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Plant Molecular Biology

, Volume 62, Issue 4–5, pp 529–545 | Cite as

Molecular cloning, expression, and cytokinin (6-benzylaminopurine) antagonist activity of peanut (Arachis hypogaea) lectin SL-I

  • Monika Pathak
  • Bharat Singh
  • Amit Sharma
  • Praveen Agrawal
  • Santosh B. Pasha
  • Hasi R. Das
  • Rakha H. Das
Original Paper

Abstract

Isolation and purification of a α-methyl-mannoside specific lectin (SL-I) of peanut was reported earlier [Singh and Das (1994) Glycoconj J 11:282–285]. Native SL-I is a glycoprotein having ∼31 kDa subunit molecular mass and forms dimer. The gene encoding this lectin is identified from a 6-day old peanut root cDNA library by anti-SL-I antibody and N-terminal amino acid sequence homology to the native lectin. Nucleotide sequence derived amino acid sequence of the re-SL-I shows amino acid sequence homology with the N-terminal and tryptic digests’ amino acid sequence of the native SL-I (nSL-I). Presence of a putative glycosylation (QNPS) site and a hydrophobic adenine-binding (VLVSYDANS) site is also identified in SL-I. Homology modeling of the lectin suggests it to be an archetype of legume lectins. It is expressed as a ~30 kDa apoprotein in E. coli and has the carbohydrate specificity and secondary structure identical to its natural counterpart. The lectin SL-I inhibits cytokinin 6-benzylaminopurine (BA)-induced “delayed leaf senescence” and “cotyledon expansion”. Equilibrium dialysis revealed a single high-affinity binding site for adenine (7.6 × 10−6 M) and BA (1.09 × 10−5 M) in the SL-I dimer and thus suggesting that the cytokinin antagonist effect of SL-I is mediated by the direct interaction of SL-I with BA.

Keywords

Peanut lectin SL-I Gene expression Cytokinin 6-benzylaminopurine Cytokinin antagonist activity 

Abbreviations

PBS

phosphate-buffered saline

nSL-I/SL-I

native peanut stem lectin

re-SL-I

recombinant SL-I

Con A

Concanavalin A

LBL

lima bean lectin

WBA I and WBA II

winged bean basic- and acidic- agglutinin respectively

PHA-E

Phaseolus vulgaris erythroagglutinin

PAL

Pterocarpus angolensis man/glc specific seed lectin

SBA

soybean lectin

ANS

1,8-anilino-1-naphthalenesulfonic acid

BA

6-benzylaminopurine

PMSF

phenylmethylsulfonylfluoride

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Notes

Acknowledgements

Authors gratefully acknowledge Dr. Souvik Maiti for his guidance in fluorescence studies. Authors also acknowledge the Director of IGIB for providing facilities to carry out this work.

References

  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Assaying the quality of cDNA libraries. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  2. Bewly JD, Black M (1985) Seeds physiology of development and germination. Plenum Press, New York and LondonGoogle Scholar
  3. Bogoeva VP, Radeva MA, Atanasova LY, Stoitsova SR, Boteva RN (2004) Fluorescence analysis of hormone binding activities of wheat germ agglutinin. Biochim Biophys Acta 1698:213–218PubMedGoogle Scholar
  4. Bourne Y, Abergel C, Cambillau C, Frey M, Rouge P, Fontecilla-Camps JC (1990) X-ray crystal structure determination and refinement at 1.9 Å resolution of isolectin I from the seeds of Lathyrus ochrus. J Mol Biol 214:571–584PubMedCrossRefGoogle Scholar
  5. Chothia C, Lesk AM (1986) The relation between the divergence of sequence and structure in proteins. Embo J 5:823–826PubMedGoogle Scholar
  6. DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Crystal structure of arcelin-5, a lectin-like defense protein from Phaseolus vulgaris. Anal Chem 28:350–356CrossRefGoogle Scholar
  7. Edelman GM, Cunningham BA, Reeke GN Jr, Becker JW, Waxdal MJ, Wang JL (1972) Crystal structure of peanut lectin, a protein with an unusual quaternary structure. Proc Natl Acad Sci USA 69:2580–2584PubMedCrossRefGoogle Scholar
  8. Einspahr H, Parks EH, Suguna K, Subramanian E, Suddath FL (1986) The crystal structure of pea lectin at 3.0 Å resolution. J Biol Chem 261:16518–16527PubMedGoogle Scholar
  9. Etzler ME (1985) Plant lectins: Molecular and biological aspects. Ann Rev Plant Physiol 36:209–234Google Scholar
  10. Farmer TB, Caprioli RM (1998) Determination of protein–protein interactions by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry. J Mass Spectrom 33:697–704PubMedCrossRefGoogle Scholar
  11. Green JF, Muir RM (1978) The effect of potassium on cotyledon expansion induced by cytokinins. Physiol Plant 43:213–216CrossRefGoogle Scholar
  12. Gegg CV, Roberts DD, Segel IH, Etzler ME (1992) Characterization of the adenine binding sites of two Dolichos biflorus lectins. Biochemistry 31:6938–6942PubMedCrossRefGoogle Scholar
  13. Gegg CV, Etzler ME (1994) Photoaffinity labeling of the adenine binding sites of two Dolichos biflorus lectins. J Biol Chem 8:5687–5692Google Scholar
  14. Ha CE, Petersen CE, Park DS, Harohalli K, Bhagavan NV (2000) Investigations of the effects of ethanol on warfarin binding to human serum albumin. J Biomed Sci 7:114–121PubMedCrossRefGoogle Scholar
  15. Hagen FS, Gray CL, Kuijper JL (1988) Assaying the quality of cDNA libraries. Biotechniques 6:340–345PubMedGoogle Scholar
  16. Hamelryck TW, Loris R, Bouckaert J, Dao-Thi MH, Strecker G, Imberty A, Fernandez E, Wyns L, Etzler ME (1999) Carbohydrate binding, quaternary structure and a novel hydrophobic binding site in two legume lectin oligomers from Dolichos biflorus. J Mol Biol 4:1161–1177CrossRefGoogle Scholar
  17. Inskeep WP, Bloom PR (1985) Extinction coefficients of Chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol 77:483–485PubMedCrossRefGoogle Scholar
  18. Kalsi G, Das HR, Babu CR, Das RH (1992) Isolation and characterization of lectin from peanut roots. Biochim Biophys Acta 1117:114–119PubMedGoogle Scholar
  19. Kalsi G, Das HR, Babu CR (1993) Further characterization of glucose-specific peanut root lectin (PRA II). Indian J Biochem Biophys 30:400–404Google Scholar
  20. Kishinevsky BD, Law IJ, Strijdom BW (1988) Detection of lectins in nodulated peanut and soyabean plants. Planta 176:10–18CrossRefGoogle Scholar
  21. Kozak M (1983) Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev 47:1–45PubMedGoogle Scholar
  22. Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132PubMedCrossRefGoogle Scholar
  23. Letham DS (1971) Regulators of cell division in plant tissues. XII. A cytokinin bioassay using excised radish cotyledons. Physiol Plant 25:391–396CrossRefGoogle Scholar
  24. Longo G, Pedretti M, Rossi G, Longo C (1979) Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons. Planta 145:209–217CrossRefGoogle Scholar
  25. Loris R, Hamelryck T, Bouckaert J, Wyns L (1998) Legume lectin structure. Biochim Biophys Acta 1:9–36Google Scholar
  26. Loris R, Imberty A, Beeckmans S, Van Driessche E, Read JS, Bouckaert J et al. (2003) Crystal structure of Pterocarpus angolensis lectin in complex with glucose, sucrose, and turanose. J Biol Chem 278:16297–16303PubMedCrossRefGoogle Scholar
  27. Loris R, Van Walle I, De Greve H, Beeckmans S, Deboeck F, Wyns L, Bouckaert J (2004) Structural basis of oligomannose recognition by the Pterocarpus angolensis seed lectin. J␣Mol Biol 335:1227–1240Google Scholar
  28. Loris R, Steyaert J, Maes D, Lisgarten J, Pickersgill R, Wyns L (1993) Crystal structure determination and refinement at 2.3-Å resolution of the lentil lectin. Biochemistry 32:8772–8781PubMedCrossRefGoogle Scholar
  29. Lotan R, Skutelsky E, Danon D, Sharon N (1975) The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea). J Biol Chem 250:8518–8523PubMedGoogle Scholar
  30. Maliarik MJ, Goldstein IJ (1988) Photoaffinity labeling of the adenine binding site of the lectins from lima bean, Phaseolus lunatus, and the kidney bean Phaseolus vulgaris. J Biol Chem 263:11274–11279PubMedGoogle Scholar
  31. Maliarik M, Plessas NR, Goldstein IJ, Musci G, Berliner LJ (1989) ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue. Biochemistry 28:912–917PubMedCrossRefGoogle Scholar
  32. Matsugi J, Murao K (2001) Study on construction of a cDNA library corresponding to an amino acid-specific tRNA and influence of the modified nucleotide upon nucleotide misincorporations in reverse transcription. J Biochim Biophys Acta 1521:81–88Google Scholar
  33. Moran R, Porath D (1980) Chlorophyll determination in intact tissues using N,N-dimethylformamide. Plant Physiol 65:478–479PubMedGoogle Scholar
  34. Orr A, Ivanova VS, Bonner WM 1995 ‘Water-bug’ dialysis technique. Biotechniques 19/2:204Google Scholar
  35. Richmond AE, Lang A (1957) Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125:650–651CrossRefGoogle Scholar
  36. Roberts DD, Goldstein IJ (1982) Hydrophobic binding properties of the lectin from lima beans (Phaseolus lunatus). J Biol Chem 257:11274–11277PubMedGoogle Scholar
  37. Roberts DD, Goldstein IJ (1983) Adenine binding sites of the lectin from lima beans (Phaseolus lunatus). J Biol Chem 22:13820–13824Google Scholar
  38. Rost B (1996) PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266:525–539PubMedCrossRefGoogle Scholar
  39. Rozwarski DA, Swami BM, Brewer CF, Sacchettini JC (1998) Crystal structure of the lectin from Dioclea grandiflora complexed with core trimannoside of asparagine-linked carbohydrates. J Biol Chem 273:32818–32825PubMedCrossRefGoogle Scholar
  40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, New York, pp 18–24Google Scholar
  41. Sanz-Aparicio J, Hermoso J, Grangeiro TB, Calvete JJ, Cavada BS (1997) The crystal structure of Canavalia brasiliensis lectin suggests a correlation between its quaternary conformation and its distinct biological properties from Concanavalin A. FEBS Lett 405:114–118PubMedCrossRefGoogle Scholar
  42. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS MODEL—an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385PubMedCrossRefGoogle Scholar
  43. Shanker S, Das RH (2001) Identification of a cDNA clone encoding for a galactose-binding lectin from peanut (Arachis hypogaea) seedling roots. Biochim Biophys Acta 1568:105–110PubMedGoogle Scholar
  44. Sharon N, Lis H (1986) Lectin biochemistry. New way of protein maturation. Nature 323:203–204PubMedCrossRefGoogle Scholar
  45. Singh R, Das HR (1994) Purification of lectins from the stems of peanut plants. Glycoconj J 11:282–285PubMedCrossRefGoogle Scholar
  46. Singh R, Gautam H, Jayaraman V, Nair G, Das HR (1997) Vegetative tissue lectins of peanut (A. hypogaea). Indian J Biochem Biophys 34:72–75PubMedGoogle Scholar
  47. Srinivas VR, Acharya S, Rawat S, Sharma V, Surolia A (2000) The primary structure of the acidic lectin from winged bean (Psophocarpus tetragonolobus): insights in carbohydrate recognition, adenine binding and quaternary association. FEBS Lett 474:76–82PubMedCrossRefGoogle Scholar
  48. Stoitsova SR, Boteva RN, Doyle RJ (2003) Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin. Biochim Biophys Acta 1619:213–219PubMedGoogle Scholar
  49. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  50. Wah DA, Romero A, Gallego del Sol F, Cavada BS, Ramos MV, Grangeiro TB, Sampaio AH, Calvete JJ (2001) Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer–tetramer association. J Mol Biol 310:885–894PubMedCrossRefGoogle Scholar
  51. Zacharius RM, Zell TE, Morrison JH, Woodlock JJ (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 1:148–152CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Monika Pathak
    • 1
  • Bharat Singh
    • 1
  • Amit Sharma
    • 1
  • Praveen Agrawal
    • 1
  • Santosh B. Pasha
    • 1
  • Hasi R. Das
    • 1
  • Rakha H. Das
    • 1
  1. 1.Unit of Proteomics and Comparative Genomics, Institute of Genomics and Integrative BiologyDelhi University CampusDelhiIndia

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