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Glycoconjugate Journal

, Volume 35, Issue 6, pp 511–523 | Cite as

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala

  • Mamta Sharma
  • Prajna Hegde
  • Kavita Hiremath
  • Vishwanath Reddy H
  • A. S. Kamalanathan
  • Bale M. Swamy
  • Shashikala R. Inamdar
Original Article
  • 114 Downloads

Abstract

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 μg/ml. AHL can be explored for its clinical potential.

Keywords

Adenia hondala lectin N-Acetylgalactosamine Poly-LacNAc Passifloraceae Glycan microarray HepG2 cells 

Notes

Author contributions

Conceived and designed the experiments: SRI, AHL purification and characterization: MS, cell culture and Flow cytometry: PH and MS, glycan array and analyzed the data: SRI and VRH, DNase activity: KH, ESI/MS: KAS Contributed reagents/ materials/analysis tools: SRI, and BMS. Wrote the paper: SRI, BMS and MS.

Funding

SRI would like to thank Protein-Glycan Interaction Resource of the CFG (supporting grant R24 GM098791) and the National Center for Functional Glycomics (NCFG) at Beth Israel Deaconess Medical Center, Harvard Medical School (supporting grant P41 GM103694) for the glycan array analysis of AHL. The work was supported by the funding from University Grant Commission under UPE (F.NO.14 3/2012 (NS/PE) project.

Compliance with ethical standards

Conflict of interest

Authors don’t have any conflict of interest to declare concerning to the present work.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10719_2018_9843_MOESM1_ESM.pdf (296 kb)
ESM 1 (PDF 295 kb)

References

  1. 1.
    Quattrocchi, U: CRC World Dictionary of Medicinal and Poisonous Plants: Common Names, Scientific Names, Eponyms, Synonyms, and Etymology, 5 Volume Set. CRC Press, Boca Raton (2012)Google Scholar
  2. 2.
    Polito, L., Bortolotti, M., Maiello, S., Battelli, M.G., Bolognesi, A.: Plants producing ribosome-inactivating proteins in traditional medicine. Molecules. (2016).  https://doi.org/10.3390/molecules21111560 CrossRefGoogle Scholar
  3. 3.
    Stirpe, F., Battelli, M.G.: Ribosome-inactivating proteins: progress and problems. Cell. Mol. Life Sci. 63, 1850–1866 (2006)CrossRefGoogle Scholar
  4. 4.
    Shih, N.R., McDonald, K., Jackman, A., Girbes, T., Iglesias, R.: Bifunctional plant defence enzymes with chitinase and ribosome inactivating activities from Trichosanthes kirilowii cell cultures. Plant Sci. 130, 145–150 (1997)CrossRefGoogle Scholar
  5. 5.
    Li, X.D., Chen, W.F., Liu, W.Y., Wang, G.H.: Large-scale preparation of two new ribosome-inactivating proteins-cinnamomin and camphorin from the seeds of Cinnamomum camphora. Protein Expr. Purif. 10, 27–31 (1997)CrossRefGoogle Scholar
  6. 6.
    Roncuzzi, L., Gasperi-Campani, A.: DNA-nuclease activity of the single-chain ribosome-inactivating proteins dianthin 30, saporin 6 and gelonin. FEBS Lett. 392, 16–20 (1996)CrossRefGoogle Scholar
  7. 7.
    Lombard, S., Helmy, M.E., Pieroni, G.: Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids. Biochem. J. 358, 773–781 (2001)CrossRefGoogle Scholar
  8. 8.
    Hey, T.D., Hartley, M., Walsh, T.A.: Maize ribosome-inactivating protein (b-32). Homologs in related species, effects on maize ribosomes, and modulation of activity by pro-peptide deletions. Plant Physiol. 107, 1323–1332 (1995)CrossRefGoogle Scholar
  9. 9.
    Reinbothe, S., Reinbothe, C., Lehmann, J., Becker, W., Apel, K., Parthier, B.: JIP60, a methyl jasmonate-induced ribosome-inactivating protein involved in plant stress reactions. Proc. Natl. Acad. Sci. U. S. A. 91, 7012–7016 (1994)CrossRefGoogle Scholar
  10. 10.
    Stirpe, F., Barbieri, L.: Ribosome-inactivating proteins up to date. FEBS Lett. 195, 1–8 (1986)CrossRefGoogle Scholar
  11. 11.
    Barbieri, L., Ciani, M., Girbes, T., Liu, W.Y., Van Damme, E.J., Peumans, W.J., Stirpe, F.: Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins. FEBS Lett. 563, 219–222 (2004)CrossRefGoogle Scholar
  12. 12.
    Tomatsu, M., Ohnishi-Kameyama, M., Shibamoto, N.: Aralin, a new cytotoxic protein from Aralia elata, inducing apoptosis in human cancer cells. Cancer Lett. 199, 19–25 (2003)CrossRefGoogle Scholar
  13. 13.
    Pelosi, E., Lubelli, C., Polito, L., Barbieri, L., Bolognesi, A., Stirpe, F.: Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae). Toxicon. 46, 658–663 (2005)CrossRefGoogle Scholar
  14. 14.
    Nnamani, C.V., Oselebe, H.O., Agbatutu, A.: Assessment of nutritional values of three underutilized indigenous leafy vegetables of Ebonyi State, Nigeria. Afr. J. Biotechnol. 8, 2321–2324 (2009)Google Scholar
  15. 15.
    Stirpe, F., Bolognesi, A., Bortolotti, M., Farini, V., Lubelli, C., Pelosi, E., Polito, L., Dozza, B., Strocchi, P., Chambery, A., Parente, A., Barbieri, L.: Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae). Toxicon. 50, 94–105 (2007)CrossRefGoogle Scholar
  16. 16.
    Schrot, J., Weng, A., Melzig, M.F.: Ribosome-inactivating and related proteins. Toxins (Basel). 7, 1556–1615 (2015)CrossRefGoogle Scholar
  17. 17.
    Goldstein, I.J., Hughes, R.C., Monsigny, M., Osawa, T., Sharon, N.: What should be called a lectin? Nature. 285, 66 (1980)CrossRefGoogle Scholar
  18. 18.
    Yuan, Y., Qiu, H., Gao, J., Wang, Z., Liu, C., Liu, Z., Jiang, Z., Li, Y., Wu, S.: Triptolide inhibits MCF-7 and HepG2 cells invasion and migration by inhibiting the synthesis of Polylactosamine chains. J. Anal. Oncol. 5(3), 102–109 (2016)CrossRefGoogle Scholar
  19. 19.
    Srinivasan, N., Bane, S.M., Ahire, S.D., Ingle, A.D., Kalraiya, R.D.: Poly N-acetyllactosamine substitutions on N-and not O-oligosaccharides or Thomsen–Friedenreich antigen facilitate lung specific metastasis of melanoma cells via galectin-3. Glycoconj. J. 26(4), 445–456 (2009)CrossRefGoogle Scholar
  20. 20.
    Miyake, M., Kohno, N., Nudelman, E.D., Hakomori, S.-I.: Human IgG3.Monoclonal Antibody Directed to an Unbranched Repeating Type 2 Chain (Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→R) Which Is Highly Expressed in Colonic and Hepatocellular Carcinoma. Cancer Res. 49, 5689–5695 (1989)PubMedGoogle Scholar
  21. 21.
    Spiro, R.G., Bhoyroo, V.D.: Structure of the O-glycosidically linked carbohydrate units of fetuin. J. Biol. Chem. 249, 5704–5717 (1974)PubMedGoogle Scholar
  22. 22.
    March, S.C., Parikh, I., Cuatrecasas, P.: A simplified method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem. 60, 149–152 (1974)CrossRefGoogle Scholar
  23. 23.
    Liener, I.E., Hill, E.G.: The effect of heat treatment on the nutritive value and hemagglutinating activity of soybean oil meal. J. Nutr. 49, 609–620 (1953)CrossRefGoogle Scholar
  24. 24.
    Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)PubMedGoogle Scholar
  25. 25.
    DuBois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem. (1965).  https://doi.org/10.1021/ac60111a017 CrossRefGoogle Scholar
  26. 26.
    Goldman, M.: Fluorescent Antibody Methods. Academic Press, New York (1968)Google Scholar
  27. 27.
    Duk, M., Lisowska, E., Wu, J.H., Wu, A.M.: The biotin/avidin-mediated microtiter plate lectin assay with the use of chemically modified glycoprotein ligand. Anal. Biochem. 221, 266–272 (1994)CrossRefGoogle Scholar
  28. 28.
    Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature. 227, 680–685 (1970)CrossRefGoogle Scholar
  29. 29.
    Chevallet, M., Luche, S., Rabilloud, T.: Silver staining of proteins in polyacrylamide gels. Nat. Protoc. 1, 1852–1858 (2006)CrossRefGoogle Scholar
  30. 30.
    Zacharius, R.M., Zell, T.E., Morrison, J.H., Woodlock, J.J.: Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148–152 (1969)CrossRefGoogle Scholar
  31. 31.
    Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J.: Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101, 17033–17038 (2004)CrossRefGoogle Scholar
  32. 32.
    Shang, C., Van Damme, E.J.: Comparative analysis of carbohydrate binding properties of Sambucus nigra lectins and ribosome-inactivating proteins. Glycoconj. J. 31, 345–354 (2014)CrossRefGoogle Scholar
  33. 33.
    Wu, A.M., Wu, J.H., Singh, T., Lai, L.J., Yang, Z., Herp, A.: Recognition factors of Ricinus communis agglutinin 1 (RCA (1)). Mol. Immunol. 43, 1700–1715 (2006)CrossRefGoogle Scholar
  34. 34.
    Carrillo, C., Cordoba-Diaz, D., Cordoba-Diaz, M., Girbes, T., Jimenez, P.: Effects of temperature, pH and sugar binding on the structures of lectins ebulin f and SELfd. Food Chem. 220, 324–330 (2017)CrossRefGoogle Scholar
  35. 35.
    singh, A.p., Saxena, K.D.: Effect of temperature, pH and denaturing agents on biological activity of MCJ lectin. Chem. Sci. Trans. 2(4), 1508–1512 (2013)Google Scholar
  36. 36.
    Lyimo, B., Funakuma, N., Minami, Y., Yagi, F.: Characterization of a new alpha-galactosyl-binding lectin from the mushroom Clavaria purpurea. Biosci. Biotechnol. Biochem. 76, 336–342 (2012)CrossRefGoogle Scholar
  37. 37.
    Green, E.D., Adelt, G., Baenziger, J.U., Wilson, S., Van Halbeek, H.: The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. J. Biol. Chem. 263(34), 18253–18268 (1988)PubMedGoogle Scholar
  38. 38.
    Battelli, M.G., Scicchitano, V., Polito, L., Farini, V., Barbieri, L., Bolognesi, A.: Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin. Biochim. Biophys. Acta. 1800, 1276–1282 (2010)CrossRefGoogle Scholar
  39. 39.
    Sharma, A., Ng, T.B., Wong, J.H., Lin, P.: Purification and characterization of a lectin from Phaseolus vulgaris cv. (Anasazi beans). J Biomed Biotechnol. 2009, 929568 (2009)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mamta Sharma
    • 1
  • Prajna Hegde
    • 1
  • Kavita Hiremath
    • 1
  • Vishwanath Reddy H
    • 1
  • A. S. Kamalanathan
    • 2
  • Bale M. Swamy
    • 1
  • Shashikala R. Inamdar
    • 1
  1. 1.Department of Studies in BiochemistryKarnatak UniversityDharwadIndia
  2. 2.Centre for Bioseparation TechnologyVIT UniversityVelloreIndia

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