Skip to main content

Advertisement

SpringerLink
Log in

WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

A novel chymotrypsin inhibitor, detected in the endosperm of Triticum aestivum, was purified and characterized with respect to the main physical–chemical properties. On the basis of its specificity, this inhibitor was named WCI (wheat chymotrypsin inhibitor). WCI is a monomeric neutral protein made up of 119 residues and molecular mass value of 12,933.40 Da. Automated sequence and mass spectrometry analyses, carried out on several samples of purified inhibitor, evidenced an intrinsic molecular heterogeneity due to the presence of the isoform [des-(Thr)WCI], accounting for about 40% of the total sample. In vitro, WCI acted as a strong inhibitor of bovine pancreatic chymotrypsin as well as of chymotryptic-like activities isolated from the midgut of two phytophagous insects, Helicoverpa armigera (Hüb.) and Tenebrio molitor L., respectively. No inhibitory activities were detected against bacterial subtilisins, bovine pancreatic trypsin, porcine pancreatic elastase or human leukocyte elastase. The primary structure of WCI was significantly similar (45.7–89.1%) to those of several proteins belonging to the cereal trypsin/α-amylase inhibitor super-family and showed the typical sequence motif of this crowed protein group. The cDNA of the inhibitor (wci-cDNA) was isolated from wheat immature caryopses and employed to obtain a recombinant product in E. coli. Experimental evidences indicated that the recombinant inhibitor was localized in the inclusion bodies from which it was recovered as soluble and partially active protein by applying an appropriate refolding procedure. WCI reactive site localization, as well as its inhibitory specificity, was investigated by molecular modeling approach.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

BTEE:

N-Benzoyl-l-tyrosine ethyl ester

CNBr:

Cyanogen bromide

DTT:

Dithiothreitol

IPTG:

Isopropyl-β-d-1-thiogalactopyranoside

MALDI-TOF:

Matrix-assisted laser desorption/ionisation-time of flight

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

SDS:

Sodium dodecyl sulphate

TAME:

N-α-p-Toluene-sulphonyl-l-arginine methyl ester

TFA:

Trifluoroacetic acid

TLCK:

N-p-tosyl-l-Lysine chloromethyl ketone

TPCK:

N-p-tosyl-l-Phenylalanine chloromethyl ketone

References

  • Alfonso-Rubi J, Ortego F, Castanera P, Carbonero P, Diaz I (2003) Transgenic expression of trypsin inhibitor CMe from barley in indica and japonica rice, confers resistance to the rice weevil Sitophilus oryzae. Transgenic Res 12:23–31

    Article  PubMed  CAS  Google Scholar 

  • Barber D, Sàncez-Monge R, Garcia-Olmedo F, Salcedo G, Mendez E (1986) Evolutionary implications of sequential homologies among members of the trypsin/α-amylase inhibitor family (CM proteins) in wheat and barley. Biochim Biophys Acta 873:147–151

    Article  CAS  Google Scholar 

  • Behnke CA, Yee VC, Trong IL, Pedersen LC, Stenkamp RE, Kim SS, Reeck GR, Teller DC (1998) Structural determinants of the bifunctional corn Hageman factor inhibitor: X-ray crystal structure at 1.95 Å resolution. Biochemistry 37:15277–15288

    Article  PubMed  CAS  Google Scholar 

  • Birk Y (2003) Plant protease inhibitors: significance in nutrition, plant protection, cancer prevention and genetic engineering. Springer, Berlin

    Google Scholar 

  • Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451

    Article  PubMed  CAS  Google Scholar 

  • Boisen S, Djurtoft R (1981) Trypsin inhibitor from wheat endosperm: purification and characterization. Cereal Chem 58:460–463

    CAS  Google Scholar 

  • Bowman DE (1948) Further identification of bean trypsin inhibiting factors. Arch Biochem Biophys 16:109–113

    CAS  Google Scholar 

  • Buonocore V, Gramenzi F, Pace W, Petrucci T, Poerio E, Silano V (1980) Interaction of wheat monomeric and dimeric protein inhibitors with alpha-amylase from yellow mealworm (Tenebrio molitor L. larva). Biochem J 187:637–645

    PubMed  CAS  Google Scholar 

  • Cha S (1975) Tight-binding inhibitors-I. Kinetic behavior. Biochem Pharmacol 24:2177–2185

    Article  PubMed  CAS  Google Scholar 

  • Del Vecchio Blanco F, Cafaro V, Di Maro A, Scognamiglio R, Siniscalco G, Parente A, Di Donato A (1998) A recombinant ribosome-inactivating protein from the plant Phytolacca dioica L. produced from a synthetic gene. FEBS Lett 437:241–245

    Article  PubMed  CAS  Google Scholar 

  • Di Gennaro S, Ficca AG, Panichi D, Poerio E (2005) cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests. Biol Chem 386:383–389

    Article  PubMed  CAS  Google Scholar 

  • Di Maro A, Ferranti P, Mastronicola M, Polito L, Bolognesi A, Stirpe F, Malorni A, Parente A (2001) Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation. J Mass Spectrom 36:38–46

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Casado G, Sanchez-Monge R, Lopez-Otin C, Salcedo G (1994) Rye inhibitors of animal alpha-amylases show different specificities, aggregative properties and IgE-binding capacities than their homologues from wheat and barley. Eur J Biochem 224:525–531

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Olmedo F, Salcedo v, Sanchez-Monge R, Gomez L, Royo J, Carbonero P (1987) Plant proteinaceous inhibitors of proteinases and alpha-amylases. Oxford Surv Plant Mol Cell Biol 4:275–334

    CAS  Google Scholar 

  • Gautier MF, Alary R, Joudrier P (1990) Cloning and characterization of a cDNA encoding the wheat (Triticum durum Desf.) CM16 protein. Plant Mol Biol 14:313–322

    Article  PubMed  CAS  Google Scholar 

  • Gourinath S, Alam N, Srinivasan A, Betzel C, Singh TP (2000) Structure of the bifunctional inhibitor of trypsin and α-amylase from ragi seeds at 2.2 Å resolution. Acta Crystallogr D Biol Crystallogr 56:287–293

    Article  PubMed  CAS  Google Scholar 

  • Janoff A (1969) Alanine p-nitrophenyl esterase activity of human leucocyte granules. Biochem J 114:157–159

    PubMed  CAS  Google Scholar 

  • Oda Y, Matsunaga T, Fukuyama K, Miyazaki T, Morimoto T (1997) Tertiary and quaternary structures of 0.19 α-amylase inhibitor from wheat kernel determined by X-ray analysis at 2.06 Å resolution. Biochemistry 36:13503–13511

    Article  PubMed  CAS  Google Scholar 

  • Odani S, Koide T, Ono T (1983) A possible evolutionary relationship between plant trypsin inhibitor, alpha-amylase inhibitor, and mammalian pancreatic secretory trypsin inhibitor (Kazal). J Biochem 1993:1701–1704

    Google Scholar 

  • Parente A, Merrifield B, Geraci G, D’Alessio G (1985) Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease. Biochemistry 24:1098–1104

    Article  PubMed  CAS  Google Scholar 

  • Poerio E, Carrano L, Carzillo AM, Buonocore V (1989) A trypsin inhibitor from the water-soluble protein fraction of wheat kernel. Phytochemistry 28:1307–1311

    Article  CAS  Google Scholar 

  • Poerio E, Di Gennaro S, Di Maro A, Farisei F, Ferranti P, Parente A (2003) Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin. Biol Chem 384:295–304

    Article  PubMed  CAS  Google Scholar 

  • Prescott A, Martin C (1987) A rapid method for the quantitative assessment of levels of specific mRNAs in plants. Plant Mol Biol Rep 4:219–224

    Article  CAS  Google Scholar 

  • Rawlings ND, Tolle DP, Barrett AJ (2004a) Evolutionary families of peptidase inhibitors. Biochem J 378:705–716

    Article  PubMed  CAS  Google Scholar 

  • Rawlings ND, Barrett AJ, Bateman A (2004b) MEROPS: the peptidase database. Nucleic Acids Res 38:D227–D233

    Article  Google Scholar 

  • Richardson M (1991) Seed storage proteins: the enzyme inhibitors. In: Rogers LJ (ed) Methods in plant biochemistry, vol 5. Academic Press, New York, pp 259–305

  • Robinson LH, Juttner J, Milligan A, Lahnstein J, Eglinton JK, Evans DE (2007) The identification of a barley haze active protein that influences beer haze stability: cloning and characterisation of the barley SE protein as a barley trypsin inhibitor of the chloroform/methanol type. J Cereal Sci 45:343–352

    Article  CAS  Google Scholar 

  • Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26:1628–1635

    Article  PubMed  CAS  Google Scholar 

  • Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738

    Article  PubMed  CAS  Google Scholar 

  • Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28:425–449

    Article  CAS  Google Scholar 

  • Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  PubMed  CAS  Google Scholar 

  • Scudiero R, Capasso C, Del Vecchio-Blanco F, Savino G, Capasso A, Parente A, Parisi E (1995) Isolation and primary structure determination of a metallothionein from Paracentrotus lividus (Echinodermata, Echinoidea). Comp Biochem Phys B Biochem Mol Biol 111:329–336

    Article  CAS  Google Scholar 

  • Severino V, Chambery A, Vitiello M, Cantisani M, Galdiero S, Galdiero M, Malorni L, Di Maro A, Parente A (2010) Proteomic analysis of human U937 cell line activation mediated by Haemophilus influenzae type b P2 porin and its surface-exposed loop 7. J Proteome Res 9:1050–1062

    Article  PubMed  CAS  Google Scholar 

  • Shewry PR (1995) Plant storage proteins. Biol Rev 70:375–426

    Article  PubMed  CAS  Google Scholar 

  • Strobl S, Muhlhahn P, Bernstein R, Wiltscheck R, Maskos K, Wunderlich M, Huber R, Glockshuber R, Holak TA (1995) Determination of the three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds by NMR spectroscopy. Biochemistry 34:8281–8293

    Article  PubMed  CAS  Google Scholar 

  • Strobl S, Maskos K, Betz M, Wiegand G, Huber R, Gomis-Ruth FX, Glockshuber R (1998) Crystal structure of yellow meal worm α-amylase at 1.64 Å resolution. J Mol Biol 278:617–628

    Article  PubMed  CAS  Google Scholar 

  • Ussuf KK, Laxmi NH, Mitra R (2001) Proteinase inhibitors: plant-derived genes of insecticidal protein for developing insect-resistant transgenic plants. Curr Sci 80:847–853

    CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the Grant COFIN 2006, from the Italian Ministero Istruzione, Università e Ricerca (MIUR) and funds from Università degli Studi della Tuscia and the Seconda Università degli Studi di Napoli. The post-doc fellowships to Dr. Natalia Bruni and Valeria Capuzzi were funded by the Università degli Studi della Tuscia. The post-doc fellowship to Dr. Francesca Tedeschi was funded by the Italian Ministero Istruzione, Università e Ricerca (MIUR) and Seconda Università degli Studi di Napoli, with the grant Fondo Sostegno Giovani, E.F. 2008.

Author information

Authors and Affiliations

  1. Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, 81100, Caserta, Italy

    Antimo Di Maro & Valeria Severino

  2. Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via S.Camillo de Lellis snc, 01100, Viterbo, Italy

    Francesca Farisei, Daniela Panichi, Natalia Bruni, Valeria Capuzzi, Francesca Tedeschi & Elia Poerio

  3. Dipartimento di Scienze Ambientali, Università degli Studi della Tuscia, 01100, Viterbo, Italy

    Anna Grazia Ficca

  4. Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli “Federico II”, 80055, Portici, Italy

    Pasquale Ferranti

Authors
  1. Antimo Di Maro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesca Farisei
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniela Panichi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Valeria Severino
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Natalia Bruni
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anna Grazia Ficca
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Pasquale Ferranti
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Valeria Capuzzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Francesca Tedeschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Elia Poerio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Elia Poerio.

Additional information

This paper was dedicated by Elia Poerio to the memory of Dr. Hiroshi Taniuchi, distinguished scientist of remarkable intellectual integrity.

The protein and nucleotide sequence data reported in this paper were deposited in the UniProt Knowledgebase and NBCI data bank under the following accession numbers: P83207 and AJ422078, respectively.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Di Maro, A., Farisei, F., Panichi, D. et al. WCI, a novel wheat chymotrypsin inhibitor: purification, primary structure, inhibitory properties and heterologous expression. Planta 234, 723–735 (2011). https://doi.org/10.1007/s00425-011-1437-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-011-1437-5

Keywords

Search

Navigation