Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing

Marko Nykänen, Marjatta Raudaskoski, Helena Nevalainen, Anita Mikkonen

Research output: Contribution to journalArticleScientificpeer-review

9 Citations (Scopus)

Abstract

Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy.
The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium.
The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB.
Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein.
This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically.
Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay.
Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus.
In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley
Original languageEnglish
Pages (from-to)138-150
JournalCanadian Journal of Microbiology
Volume48
Issue number2
DOIs
Publication statusPublished - 2002
MoE publication typeA1 Journal article-refereed

Fingerprint

Cysteine Endopeptidases
Trichoderma
Hordeum
Tunicamycin
Monensin
Peptides
Enzymes
Gels
Brefeldin A
endopeptidase B
Hyphae
Glycosylation
Gold
Culture Media
Microscopy
Amino Acid Sequence
Peptide Hydrolases
Fungi

Keywords

  • cysteine proteinase
  • secretion
  • Kex2p
  • glycosylation
  • modified Golgi-like body

Cite this

Nykänen, Marko ; Raudaskoski, Marjatta ; Nevalainen, Helena ; Mikkonen, Anita. / Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing. In: Canadian Journal of Microbiology. 2002 ; Vol. 48, No. 2. pp. 138-150.
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abstract = "Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley",
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author = "Marko Nyk{\"a}nen and Marjatta Raudaskoski and Helena Nevalainen and Anita Mikkonen",
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Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing. / Nykänen, Marko; Raudaskoski, Marjatta; Nevalainen, Helena; Mikkonen, Anita.

In: Canadian Journal of Microbiology, Vol. 48, No. 2, 2002, p. 138-150.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing

AU - Nykänen, Marko

AU - Raudaskoski, Marjatta

AU - Nevalainen, Helena

AU - Mikkonen, Anita

PY - 2002

Y1 - 2002

N2 - Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley

AB - Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic inhibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley

KW - cysteine proteinase

KW - secretion

KW - Kex2p

KW - glycosylation

KW - modified Golgi-like body

U2 - 10.1139/w01-144

DO - 10.1139/w01-144

M3 - Article

VL - 48

SP - 138

EP - 150

JO - Canadian Journal of Microbiology

JF - Canadian Journal of Microbiology

SN - 0008-4166

IS - 2

ER -