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Planta

, Volume 245, Issue 2, pp 343–353 | Cite as

A Bowman–Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid

  • Martín Indarte
  • Cristian M. Lazza
  • Diego Assis
  • Néstor O. Caffini
  • María A. Juliano
  • Francesc X. Avilés
  • Xavier Daura
  • Laura M. I. López
  • Sebastián A. Trejo
Original Article
  • 293 Downloads

Abstract

Main conclusion

A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family.

In this work, we report a Bowman–Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3′RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M−1 cm−1. MpBBI inhibits strongly trypsin with K i in the 10−10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Keywords

BBI-type protease inhibitor Cloning Homology modeling Loop Three-dimensional structure Trypsin inhibition 

Abbreviations

BBI

Bowman–Birk inhibitor

MpBBI

Maclura pomifera Bowman–Birk inhibitor

PMF

Peptide mass fingerprinting

Notes

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-Project-12/50191-4R, D.A. and M.A.J.), CYTED (Red temática PROMAL 210RT0398, C.M.L., N.O.C., F.X.A., L.M.I.L. and S.A.T.) and CONICET (PIP 0297, L.M.I.L. and S.A.T.). The MALDI-TOF MS analyses were carried out in the Proteomics and Bioinformatics Facility of the Universitat Autònoma de Barcelona (SePBioEs-UAB) by S.A.T.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

425_2016_2611_MOESM1_ESM.tiff (1.2 mb)
Supplementary material 1 (TIFF 1243 kb)

References

  1. Baici A, Schenker P, Wächter M, Rüedi P (2009) 3-Fluoro-2,4-dioxa-3-phosphadecalins as inhibitors of acetylcholinesterase. A reappraisal of kinetic mechanisms and diagnostic. Methods Chem Biodiv 6:261–282CrossRefGoogle Scholar
  2. Birk Y (1985) The Bowman–Birk inhibitor. Int J Pept Protein Res 25:113–131CrossRefPubMedGoogle Scholar
  3. Birk Y, Gertler A, Khalef S (1963) A pure trypsin inhibitor from soya beans. Biochem J 87:281–284CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bowman DE (1946) Differentiation of soy bean antitryptic factors. Proc Soc Exp Biol Med 63:547–550CrossRefPubMedGoogle Scholar
  5. Brauer AB, Nievo M, McBride JD, Leatherbarrow RJ (2003) The structural basis of a conserved P2 threonine in canonical serine proteinase inhibitors. J Biomol Struct Dyn 20:645–656CrossRefPubMedGoogle Scholar
  6. Dai H, Ciric B, Zhang G-X, Rostami A (2012) Interleukin-10 plays a crucial role in suppression of experimental autoimmune encephalomyelitis by Bowman–Birk inhibitor. J Neuroimmunol 245:1–7CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dantzger M, Ilka MV, Scorsato V, Marangoni S, Rodrigues Macedo ML (2015) Bowman–Birk proteinase inhibitor from Clitoria fairchildiana seeds: isolation, biochemical properties and insecticidal potential. Phytochemistry 118:224–235CrossRefPubMedGoogle Scholar
  8. Deshimaru M, Hanamoto R, Kusano C, Yoshimi S, Terada S (2002) Purification and characterization of proteinase inhibitors from wild soja (Glycine soja) seeds. Biosci Biotechnol Biochem 66:1897–1903CrossRefPubMedGoogle Scholar
  9. Erlanger B, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278CrossRefPubMedGoogle Scholar
  10. Fereidunian A, Sadeghalvad M, Oscoie MO, Mostafaie A (2014) Soybean Bowman–Birk protease inhibitor (BBI): identification of the mechanisms of BBI suppressive effect on growth of two adenocarcinoma cell lines: AGS and HT29. Arch Med Res 45:455–461CrossRefPubMedGoogle Scholar
  11. Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RMM (1966) Hydrogen ion buffers for biological research. Biochemistry 5:467–477CrossRefPubMedGoogle Scholar
  12. Joshi RS, Mishra M, SureshCG GuptaVS, Giri AP (2013) Complementation of intramolecular interactions for structural-functional stability of plant serine proteinase inhibitors. Biochim Biophys Acta 1830:5087–5094CrossRefPubMedGoogle Scholar
  13. Kalume E, Sousa MV, Morhy L (1995) Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the Brazilian tree Dipteryxalata (Leguminosae). J Protein Chem 14:685–693CrossRefPubMedGoogle Scholar
  14. Kennedy AR (1998) Chemopreventive agents: protease inhibitors. Pharmacol Ther 78:167–209CrossRefPubMedGoogle Scholar
  15. Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan ML, Juliano L, Gautier F (2008) Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 3:1–9CrossRefGoogle Scholar
  16. Larionova NI, Gladysheva IP, Tikhonova TV, Kazanskaia NF (1993) Inhibition of cathepsin G and elastase from human granulocytes by multiple forms of the Bowman–Birk type of soy inhibiton. Biokhimiia 58:1437–1444PubMedGoogle Scholar
  17. Laskowski M, Kato I (1980) Protein inhibitors of proteinases. Annu Rev Biochem 49:593–626CrossRefPubMedGoogle Scholar
  18. Lazza CM, Trejo SA, Obregón WD, Pistaccio LG, Caffini NO, López LMI (2010) A novel trypsin and α-chymotrypsin inhibitor from Maclura pomífera seeds. Lett Drug Design Discov 7:244–249CrossRefGoogle Scholar
  19. Losso JN (2008) The biochemical and functional food properties of the Bowman–Birk inhibitor. Crit Rev Food Sci Nutr 48:94–118CrossRefPubMedGoogle Scholar
  20. Marin-Manzano MC, Ruiz R, Jimenez E, Rubio LA, Clemente A (2009) Anti-carcinogenic soybean Bowman–Birk inhibitors survive fecal fermentation in their active form and do not affect the microbiota composition in vitro. Br J Nutr 101:967–971CrossRefPubMedGoogle Scholar
  21. McBride JD, Leatherbarrow RJ (2001) Synthetic peptide mimics of the Bowman–Birk inhibitor protein. Curr Med Chem 8:909–917CrossRefPubMedGoogle Scholar
  22. McBride JD, Watson EM, Brauer AB, Jaulent AM, Leatherbarrow RJ (2002) Peptide mimics of the Bowman–Birk inhibitor reactive site loop. Biopolymers 66:79–92CrossRefPubMedGoogle Scholar
  23. Osman MA, Reid PM, Weber CW (2002) Thermal inactivation of tepary bean (Phaseolus acutifolius), soybean and lima bean protease inhibitors: effect of acidic and basic pH. Food Chem 78:419–423CrossRefGoogle Scholar
  24. Park JH, Jeong HJ, de Lumen BO (2007) In vitro digestibility of the cancer-preventive soy peptides lunasin and BBI. J Agric Food Chem 55:10703–10706CrossRefPubMedGoogle Scholar
  25. Ponder JW, Case DA (2003) Force fields for protein simulations. Adv Protein Chem 66:27–85CrossRefPubMedGoogle Scholar
  26. Prasad ER, Dutta-Gupta A, Padmasree K (2010) Purification and characterization of a Bowman–Birk proteinase inhibitor from the seeds of black gram (Vigna mungo). Phytochemistry 71:363–372CrossRefPubMedGoogle Scholar
  27. Qi RF, Song ZW, Chi CW (2005) Structural features and molecular evolution of Bowman–Birk protease inhibitors and their potential application. Acta Biochim Biophys Sinica 37:283–292CrossRefGoogle Scholar
  28. Rawlings ND, Tolle DP, Barrett AJ (2004) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rawlings ND, Barrett AJ, Bateman A (2012) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 40:D343–D350CrossRefPubMedGoogle Scholar
  30. Souza LDC, Camargo R, Demasi M, Santana JM, Sa´ CM, de Freitas SM (2014) Effects of an anticarcinogenic Bowman–Birk protease inhibitor on purified 20S proteasome and MCF-7 breast cancer cells. PLoS One 9(1):e86600CrossRefPubMedCentralGoogle Scholar
  31. Swathi M, Lokya V, Swaroop V, Mallikarjuna N, Kannan M, Gupta AD, Padmasree K (2014) Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118). Plant Physiol Biochem 83:77–87CrossRefPubMedGoogle Scholar
  32. Tashiro M, Hashino K, Shiozaki M, Ibuki F, Maki Z (1987) The complete amino acid sequence of rice bran trypsin inhibitor. J Biochem 102:297–306PubMedGoogle Scholar
  33. Touil T, Ciric B, Ventura E, Shindler KS, Gran B, Rostami A (2008) Bowman–Birk inhibitor suppresses autoimmune inflammation and neuronal loss in a mouse model of multiple sclerosis. J Neurol Sci 271:191–202CrossRefPubMedPubMedCentralGoogle Scholar
  34. Utrilla MP, Peinado MJ, Ruiz R, Rodriguez-Nogales A, Algieri F, Rodriguez-Cabezas ME, Clemente A, Galvez J, Rubio LA (2015) Pea (Pisum sativum L.) seed albumin extracts show anti-inflammatory effect in the DSS model of mouse colitis. Mol Nutr Food Res 59:807–819CrossRefPubMedGoogle Scholar
  35. Wang J, Li X, Xia X, Li H, Liu J, Li QX, Li J, Xu T (2014) Extraction, purification, and characterization of a trypsin inhibitor from cowpea seeds (Vigna unguiculata). Prep Biochem Biotechnol 44:1–15CrossRefPubMedGoogle Scholar
  36. Ware JH, Wan XS, Rubin H, Schechter NM, Kennedy AR (1997) Soybean Bowman–Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase. Arch Biochem Biophys 344:133–138CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Martín Indarte
    • 1
  • Cristian M. Lazza
    • 2
  • Diego Assis
    • 3
  • Néstor O. Caffini
    • 2
  • María A. Juliano
    • 3
  • Francesc X. Avilés
    • 4
  • Xavier Daura
    • 4
  • Laura M. I. López
    • 5
    • 6
  • Sebastián A. Trejo
    • 7
  1. 1.PHusisTherapeuticsSan DiegoUSA
  2. 2.Centro de Investigación de Proteínas Vegetales, Facultad de Ciencias ExactasUniversidad Nacional de La PlataLa PlataArgentina
  3. 3.Departamento de BiofisicaUniversidade Federal de São PauloSão PauloBrazil
  4. 4.Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de BarcelonaBarcelonaSpain
  5. 5.Instituto de Ciencias de la Salud, Universidad Nacional Arturo JauretcheBuenos AiresArgentina
  6. 6.CITECBuenos AiresArgentina
  7. 7.Laboratorio de Neurofisiología del Instituto Multidisciplinar de Biología Celular (IMBICE), La PlataBuenos AiresArgentina

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