Applied Biochemistry and Biotechnology

, Volume 187, Issue 1, pp 1–13 | Cite as

Biochemical and Molecular Study of Trichoderma harzianum Enriched Secretome Protein Profiles Using Lectin Affinity Chromatography

  • Stephanie Nauom
  • Benedito Rodrigues da Silva Neto
  • Marcela Suriani Ribeiro
  • Wellington Ramos Pedersoli
  • Cirano José Ulhoa
  • Roberto N. Silva
  • Valdirene Neves MonteiroEmail author


Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum. This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.


Glycoprotein Trichoderma harzianum Enriched secretome Phytopathogens 


Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12010_2018_2795_MOESM1_ESM.docx (27 kb)
Table S1 (DOCX 27 kb)
12010_2018_2795_MOESM2_ESM.docx (12 kb)
Table S2 (DOCX 11 kb)


  1. 1.
    Almeida, F. B., Cerqueira, F. M., Silva, R. N., Ulhoa, C. J., & Lima, A. L. (2007). Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production. Biotechnology Letters, 29(8), 1189–1193.Google Scholar
  2. 2.
    Bartnicki-Garcia, S. (1968). Cell wall chemistry, morphogenesis, and taxonomy of Fungi. Annual Review of Microbiology, 22(1), 87–108.Google Scholar
  3. 3.
    Bowman, S. M., & Free, S. J. (2006). The structure and synthesis of the fungal cell wall. BioEssays, 28(8), 799–808.Google Scholar
  4. 4.
    Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., & Henrissat, B. (2009). The carbohydrate-active enZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research, 37, D233–D238.Google Scholar
  5. 5.
    Charney, M. S., & Tomarelli, R. M. (1947). A colorimetric method for the determination of the proteolytic activity of duodenal juice. The Journal of Biological Chemistry, 171, 501–505.Google Scholar
  6. 6.
    Chen, C.-C., Su, W.-C., Huang, B.-Y., Chen, Y.-J., Tai, H.-C., & Obena, R. P. (2014). Interaction modes and approaches to glycopeptides and glycoprotein enrichment. Analyst. Journal The Royal Society of Chemistry, 139, 688–704.Google Scholar
  7. 7.
    Chen, W., Smeekens, J. M., & Wu, R. (2014). A universal chemical enrichment method for mapping the yeast N-glycoproteome by mass spectrometry (MS). Molecular & Cellular Proteomics, 13(6), 1563–1572.Google Scholar
  8. 8.
    Cohen-Kupiec, R., Broglie, K. E., Friesem, D., Broglie, R. M., & Chet, I. (1999). Molecular characterization of a novelb-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum. Gene, 226(2), 147–154.Google Scholar
  9. 9.
    Darula, Z., & Medzihradszky, K. F. (2009). Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Molecular and Cellular Proteomics, 8(11), 2515–2526.Google Scholar
  10. 10.
    De la Cruz, J., Pintor-Toro, J. A., Benítez, T., Llobell, A., & Romero, L. C. (1995). Novel endo-β-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. Journal of Bacteriology, 177(23), 6937–6945.Google Scholar
  11. 11.
    De Pourcq, K., De Schutter, K., & Callewaert, N. (2010). Engineering of glycosylation in yeast and other fungi: current state and perspectives. Applied Microbiology and Biotechnology, 87, 1617–1631.Google Scholar
  12. 12.
    Delabona, P. S., Cota, J., Hoffmam, Z. B., Paixão, D. A. A., Farinas, C. S., Cairo, J. P. L. F., & da Cruz Pradella, J. G. (2013). Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase. Bioresource Technology, 131, 500–507.Google Scholar
  13. 13.
    Dos Santos, C. L., Pedersoli, W. R., Antoniêto, A. C., Steindorff, A. S., Silva-Rocha, R., Martinez-Rossi, N. M., Rossi, A., Brown, N., Goldman, G. H., Faça, V. M., Persinoti, G. F., & Silva, R. N. (2014). Comparative metabolism of cellulose, sophorose and glucose in Trichoderma reesei using high-throughput genomic and proteomic analyses. Biotechnology for Biofuels, 7(1), 41.Google Scholar
  14. 14.
    Druzhinina, I. S., Seidl-Seiboth, V., Herrera-Estrella, A., Horwitz, B. A., Kenerley, C. M., Monte, E., Mukherjee, P. K., Zeilinger, S., Grigoriev, I. V., & Kubicek, C. P. (2011). Trichoderma: the genomics of opportunistic success. Nature Reviews Microbiology, 16, 749–759.Google Scholar
  15. 15.
    Duo-Chuan, L. (2006). Review of fungal chitinases. Mycopathologia, 161(6), 345–360.Google Scholar
  16. 16.
    Durham, M., & Regnier, F. E. (2006). Targetedglycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome. Journal of Chromatography A, 1132(1-2), 165–173.Google Scholar
  17. 17.
    Gaikwad, S. M., Keskar, S. S., & Kha, M. I. (1995). Purification and characterization of a-D mannosidase from Aspergillus sp. Biochimica et Biophysica Acta, 1250, 144–148.Google Scholar
  18. 18.
    Goto, M. (2007). Protein O-glycosylation in fungi: diverse structures and multiple functions. Bioscience, Biotechnology, and Biochemistry, 71(6), 1415–1427.Google Scholar
  19. 19.
    Horton, P., Park, K. J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., & Nakai, K. (2007). WoLF PSORT: protein localization predictor. Nucleic Acids Research, 35(Web Server issue), W585–W587.Google Scholar
  20. 20.
    Kaneko, S., Kuno, A., Matsuo, N., Ishii, T., Kobayashi, H., Hayashi, K., & Kusakabe, I. (1998). Substrate Specificity of the α-L-Arabinofuranosidase from Trichoderma reesei. Bioscience, Biotechnology, and Biochemistry, 62, 11.Google Scholar
  21. 21.
    Keller, A., Nesvizhskii, A. I., Kolker, E., & Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Analytical Chemistry, 74(20), 5383–5392.Google Scholar
  22. 22.
    Kubicek, C. P., Mach, R. L., Peterbauer, C. K., & Lorito, M. (2001). Trichoderma: from genes to biocontrol. Journal of Plant Pathology, 83(2), 11–23.Google Scholar
  23. 23.
    Laemmli, U. K. (1970). Cleavage of structural proteins assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.Google Scholar
  24. 24.
    Levasseur, A., Drula, E., Lombard, V., Coutinho, P. M., & Henrissat, B. (2013). Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnology for Biofuels, 6(1), 41.Google Scholar
  25. 25.
    Liu, Z., Wei, R., He, W., Ruan, Y., & Liu, C. (2015). Characterization of an extracellularly derived α-mannosidase from the liquid exudate of the sclerotia of Sclerotiniasclerotiorum (Lib.) de Bary. Antonie Van Leeuwenhoek, 108(1), 107–115.Google Scholar
  26. 26.
    Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods, 25(4), 402–408.Google Scholar
  27. 27.
    MacLean, B., Eng, J. K., Beavis, R. C., & McIntosh, M. (2006). General framework for developing and evaluating database scoring algorithms using the TANDEM search engine. Bioinformatics, 22(22), 2830–2832.Google Scholar
  28. 28.
    Markovich, N. A., & Kononova, G. L. (2003). Lytic enzymes of Trichoderma and their role in protecting plants from fungal diseases. Prikladnaia Biokhimiia i Mikrobiologiia, 39(4), 389–400.Google Scholar
  29. 29.
    Martin, K., McDougall, B. M., McIlroy, S., Jayus, Chen, J., & Seviour, R. J. (2007). Biochemistry and molecular biology of exocellular fungal β-(1,3)- and β-(1,6)-glucanases. FEMS Microbiology Reviews, 31(2), 168–192.Google Scholar
  30. 30.
    Monteiro, V. N., Silva, R. N., Steindorff, A. S., Costa, F. T., Noronha, E. F., Ricart, C. A. O., Sousa, M. V., Vainstein, M. H., & Ulhoa, C. J. (2010). New insights in Trichoderma harzianumAntagonism of fungal plant pathogens by secreted protein analysis. Current Microbiology, 61, 298–305.Google Scholar
  31. 31.
    Nesvizhskii, A. I., Keller, A., Kolker, E., & Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Analytical Chemistry, 75(17), 4646–4658.Google Scholar
  32. 32.
    Ramada, M. H. S., Steindorff, A. S., Bloch Jr., C., & Ulhoa, C. J. (2016). Secretome analysis of the mycoparasitic fungus Trichoderma harzianum ALL 42 cultivated in different media supplemented with Fusarium solani cell wall or glucose. Proteomics, 16(3), 477–490.Google Scholar
  33. 33.
    Roche, N., Berna, P., Desgranges, C., & Durand, A. (1995). Substrate use and production of α-L-arabinofuranosidase during solid-state culture of Trichoderma reesei on sugar beet pulp. Enzyme and Microbial Technology, 17(10), 935–941.Google Scholar
  34. 34.
    Ruiz-Herrera, J. (1992). Fungal cell wall: structure, synthesis, and assembly (p. 248). Boca Raton: CRC Press.Google Scholar
  35. 35.
    Schmoll, M., et al. (2016). The genome of three uneven siblings: footprints of the lifestyles of three Trichoderma species. Microbiology and Molecular Biology Reviews, 80(1), 205–327.Google Scholar
  36. 36.
    Selinheimo, E., Saloheimo, M., Ahola, E., Westerholm-Parvinen, A., Kalkkinen, N., Buchert, J., & Kruus, K. (2006). Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei. FEBS Journal, 273, 4322–4335.Google Scholar
  37. 37.
    Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., & Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76–85.Google Scholar
  38. 38.
    Steindorff, A. S., Silva, R. N., Coelho, A. S. G., Noronha, E. F., & Ulhoa, C. J. (2012). Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against F. solani. Biological Control, 61(2), 134–140.Google Scholar
  39. 39.
    Steindorff, A. S., Ramada, M. H. S., Coelho, A. S. G., Miller, R. N., Júnior, G., Pappas, G. J., Ulhoa, C. J., & Noronha, E. F. (2014). Identification of mycoparasitism-related genes against the phytopathogenSclerotiniasclerotiorum through transcriptome and expression profile analysis in Trichoderma harzianum. BMC Genomics, 15(1), 204.Google Scholar
  40. 40.
    Tachibana, K., Nakamura, S., Wang, H., Iwasaki, H., Tachibana, K., Maebara, K., Cheng, L., Hirabayashi, J., & Narimatsu, H. (2006). Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography. Glycobiology, 16(1), 46–53.Google Scholar
  41. 41.
    Troian, R. F., Steindorff, A. S., Ramada, M. H. S., Arruda, W., & Ulhoa, C. J. (2014). Mycoparasitism studies of Trichoderma harzianum against Sclerotiniasclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes. Biotechnology Letters, 36(10), 2095–2101.Google Scholar
  42. 42.
    Viterbo, A., Ramot, O., Chernin, L., & Chet, I. (2002). Significance of lytic enzymes from Trichoderma ssp. in the biocontrol of fungal plant pathogens. Antonie von Leewenhoek, 81(1), 549–556.Google Scholar
  43. 43.
    Yip, V. L., & Withers, S. G. (2006). Family 4 glycosidases carry out efficient hydrolysis of thioglycosides by an alpha, beta-elimination mechanism. Angewandte Chemie International Edition in English, 45(37), 6179–6182.Google Scholar
  44. 44.
    Zaidi, K. U., Ali, A. S., & Ali, S. A. (2014). Purification and characterization of melanogenic enzyme tyrosinase from button mushroom. Enzyme Research, 2014, 1–6.Google Scholar

Copyright information

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

Authors and Affiliations

  • Stephanie Nauom
    • 1
  • Benedito Rodrigues da Silva Neto
    • 1
  • Marcela Suriani Ribeiro
    • 2
  • Wellington Ramos Pedersoli
    • 3
  • Cirano José Ulhoa
    • 2
  • Roberto N. Silva
    • 3
  • Valdirene Neves Monteiro
    • 1
    Email author return OK on get
  1. 1.Campus of Exact Sciences and Technologies, Campus Henrique SantilloState University of Goiás (UEG)AnápolisBrazil
  2. 2.Department of Biochemistry and Cellular Biology, Biological Sciences Institute, Campus SamambaiaFederal University of Goiás (UFG)GoiâniaBrazil
  3. 3.Department of Biochemistry and Immunology, Ribeirão Preto Medical SchoolUniversity of São Paulo (USP)Ribeirão PretoBrazil

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