, pp. 163-177
  • Vu, B., Chen, M., Crawford, R.J., Ivanova, E.P., Bacterial extracellular polysaccharides involved in biofilm formation (2009) Molecules, 14, pp. 2535-2554
  • Ghafoor, A., Hay, I.D., Rehm, B.H., Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture (2011) Appl. Environ. Microbiol., 77, pp. 5238-5246
  • Begun, J., Gaiani, J.M., Rohde, H., MacK, D., Calderwood, S.B., Ausubel, F.M., Sifri, C.D., Staphylococcal biofilm exopolysaccharide protects against Caenorhabditis elegans immune defenses (2007) PLoS Pathog, 3, pp. e57


---------- APA ----------
Abdian, P.L., Caramelo, J.J., Ausmees, N. & Zorreguieta, A. (2013) . RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize rhizobium leguminosarum acidic exopolysaccharides. Journal of Biological Chemistry, 288(4), 2893-2904.
---------- CHICAGO ----------
Abdian, P.L., Caramelo, J.J., Ausmees, N., Zorreguieta, A. "RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize rhizobium leguminosarum acidic exopolysaccharides" . Journal of Biological Chemistry 288, no. 4 (2013) : 2893-2904.
---------- MLA ----------
Abdian, P.L., Caramelo, J.J., Ausmees, N., Zorreguieta, A. "RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize rhizobium leguminosarum acidic exopolysaccharides" . Journal of Biological Chemistry, vol. 288, no. 4, 2013, pp. 2893-2904.
---------- VANCOUVER ----------
Abdian, P.L., Caramelo, J.J., Ausmees, N., Zorreguieta, A. RapA2 is a calcium-binding lectin composed of two highly conserved cadherin-like domains that specifically recognize rhizobium leguminosarum acidic exopolysaccharides. J. Biol. Chem. 2013;288(4):2893-2904.