Skip to main content

Advertisement

SpringerLink
Log in

Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds

  • Published:
Journal of Biosciences Aims and scope Submit manuscript

Abstract

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains ~39% β-sheets but only ~5% α-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at ~70°C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed ~24 and ~25% sequence identity with α-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149–Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44–Cys91, conserved in all Kunitz-type TIs, is also identified in the model.

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

Abbreviations

ACN:

acetonitrile

BAEE:

N-α-benzoyl-l-arginine ethyl ester

CID:

collision induced dissociation

CD:

circular dichroism

DSF:

differential scanning fluorimetry

DTT:

dithiothreitol

OPI:

okra protease inhibitor

PI:

protease inhibitor

RP-HPLC:

reverse-phase high-performance liquid chromatography

RT:

retention time

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SKTI:

soybean Kunitz-trypsin inhibitor

TFA:

trifluoroacetic acid

TI:

trypsin inhibitor

References

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W and Lipman DJ 1997 Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25 3389–3402

    Article  CAS  Google Scholar 

  • Azarkan M, Dibiani R, Goormaghtigh E, Raussens V and Baeyens-Volant D 2006 The papaya Kunitz-type trypsin inhibitor is a highly stable β-sheet glycoprotein. Biochim. Biophys. Acta 1764 1063–1072

    Article  CAS  Google Scholar 

  • Bhattacharyya A and Babu CR 2009 Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: Insight into structural and antimalarial features. Phytochemistry 70 703–712

    Article  CAS  Google Scholar 

  • Bobbili KB, Pohlentz G, Narahari A, Sharma K, Surolia A, Mormann M and Swamy MJ 2018 Coccinia indica agglutinin, a 17 kDa PP2 like phloem lectin: Affinity purification, primary structure and formation of self-assembled filaments. Int. J. Biol. Macromol. 108 1227–1236

    Article  CAS  Google Scholar 

  • Datta D and Swamy MJ 2017 Fluorescence and circular dichroism studies on the accessibility of tryptophan residues and unfolding of a jacalin-related α-d-galactose-specific lectin from mulberry (Morus indica). J. Photochem. Photobiol. B 170 108–117

    Article  CAS  Google Scholar 

  • Datta D, Pohlentz G, Schulte M, Kaiser M, Goycoolea FM, Müthing J, Mormann M and Swamy MJ 2016 Physico-chemical characteristics and primary structure of an affinity-purified α-D-galactose-specific, jacalin-related lectin from the latex of mulberry (Morus indica). Arch. Biochem. Biophys. 609 59–68

    Article  CAS  Google Scholar 

  • De Leo F, Volpicella M, Licciulli F, Liuni S, Gallerani R and Ceci LR 2002 PLANT-PIs: A database for plant protease inhibitors and their genes. Nucleic Acids Res. 30 347–348

    Article  Google Scholar 

  • García-Olmedo F, Salcedo G, Sánchez-Monge R, Gómez L, Royo J and Carbonero P 1987 Plant proteinaceous inhibitors of proteinases and alpha-amylases. Oxford Surv. Plant Mol. Cell Biol. 4 275–334

    Google Scholar 

  • Habib H and Fazili KM 2007 Plant protease inhibitors: A defense strategy in plants. Biotechnol. Mol. Biol. Rev. 2 68–85

    Google Scholar 

  • Kessler A and Baldwin IT 2002 Plant responses to insect herbivory: The emerging molecular analysis. Annu. Rev. Plant Biol. 53 299–328

    Article  CAS  Google Scholar 

  • Kochhar S, Gartenmann K and Juillerat MA 2000 Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry. J. Agric. Food Chem. 48 5593–5599

    Article  CAS  Google Scholar 

  • Kumar G, Pohlentz G, Schulte M, Mormann M and Kumar NS 2014 Jack bean α-mannosidase: amino acid sequencing and N-glycosylation analysis of a valuable glycomics tool. Glycobiology 24 252–261

    Article  Google Scholar 

  • Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 680–685

    Article  CAS  Google Scholar 

  • Laskowski M Jr and Kato I 1980 Protein inhibitors of proteinases. Annu. Rev. Biochem. 49 593–626

    Article  CAS  Google Scholar 

  • Laskowski M and Qasim MA 2000 What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim. Biophys. Acta. 1477 324–337

    Article  CAS  Google Scholar 

  • Lawrence PK and Koundal KR 2002 Plant protease inhibitors in control of phytophagous insects. Electron. J. Biotechnol. 5 93–109

    Article  Google Scholar 

  • Lopez-Otin C and Bond JS 2008 Proteases: Multifunctional enzymes in life and disease. J. Biol. Chem. 283 30433–30437

    Article  CAS  Google Scholar 

  • Mormann M, Eble J, Schwöppe C, Mesters RM, Berdel WE, Peter-Katalinić J and Pohlentz G 2008 Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation. Anal. Bioanal. Chem. 392 831–838

    Article  CAS  Google Scholar 

  • Mosolov VV and Valueva TA 2005 Proteinase inhibitors and their function in plants: A review. Appl. Biochem. Microbiol. 41 227–246

    Article  CAS  Google Scholar 

  • Ogata F, Imamura H, Hirayama K and Makisumi S 1986 Purification and characterization of four trypsin inhibitors from seeds of okra, Abelmoschus esculentus L. Agric. Biol. Chem. 50 2325–2333

    Article  CAS  Google Scholar 

  • Oliva MLV, Silva MC, Sallai RC, Brito MV and Sampaio MU 2010 A novel subclassification for Kunitz proteinase inhibitors from leguminous seeds. Biochimie. 92 1667–1673

    Article  CAS  Google Scholar 

  • Pohlentz G, Marx K and Mormann M 2016 Characterization of protein N-glycosylation by analysis of ZIC-HILIC-enriched intact proteolytic glycopeptides; In: Methods in molecular biology, proteomics in systems biology: Methods and protocols (ed) Reinders J Vol 1394, (Heidelberg: Springer) Chapter 12, pp 163–179

  • Ramasarma PR, Appu Rao AG and Rao DR 1995 Role of disulfide linkages in structure and activity of proteinase inhibitor from horsegram (Dolichos biflorus). Biochim. Biophys. Acta 1248 35–42

    Article  Google Scholar 

  • Read SM and Northcote DH 1983 Subunit structure and interactions of the phloem proteins of Cucurbita maxima (pumpkin). Eur. J. Biochem. 134 561–569

    Article  CAS  Google Scholar 

  • Roy A, Shrivastava SL and Mandal SM 2014 Functional properties of Okra Abelmoschus esculentus L.(Moench): Traditional claims and scientific evidences. Plant Sci. Today 1 121–130

    Article  Google Scholar 

  • Roychaudhuri R, Sarath G, Zeece M and Markwell J 2003 Reversible denaturation of the soybean Kunitz trypsin inhibitor. Arch. Biochem. Biophys. 412 20–26

    Article  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 

  • Šali A and Blundell TL 1993 Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234 779–815

    Article  Google Scholar 

  • Shevchenko A, Tomas H, Havli J, Olsen JV and Mann M 2006 In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1 2856–2860

    Article  CAS  Google Scholar 

  • Shukla E, Agrawal SB and Gaikwad SM 2017 Conformational and functional transitions and in silico analysis of a serine protease from Conidiobolus brefeldianus (MTCC 5185). Int. J. Biol. Macromol. 98 387–397

    Article  CAS  Google Scholar 

  • Sievers F and Higgins DG 2014 Clustal omega. Curr. Protoc. Bioinf. 48 3–13

    Google Scholar 

  • Song HK and Suh SW 1998 Kunitz-type soybean trypsin inhibitor revisited: Refined structure of its complex procine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator. J. Mol. Biol. 275 347–363

    Article  CAS  Google Scholar 

  • Spencer ME and Hodge R 1991 Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao. Planta 183 528–535

    Article  CAS  Google Scholar 

  • Sreerama N and Woody RW 2000 Estimation of protein secondary structure from CD spectra: comparison of CONTIN, SELCON and CDSSTR methods with an expanded reference set. Anal. Biochem. 287 252–260

    Article  CAS  Google Scholar 

  • Sultan NAM, Kenoth R and Swamy MJ 2004 Purification, physicochemical characterization, saccharide specificity, and chemical modification of a Gal/GalNAc specific lectin from the seeds of Trichosanthes dioica. Arch. Biochem. Biophys. 432 212–221

    Article  CAS  Google Scholar 

  • Tai H, McHenry L, Fritz PJ and Furtek DB 1991 Nucleic acid sequence of a 21 kDa cocoa seed protein with homology to the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Plant Mol. Biol. 16 913–915

    Article  CAS  Google Scholar 

  • Thyrock A, Ossendorf E, Stehling M, Kail M, Kurtz T, Pohlentz G, Waschbüsch D, Eggert S, Formstecher E, Müthing J and Dreisewerd K 2013 A new Mint1 isoform, but not the conventional Mint1, interacts with the small GTPase Rab6. PLOS One 8 e64149

    Article  CAS  Google Scholar 

  • Yanes O, Villanueva J, Querol E and Aviles FX 2007 Detection of non-covalent protein interactions by ‘intensity fading’ MALDI-TOF mass spectrometry: Applications to proteases and protease inhibitors. Nat. Protoc. 2 119–130

    Article  CAS  Google Scholar 

  • Zhou D, Lobo YA, Batista IF, Marques-Porto R, Gustchina A, Oliva ML and Wlodawer A 2013 Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin. PLOS One 8 e62252

    Article  CAS  Google Scholar 

  • Zhu-Salzman K and Zeng R. 2015 Insect response to plant defensive protease inhibitors. Annu. Rev. Entomol. 60 233–252

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a research grant from the Department of Biotechnology (India) to MJS. We thank the University Grants Commission (India) for its support through the UPE-II grant to the University of Hyderabad and the CAS program to the School of Chemistry. Support from the Department of Science and Technology under the FIST and PURSE programs is gratefully acknowledged. DD was supported by Senior Research Fellowship from the Council of Scientific and Industrial Research (CSIR), India. The authors are thankful to Nano Temper technologies, Bangalore, India for the use of Prometheus NT.48 instrument and their application specialist, Ms. Saji Menon for help with the DSF experiments. This project was carried out under IRTG-MCGS (GRK 1549) financed by DFG in Germany and UGC in India.

Author information

Authors and Affiliations

  1. School of Chemistry, University of Hyderabad, Hyderabad, 500046, India

    Debparna Datta, Saradamoni Mondal, M Bala Divya, Lalitha Guruprasad & Musti J Swamy

  2. Institute for Hygiene, University of Münster, 48149, Münster, Germany

    Gottfried Pohlentz & Michael Mormann

Authors
  1. Debparna Datta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gottfried Pohlentz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Saradamoni Mondal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Bala Divya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lalitha Guruprasad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Mormann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Musti J Swamy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Musti J Swamy.

Additional information

Communicated by BJ Rao.

Corresponding editor: BJ Rao

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 72 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Datta, D., Pohlentz, G., Mondal, S. et al. Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds. J Biosci 44, 35 (2019). https://doi.org/10.1007/s12038-019-9859-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12038-019-9859-5

Keywords

Search

Navigation