Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure

BMC Biochemistry - Tập 5 - Trang 1-7 - 2004
Frédéric Gressent1,2, Julie V Cullimore3, Raoul Ranjeva1, Jean-Jacques Bono1,3
1Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, Castanet-Tolosan, France
2Biologie Fonctionnelle, Insectes et Interactions, UMR INRA-INSA de Lyon, INSA Bat L. Pasteur, Villeurbanne Cedex, France
3Laboratoire des Interactions Plantes-Microorganismes, UMR INRA-CNRS 215, Castanet-Tolosan, France

Tóm tắt

The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). The aim of this work was to optimize the radiolabelling of Nod factors with 35S. The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step). By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti. By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci.mmol-1 with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules.

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Tài liệu tham khảo

Roche P, Debellé F, Maillet F, Lerouge P, Faucher C, Truchet G, Dénarié J, Promé JC: Molecular basis of symbiotic host specificity in Rhizobium meliloti nodH and nodPQ genes encode the sulfation of lipo-chitooligosaccharides signals. Cell. 1991, 67: 1131-1143.

Imai Y, Lasky LA, Rosen SD: Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin. Nature. 1993, 361: 555-557. 10.1038/361555a0.

Pye DA, Vives RR, Turnbull JE, Hyde P, Gallagher JT: Heparan sulfate oligosaccharides require 6-O-sulfation for promotion of basic fibroblast growth factor mitogenic activity. J Biol Chem. 1998, 273: 22936-22942. 10.1074/jbc.273.36.22936.

Bouarab K, Potin P, Correa J, Kloareg B: Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte. The Plant Cell. 1999, 11: 1635-1650. 10.1105/tpc.11.9.1635.

Bowman KG, Bertozzi CR: Carbohydrate sulfotransferase: mediators of extracellular communication. Curr Biol. 1999, 6: R9-R22. 10.1016/S1074-5521(99)80014-3.

Schwedock JS, Long SR: ATP sulphurylase activity of the nodP and nodQ gene products of Rhizobium meliloti. Nature. 1990, 348: 644-647. 10.1038/348644a0.

Ehrhardt DW, Atkinson EM, Faull KF, Freedberg DI, Sutherlin DP, Armstrong R, Long SR: In vitro sulfotransferase activity of NodH, a nodulation protein of Rhizobium meliloti required for host-specific nodulation. J Bact. 1995, 177: 6237-6245.

Bourdineaud JP, Bono JJ, Ranjeva R, Cullimore JV: Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti. Biochem J. 1995, 306: 259-264.

Lin C, Shen G, Garcia-Junceda E, Wong C: Enzymatic synthesis and regeneration of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) for regioselective sulfation of oligosaccharides. J Am Chem Soc. 1995, 117: 8031-8032.

Truchet G, Roche P, Lerouge P, Vasse J, Camut S, de Billy F, Promé JC, Dénarié J: Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature. 1991, 351: 670-673. 10.1038/351670a0.

Gressent F, Drouillard S, Mantegazza N, Samain E, Geremia RA, Canut H, Niebel A, Driguez H, Cullimore JV, Ranjeva R, Bono JJ: Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures. Proc Natl Acad Sci USA. 1999, 96: 4704-4709. 10.1073/pnas.96.8.4704.

Dénarié J, Debellé F, Promé JC: Rhizobium lipo-oligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem. 1996, 65: 503-535. 10.1146/annurev.bi.65.070196.002443.

Schwedock JS, Liu C, Leyh TS, Long SR: Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity. J Bact. 1994, 176: 7055-7064.

Leyh TS, Suo Y: GTPase-mediated activation of ATP sulfurylase. J Biol Chem. 1992, 267: 542-545.

Foster BA, Thomas SM, Mahr JA, Renosto F, Patel HC, Segel IH: Cloning and sequencing of ATPsulfurylase from Penicillium chrysogenum. J Biol Chem. 1994, 269: 19777-19786.

Lyle S, Geller DH, Ng K, Westley J, Schartz N: Kinetic mechanism of ATP-sulfurylase from rat chondrosarcoma. Biochem J. 1994, 301: 349-354.

Sawhney SK, Nicholas DJ: Effects of adenine nucleotides and phosphate on adenosine triphosphate sulphurylase from Anabaena cylindrica. Biochem J. 1977, 164: 161-167.

Schultze M, Staehelin C, Röhrig H, John M, Schmidt J, Kondorosi E, Schell J, Kondorosi A: In vitro sulfotransferase activity of Rhizobium meliloti NodH protein: lipochitooligosaccharide nodulation signals are sulfated after synthesis of the core structure. Proc Natl Acad Sci USA. 1995, 92: 2706-2709.

Hanin M, Jabbouri S, Quasada-Vincens D, Freiberg C, Perret X, Promé JC, Broughton WJ, Fellay R: Sulphation of Rhizobium sp. NGR234 Nod factor is dependant on noeE a new host-specificity gene. Mol Microbiol. 1997, 24: 1119-1129. 10.1046/j.1365-2958.1997.3981777.x.

Madsen EB, Madsen LH, Radutiu S, Olbryt M, Rakwalska M, Szczyglowski K, Sato S, Kaneko T, Tabata S, Sandal N, Stougaard J: A receptor-kinase gene of the LysM type is involved in perception of rhizobial signals. Nature. 2003, 425: 637-638. 10.1038/nature02045.

Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J: Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature. 2003, 425: 585-592. 10.1038/nature02039.

Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R: LysM domain receptor kinase regulating rhizobial Nod factor induced infection. Science. 2003, 302: 630-633. 10.1126/science.1090074.

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