Eur. J. Entomol. 120: 233-243, 2023 | DOI: 10.14411/eje.2023.026

Comparative kinetic characterization of the activity of glycosylated and non-glycosylated trypsin-like serine protease isolated from adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on the grain of three different cultivars of wheatOriginal article

Fernanda S. ZAVALA-IBARRA1, Aldo A. ARVIZU-FLORES ORCID...2,*, Oliviert MARTÍNEZ-CRUZ ORCID...1, Pablo S. OSUNA-AMARILLAS3, José L. CÁRDENAS-LÓPEZ ORCID...1, Carmen L. DEL-TORO-SÁNCHEZ ORCID...1, Carlos R. GONZÁLEZ-RUIZ4, José A. TAPIA-HERNÁNDEZ ORCID...1, Rey D. ITURRALDE-GARCÍA1, Francisco J. CINCO-MOROYOQUI ORCID...1,*
1 Departamento de Investigación y Posgrado en Alimentos, Grupo de Investigación en Bioquímica, Química Agrícola y Manejo Postcosecha (BioQAMPO), Universidad de Sonora, 83000 Hermosillo, Sonora, México; e-mails: a219230159@unison.mx, oliviert.martinez@unison.mx, joseluis.cardenas@unison.mx, carmen.deltoro@unison.mx, joseagustin.tapia@unison.mx, rey.iturralde@unison.mx, javier.cinco@unison.mx
2 Departamento de Ciencias Químico-Biológicas, Universidad de Sonora, Blvd. Luis Encinas y Blvd. Rosales, Hermosillo, Sonora 83000, México; e-mail: aldo.arvizu@unison.mx
3 Universidad Estatal de Sonora, Carretera Navojoa-Huatabampo km 5, Navojoa, Sonora 85874, México; e-mail: pablo.osuna@ues.mx
4 Instituto Potosino de Investigación Científica y Tecnológica A.C., Camino a la Presa de San José 2055, Lomas 4ta Sección, 78216 San Luis, S.L.P., México; e-mail: carlos.gonzalez@ipicyt.edu.mx

Rhyzopertha dominica is a pest that uses trypsin-like serine protease enzymes to hydrolyse the proteins in the cereal grains on which it feeds. The present study reveals for the first time that that there are both glycosylated and non-glycosylated serine proteases. The progeny of R. dominica reared on the grain three varieties of wheat were used to fractionate their trypsin-like serine proteases using Concanavalin A affinity chromatography. The albumin fractions from the wheat cultivars used in this study were subjected to size exclusion chromatography to fractionate the albumin inhibitors that are highly specific for the serine protease activity of R. dominica. Kinetic and thermodynamic assays were used to differentiate both types of enzymes. In general, the catalytic efficiency values Vmax/Km for glycosylated proteases were higher, indicating that glycosylation increases the affinity for the substrate. Inhibition assays using wheat albumins revealed that the glycosylated enzymes had higher Ki values, indicating a low affinity for the inhibitors than the non-glycosylated enzymes. Thermodynamic analysis indicates that glycosylation increases the activation energy Ea improving the serine proteases' catalysis. Thus it is likely that R. dominica uses glycosylated proteases in order to optimize the hydrolysis of cereal proteins and nullify the action of wheat grain protease inhibitors and increase its chances of survival.

Keywords: Glycosylation, thermodynamic parameters, enzyme kinetics, lectin, Michaelis-Menten equation

Received: September 12, 2022; Revised: June 23, 2023; Accepted: June 23, 2023; Published online: July 3, 2023  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
ZAVALA-IBARRA, F.S., ARVIZU-FLORES, A.A., MARTÍNEZ-CRUZ, O., OSUNA-AMARILLAS, P.S., CÁRDENAS-LÓPEZ, J.L., DEL-TORO-SÁNCHEZ, C.L., ... CINCO-MOROYOQUI, F.J. (2023). Comparative kinetic characterization of the activity of glycosylated and non-glycosylated trypsin-like serine protease isolated from adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) reared on the grain of three different cultivars of wheat. EJE, 120, Article 233-243. https://doi.org/10.14411/eje.2023.026
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. Abdel-Naby M.A., Fouad A.A. & El-Refai H.A. 2015: Catalytic and thermodynamic properties of glycosylated Bacillus cereus cyclodextrin glycosyltransferase. - Int. J. Biol. Macromol. 76: 132-137. Go to original source...
    2. Abdel-Naby M.A., Ibrahim M.H. & El-Refai H.A. 2016: Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin. - Int. J. Biol. Macromol. 85: 238-245. Go to original source...
    3. Abdel-Naby M.A., Ahmed S.A., Wehaidy H.R. & El-Mahdy S.A. 2017: Catalytic, kinetic, and thermodynamic properties of stabilized Bacillus stearothermophilus alkaline protease. - Int. J. Biol. Macromol. 96: 265-271. Go to original source...
    4. An H.J., Froehlich J.W. & Lebrilla C.B. 2009: Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. - Curr. Opin. Chem. Biol. 13: 421-426. Go to original source...
    5. Barrett A.J., Rawlings N.D. & Woessner J.F. 2012: Handbook of Proteolytic Enzymes. 3rd ed. Elsevier Academic Press, San Diego, CA, 984 pp.
    6. Bause E. 1984: Model studies on N-glycosylation of proteins. - Biochem. Soc. Trans. 12: 514-517. Go to original source...
    7. Bonzom C., Hüttner S., Mirgorodskaya E., Chong S.-L., Uthoff S., Steinbüchel A., Verhaert R.M.D. & Olsson L. 2019: Glycosylation influences activity, stability, and immobilization of the feruloyl esterase 1a from Myceliophthora thermophila. - AMB Express 9(126): 13 pp. Go to original source...
    8. Bradford M. 1976: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. - Anal. Biochem. 72: 248-254. Go to original source...
    9. Burlingham B.T. & Widlanski T.S. 2003: An intuitive look at the relationship of Ki and IC50: a more general use for the Dixon plot. - J. Chem. Educ. 80: 214-218. Go to original source...
    10. Chaaban H., Ioannou I., Chebil L., Slimane M., Gérardin C., Paris C., Charbonnel C., Chekir L. & Ghoul M. 2017: Effect of heat processing on thermal stability and antioxidant activity of six flavonoids. - J. Food Process. Preserv. 41: e13203, 12 pp. Go to original source...
    11. Chavaroche A., Cudi M., Giulianotti M., Houghten R.A., Fields G.B. & Minond D. 2014: Glycosylation of a disintegrin and metalloprotease 17 (ADAM17) affects its activity and inhibition. - Anal. Biochem. 449: 68-75. Go to original source...
    12. Chen H., Zhu Y.C., Whitworth R.J., Reese J.C. & Chen M.S. 2013: Serine and cysteine protease-like genes in the genome of a gall midge and their interactions with host plant genotypes. - Insect Biochem. Mol. Biol. 43: 701-711. Go to original source...
    13. Cinco-Moroyoqui F.J., Díaz-Malváez F.I., Alanís-Villa A., Barrón-Hoyos J.M., Cárdenas-López J.L., Cortez-Rocha M.O. & Wong-Corral F.J. 2008: Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae). - Comp. Biochem. Physiol. (B) 150: 153-160. Go to original source...
    14. Cuccioloni M., Mozzicafreddo M., Ali I., Bonfili L., Cecarini V., Eleuteri A.M. & Angeletti M. 2016: Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding. - Food Chem. 213: 571-578. Go to original source...
    15. Delanghe J.R., De Buyzere M.L., De Scheerder I.K., Faust U., Roger J. & Wieme R.J. 1989: Activation energy and lectin affinity chromatography of gamma-glutamyltransferase as a marker for enzyme heterogeneity. - Clin. Biochem. 22: 115-119. Go to original source...
    16. Edde P. 2012: A review of the biology and control of Rhyzo­pertha dominica (F.), the lesser grain borer. - J. Stored Prod. Res. 48: 1-18. Go to original source...
    17. Gavel Y. & von Heijne G. 1990: Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. - Protein Eng. 3: 433-442. Go to original source...
    18. Gholamzadeh C.M., Ghadamyari M. & Sharafi M. 2013: Identification and characterization of gut proteases in the fig tree skeletonizer moth, Choreutis memoranda Hübner (Lepido­ptera: Choreutidae). - Plant Protect. Sci. 46: 19-26. Go to original source...
    19. Goettig P. 2016: Effects of glycosylation on the enzymatic activity and mechanisms of proteases. - Int. J. Mol. Sci. 17(12): 1969, 24 pp. Go to original source...
    20. González-Ruiz C.R., Del-Toro-Sánchez C.L., Cornejo-Ramírez Y.I., Rodríguez-Félix F., Wong-Corral F.J., Márquez-Ríos R., Cárdenas-López J.L. & Francisco J. Cinco-Moroyoqui 2021: Differential biochemical and kinetic properties of α-amylases from Rhyzopertha dominica (F.) progenies reared on wheat varieties differing in α-amylase inhibitory activity. - J. Stored Prod. Res. 90: 101748, 9 pp. Go to original source...
    21. Goodrich J.M. & Basu N. 2012: Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals. - Toxicol. In Vitro 26: 630-635. Go to original source...
    22. Guan X., Chaffey P.K., Wei X., Gulbranson D.R., Ruan Y., Wang X., Li Y., Ouyang Y., Chen L., Zeng C. & et. al. 2018: Chemically precise glycoengineering improves human insulin. - ACS Chem. Biol. 13: 73-81. Go to original source...
    23. Han C., Wang Q., Sun Y., Ruirui Y., Liu M., Wang S., Liu Y., Zhou L. & Li D. 2020: Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites. - Biotechnol. Biofuels 13: 30, 11 pp. Go to original source...
    24. Heussen C. & Dowdle E.B. 1980: Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. - Anal. Biochem. 102: 196-202. Go to original source...
    25. Javed M.R., Rashid M.H., Nadeem H., Riaz M. & Perveen R. 2008: Catalytic and thermodynamic characterization of endoglucanase (CMCase) from Aspergillus oryzae cmc-1. - Appl. Biochem. Biotechnol. 157: 483-497. Go to original source...
    26. Julio A.H.F., Gigliolli A.A.S., Cardoso K.A.K., Drosdoski S.D., Kulza R.A., Seixas F.A.V., Ruvolo-Takasusuki M.C.C., de Souza C.G.M. & Lapenta A.S. 2017: Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2. - Comp. Biochem. Physiol. (C) 195: 7-43. Go to original source...
    27. Konarev A. 1996: Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite coevolution. - Euphytica 92: 89-94. Go to original source...
    28. Laemmli U.K. 1970: Cleavage of structural proteins during the assembly of the head bacteriophage T4. - Nature 22: 77-80. Go to original source...
    29. Lam P.V.N., Goldman R., Karagiannis K., Narsule T., Simonysn V., Doiks V. & Mazumder R. 2013: Structured-based comparative analysis and prediction of N-linked glycosylation sites in evolutionarily distant eukaryotes. - Genom. Proteom. Bioinf. 11: 96-104. Go to original source...
    30. Liebminger E., Grass J., Altmann F., Mach L. & Strasser R. 2013: Characterizing the link between glycosylation state and enzymatic activity of the endo-b1,4-glucanase KORRIGAN1 from Arabidopsis thaliana. - J. Biol. Chem. 288: 22270-22280. Go to original source...
    31. López-Otín C. & Bond J. 2008: Proteases: multifunctional enzymes in life and disease. - J. Biol. Chem. 283: 30433-30437. Go to original source...
    32. Ma B., Guan X., Li Y., Shang S., Li J. & Tan Z. 2020: Protein glycoengineering: An approach for improving protein properties. - Front. Chem. 8: 622, 14 pp. Go to original source...
    33. Mahbobe S., Moloud G., Mohammad G. & Maryam A. 2012: Identification and characterization of midgut digestive proteases from the rosaceous branch borer, Osphranteria coerulescens Redtenbacher (Coleoptera: Cerambycidae). - Rom. J. Biochem. 49: 33-47.
    34. Mehrabadi M., Bandani A.R., Saadati F. & Mahmudvand M. 2011: α-Amylase activity of stored products insects and its inhibition by medicinal plant extracts. - J. Agr. Sci. Tech. 13: 173-182.
    35. Nagai K., Ihara Y., Wada Y. & Taniguchi N. 1997: N-glycosylation is requisite for enzyme activity and Golgi retention of N-acetylglucosaminyltransferase III. - Glycobiology 7: 767-776. Go to original source...
    36. Oppert B. & Morgan T.D. 2013: Improved high-throughput bioassay for Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). - J. Stored Prod. Res. 52: 68-73. Go to original source...
    37. Oppert B., Elpidina E.N., Toutges M. & Mazumdar-Leighton S. 2010: Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors. - Comp. Biochem. Physiol. (D) 5: 280-287. Go to original source...
    38. Osuna-Amarillas P.S., Cinco-Moroyoqui F.J., Cárdenas-López J.L., Ezquerra-Brauer J.M., Sotelo-Mundo R., Cortez-Rocha M.O., Barrón-Hoyos J.M., Rouzaud-Sández O. & Borboa-Flores J. 2012: Biochemical and kinetic characterization of the digestive trypsin-like activity of the lesser grain borer Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). - J. Stored Prod. Res. 51: 41-48. Go to original source...
    39. Priya S., Kaur N., Anil K. & Gupta A.K. 2010: Purification, characterization, and inhibition studies of α-amylase of Rhyzopertha dominica. - Pestic. Biochem. Physiol. 98: 231-237. Go to original source...
    40. Pytelková J., Hubert J., Lep¹ík M., ©obotník J., ©indelka R., Køí¾ková I., Horn M. & Mare¹ M. 2009: Digestive alpha-amylases of the flour moth Ephestia kuehniella - adaptation to alkaline environment and plant inhibitors. - FEBS J. 276: 3531-3546. Go to original source...
    41. Rawlings N.D., Barrett A.J., Thomas P.D., Huang X., Bateman A. & Finn R.D. 2018: The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. - Nucl. Acids Res. 46: D624-D632. Go to original source...
    42. Rendiæ D., Wilson I.B.H. & Paschinger K. 2008: The glycosylation capacity of cells. - Croat. Chem. Acta 82: 7-21.
    43. Roth Z., Yehezkel G. & Khalaila I. 2012: Identification and quantification of protein glycosylation. - Int. J. Carbohydr. Chem. 2012: 640923, 11 pp. Go to original source...
    44. SAS Institute 2005: PROC User's Manual, 8th Ver. SAS Institute, Cary, NC.
    45. Siadat S.O.R., Mollasalehi H. & Heydarzadeh N. 2016: Substrate affinity and catalytic efficiency are improved by decreasing glycosylation sites in Trichoderma reesei cellobiohydrolase I expressed in Pichia pastoris. - Biotechnol. Lett. 38: 483-488. Go to original source...
    46. Silva C.P., Terra W.R., de Sá M.F.G., Samuels R.I., Isejima E.M., Bifano T.D. & Almeida J.S. 2001: Induction of digestive α-amylases in larvae of Zabrotes subfasciatus (Coleoptera: Bruchidae) in response to ingestion of common bean α-amylase inhibitor 1. - J. Insect Physiol. 47: 1283-1290. Go to original source...
    47. Solá R.J. & Griebenow K. 2006: Chemical glycosylation: New insights on the interrelation between protein structural mobility, thermodynamic stability, and catalysis. - FEBS Lett. 580: 1685-1690. Go to original source...
    48. Sotelo-Mundo R.R., López-Zavala A.A., García-Orozco K.D., Arvizu-Flores A.A, Velázquez-Contreras E.F., Valenzuela-Soto E.M., Rojo-Domínguez A. & Kanost M.R. 2007: The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme. - Protein Pept. Lett. 14: 774-778. Go to original source...
    49. Spiro R. 2002: Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptides bonds. - Glycobiology 12: 43R-56R. Go to original source...
    50. Srimathi S. & Jayaraman G. 2005: Effect of glycosylation on the catalytic and conformational stability of homologous α-amylases. - Protein J. 24: 79-88. Go to original source...
    51. Talat S., Thiruvikraman M., Kumari S. & Kaur K.J. 2011: Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity. - Glycoconj. J. 28: 537-555. Go to original source...
    52. Taylor C.B., Talib M.F., McCabe C., Bu L., Adney W.S., Himmel M.E., Crowley M.F. & Beckham G.T. 2012: Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module. - J. Biol. Chem. 287: 3147-3155. Go to original source...
    53. Vandenborre G., Smagghe G., Ghesquière B., Menschaert G., Nagender R., Gevaert K. & Van Damm J. 2011: Diversity in protein glycosylation among insect species. - PLoS ONE 6(2): e16682, 9 pp. Go to original source...
    54. Wei Y.D., Lee K.S., Gui Z.Z., Yoon H.J., Kim I., Je Y.H., Lee S.M., Zhang G.Z., Guo X., Sohn H.D. & Jin B.R. 2006: N-linked glycosylation of a beetle (Apriona germani) cellulose Ag-EGase II is necessary for enzymatic activity. - Insect Biochem. Mol. Biol. 36: 435-441. Go to original source...
    55. Wielkopolan B., Walczak F., Podlesny A., Nawrot R. & Obrepalska-Steplowska A. 2015: Identification and partial characterization of proteases in the cereal leaf beetle (Oulema melanopus, Chrysomelidae, Coleoptera). - Arch. Insect Biochem. Physiol. 88: 192-202. Go to original source...
    56. Zhang Z., Chen Y., Tang Y. & Sheng Z. 2008: Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acids pairs. - BCM Bioinform. 9: 101, 12 pp. Go to original source...
    57. Zhu Y.-C. & Baker J. 1999: Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae). - Insect Biochem. Mol. Biol. 29: 1053-1063. Go to original source...
    58. Zhu-Salzman K. & Zeng R. 2015: Insect response to plant defensive protease inhibitors. - Annu. Rev. Entomol. 60: 233-252. Go to original source...

    This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content