Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants

Wioletta Górka, Renata Bakowska, Markku Saloheimo, Merja Penttilä, G. Palamarczyk, Joanna S. Kruszewska

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

Abstract

The saprophytic fungus Trichoderma reesei secretes a wide range of enzymes which are of considerable biotechnological importance. It was postulated that O-mannosylation is necessary for protein secretion in T.reesei. Due to the complicated genetics of the fungus it is very difficult to study this process in T.reesei. Thus to follow an effect of O-mannosylation on the secretion of T.reesei protein CBHII (cellobiohydrolase II) containing a heavily O-mannosylated linker between the catalytic and cellulose binding domains, we have decided to use S.cerevisiae. In S.cerevisiae seven genes, PMT 1-7 ,coding for protein-O-mannosyltransferases, the first enzyme starting synthesis of O-glycosidic linkage sugars, were identified. CBHII was expressed in yeast pmt mutants and in a parental strain. Enzyme activity was detected on the plates containing medium with beta-glucan. Hydrolysis of the substrate was observed only in pmt4 mutant and in parental strain. pmtl and pmt2 mutants bearing cbh2 gene were not exhibiting CBHII activity on the plate. CBHII protein secreted by the pmt4 mutant and the parental strain was glycosylated to the same extend what means that CBHII is not glycosylated by Pmt4p. Our results indicate that most likely Pmt1,2 protein complex is needed for CBHII O-mannosylation. If the O-mannosylation is necessary for protein secretion the protein should be accumulated in the cells of pmtl and pmt2 mutants. However, immunodetection of CBHII in the cells free extracts of the pmtl and pmt2 strains indicates absence of the protein in the cells. In pmt2 mutant this effect was corrected by Trichoderma pmt gene homologous to the yeast PMT2. The latter does not confirm however direct dependence between secretion on O-mannosylation. Thus the proteolytic stability of the non glycosylated CBHII protein is also considered.
Original languageEnglish
Title of host publicationPoster Abstracts
Pages189
Publication statusPublished - 2004
Event7th European Conference on Fungal Genetics - Copenhagen, Denmark
Duration: 17 Apr 200420 Apr 2004

Conference

Conference7th European Conference on Fungal Genetics
CountryDenmark
CityCopenhagen
Period17/04/0420/04/04

Fingerprint

cellulose 1,4-beta-cellobiosidase
mutants
proteins
protein secretion
secretion
glycosidic linkages
yeasts
microbial genetics
Trichoderma reesei
genes
Trichoderma
cells
beta-glucans
enzymes
cellulose
hydrolysis
enzyme activity
sugars
fungi
synthesis

Cite this

Górka, W., Bakowska, R., Saloheimo, M., Penttilä, M., Palamarczyk, G., & Kruszewska, J. S. (2004). Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants. In Poster Abstracts (pp. 189). [VIIIp-6]
Górka, Wioletta ; Bakowska, Renata ; Saloheimo, Markku ; Penttilä, Merja ; Palamarczyk, G. ; Kruszewska, Joanna S. / Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants. Poster Abstracts. 2004. pp. 189
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abstract = "The saprophytic fungus Trichoderma reesei secretes a wide range of enzymes which are of considerable biotechnological importance. It was postulated that O-mannosylation is necessary for protein secretion in T.reesei. Due to the complicated genetics of the fungus it is very difficult to study this process in T.reesei. Thus to follow an effect of O-mannosylation on the secretion of T.reesei protein CBHII (cellobiohydrolase II) containing a heavily O-mannosylated linker between the catalytic and cellulose binding domains, we have decided to use S.cerevisiae. In S.cerevisiae seven genes, PMT 1-7 ,coding for protein-O-mannosyltransferases, the first enzyme starting synthesis of O-glycosidic linkage sugars, were identified. CBHII was expressed in yeast pmt mutants and in a parental strain. Enzyme activity was detected on the plates containing medium with beta-glucan. Hydrolysis of the substrate was observed only in pmt4 mutant and in parental strain. pmtl and pmt2 mutants bearing cbh2 gene were not exhibiting CBHII activity on the plate. CBHII protein secreted by the pmt4 mutant and the parental strain was glycosylated to the same extend what means that CBHII is not glycosylated by Pmt4p. Our results indicate that most likely Pmt1,2 protein complex is needed for CBHII O-mannosylation. If the O-mannosylation is necessary for protein secretion the protein should be accumulated in the cells of pmtl and pmt2 mutants. However, immunodetection of CBHII in the cells free extracts of the pmtl and pmt2 strains indicates absence of the protein in the cells. In pmt2 mutant this effect was corrected by Trichoderma pmt gene homologous to the yeast PMT2. The latter does not confirm however direct dependence between secretion on O-mannosylation. Thus the proteolytic stability of the non glycosylated CBHII protein is also considered.",
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Górka, W, Bakowska, R, Saloheimo, M, Penttilä, M, Palamarczyk, G & Kruszewska, JS 2004, Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants. in Poster Abstracts., VIIIp-6, pp. 189, 7th European Conference on Fungal Genetics, Copenhagen, Denmark, 17/04/04.

Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants. / Górka, Wioletta; Bakowska, Renata; Saloheimo, Markku; Penttilä, Merja; Palamarczyk, G.; Kruszewska, Joanna S.

Poster Abstracts. 2004. p. 189 VIIIp-6.

Research output: Chapter in Book/Report/Conference proceedingConference abstract in proceedingsScientific

TY - CHAP

T1 - Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants

AU - Górka, Wioletta

AU - Bakowska, Renata

AU - Saloheimo, Markku

AU - Penttilä, Merja

AU - Palamarczyk, G.

AU - Kruszewska, Joanna S.

PY - 2004

Y1 - 2004

N2 - The saprophytic fungus Trichoderma reesei secretes a wide range of enzymes which are of considerable biotechnological importance. It was postulated that O-mannosylation is necessary for protein secretion in T.reesei. Due to the complicated genetics of the fungus it is very difficult to study this process in T.reesei. Thus to follow an effect of O-mannosylation on the secretion of T.reesei protein CBHII (cellobiohydrolase II) containing a heavily O-mannosylated linker between the catalytic and cellulose binding domains, we have decided to use S.cerevisiae. In S.cerevisiae seven genes, PMT 1-7 ,coding for protein-O-mannosyltransferases, the first enzyme starting synthesis of O-glycosidic linkage sugars, were identified. CBHII was expressed in yeast pmt mutants and in a parental strain. Enzyme activity was detected on the plates containing medium with beta-glucan. Hydrolysis of the substrate was observed only in pmt4 mutant and in parental strain. pmtl and pmt2 mutants bearing cbh2 gene were not exhibiting CBHII activity on the plate. CBHII protein secreted by the pmt4 mutant and the parental strain was glycosylated to the same extend what means that CBHII is not glycosylated by Pmt4p. Our results indicate that most likely Pmt1,2 protein complex is needed for CBHII O-mannosylation. If the O-mannosylation is necessary for protein secretion the protein should be accumulated in the cells of pmtl and pmt2 mutants. However, immunodetection of CBHII in the cells free extracts of the pmtl and pmt2 strains indicates absence of the protein in the cells. In pmt2 mutant this effect was corrected by Trichoderma pmt gene homologous to the yeast PMT2. The latter does not confirm however direct dependence between secretion on O-mannosylation. Thus the proteolytic stability of the non glycosylated CBHII protein is also considered.

AB - The saprophytic fungus Trichoderma reesei secretes a wide range of enzymes which are of considerable biotechnological importance. It was postulated that O-mannosylation is necessary for protein secretion in T.reesei. Due to the complicated genetics of the fungus it is very difficult to study this process in T.reesei. Thus to follow an effect of O-mannosylation on the secretion of T.reesei protein CBHII (cellobiohydrolase II) containing a heavily O-mannosylated linker between the catalytic and cellulose binding domains, we have decided to use S.cerevisiae. In S.cerevisiae seven genes, PMT 1-7 ,coding for protein-O-mannosyltransferases, the first enzyme starting synthesis of O-glycosidic linkage sugars, were identified. CBHII was expressed in yeast pmt mutants and in a parental strain. Enzyme activity was detected on the plates containing medium with beta-glucan. Hydrolysis of the substrate was observed only in pmt4 mutant and in parental strain. pmtl and pmt2 mutants bearing cbh2 gene were not exhibiting CBHII activity on the plate. CBHII protein secreted by the pmt4 mutant and the parental strain was glycosylated to the same extend what means that CBHII is not glycosylated by Pmt4p. Our results indicate that most likely Pmt1,2 protein complex is needed for CBHII O-mannosylation. If the O-mannosylation is necessary for protein secretion the protein should be accumulated in the cells of pmtl and pmt2 mutants. However, immunodetection of CBHII in the cells free extracts of the pmtl and pmt2 strains indicates absence of the protein in the cells. In pmt2 mutant this effect was corrected by Trichoderma pmt gene homologous to the yeast PMT2. The latter does not confirm however direct dependence between secretion on O-mannosylation. Thus the proteolytic stability of the non glycosylated CBHII protein is also considered.

M3 - Conference abstract in proceedings

SP - 189

BT - Poster Abstracts

ER -

Górka W, Bakowska R, Saloheimo M, Penttilä M, Palamarczyk G, Kruszewska JS. Heterologous expression of T. reesei secretory protein in S. cerevisiae PMT mutants. In Poster Abstracts. 2004. p. 189. VIIIp-6