Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

2007 ◽  
Vol 1770 (5) ◽  
pp. 774-780 ◽  
Author(s):  
Wioletta Górka-Nieć ◽  
Renata Bańkowska ◽  
Grażyna Palamarczyk ◽  
Hubert Krotkiewski ◽  
Joanna S. Kruszewska
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trichoderma Reesei ◽  
Protein Glycosylation ◽  
Cellobiohydrolase Ii

Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation

2006 ◽  
Vol 43 (6) ◽  
pp. 422-429 ◽  
Author(s):  
Urszula Perlińska-Lenart ◽  
Renata Bańkowska ◽  
Grażyna Palamarczyk ◽  
Joanna S. Kruszewska
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trichoderma Reesei ◽  
Protein Glycosylation

scholarly journals Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

2006 ◽  
Vol 72 (12) ◽  
pp. 7778-7784 ◽  
Author(s):  
Urszula Perlińska-Lenart ◽  
Jacek Orłowski ◽  
Agnieszka E. Laudy ◽  
Ewa Zdebska ◽  
Grażyna Palamarczyk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Trichoderma Reesei ◽  
Morphological Characterization ◽  
Protein Glycosylation ◽  
Fungal Cell Wall ◽  
Ultrastructural Changes ◽  
Calcofluor White ◽  
Fungal Cell ◽  
Gene Coding

ABSTRACT Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble β-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.

scholarly journals Protein Glycosylation inAspergillus fumigatusIs Essential for Cell Wall Synthesis and Serves as a Promising Model of Multicellular Eukaryotic Development

2012 ◽  
Vol 2012 ◽  
pp. 1-21 ◽  
Author(s):  
Cheng Jin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Posttranslational Modification ◽  
Mammalian Cells ◽  
Protein Glycosylation ◽  
Cell Wall Synthesis ◽  
Fungal Cell ◽  
Multiple Functions ◽  
Significant Attention

Glycosylation is a conserved posttranslational modification that is found in all eukaryotes, which helps generate proteins with multiple functions. Our knowledge of glycosylation mainly comes from the investigation of the yeastSaccharomyces cerevisiaeand mammalian cells. However, during the last decade, glycosylation in the human pathogenic moldAspergillus fumigatushas drawn significant attention. It has been revealed that glycosylation inA. fumigatusis crucial for its growth, cell wall synthesis, and development and that the process is more complicated than that found in the budding yeastS. cerevisiae. The present paper implies that the investigation of glycosylation inA. fumigatusis not only vital for elucidating the mechanism of fungal cell wall synthesis, which will benefit the design of new antifungal therapies, but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes. This paper describes the advances in functional analysis of protein glycosylation inA. fumigatus.

scholarly journals Production of Recombinant Trichoderma reesei Cellobiohydrolase II in a New Expression System Based on Wickerhamomyces anomalus

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Dennis J. Díaz-Rincón ◽  
Ivonne Duque ◽  
Erika Osorio ◽  
Alexander Rodríguez-López ◽  
Angela Espejo-Mojica ◽  
...  
Keyword(s):  
Trichoderma Reesei ◽  
Recombinant Expression ◽  
Expression System ◽  
Enzymatic Extract ◽  
Native Strain ◽  
Wickerhamomyces Anomalus ◽  
Optimum Ph ◽  
Hydrolysis Of Cellulose ◽  
Cellobiohydrolase Ii ◽  
Hydrolysis Of

Cellulase is a family of at least three groups of enzymes that participate in the sequential hydrolysis of cellulose. Recombinant expression of cellulases might allow reducing their production times and increasing the low proteins concentrations obtained with filamentous fungi. In this study, we describe the production of Trichoderma reesei cellobiohydrolase II (CBHII) in a native strain of Wickerhamomyces anomalus. Recombinant CBHII was expressed in W. anomalus 54-A reaching enzyme activity values of up to 14.5 U L−1. The enzyme extract showed optimum pH and temperature of 5.0–6.0 and 40°C, respectively. Enzyme kinetic parameters (KM of 2.73 mM and Vmax of 23.1 µM min−1) were between the ranges of values reported for other CBHII enzymes. Finally, the results showed that an enzymatic extract of W. anomalus 54-A carrying the recombinant T. reesei CBHII allows production of reducing sugars similar to that of a crude extract from cellulolytic fungi. These results show the first report on the use of W. anomalus as a host to produce recombinant proteins. In addition, recombinant T. reesei CBHII enzyme could potentially be used in the degradation of lignocellulosic residues to produce bioethanol, based on its pH and temperature activity profile.

scholarly journals Yil102c-A is a Functional Homologue of the DPMII Subunit of Dolichyl Phosphate Mannose Synthase in Saccharomyces cerevisiae

2020 ◽  
Vol 21 (23) ◽  
pp. 8938
Author(s):  
Sebastian Piłsyk ◽  
Urszula Perlinska-Lenart ◽  
Anna Janik ◽  
Elżbieta Gryz ◽  
Marta Ajchler-Adamska ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Trichoderma Reesei ◽  
Transmembrane Domain ◽  
Human Cells ◽  
Catalytic Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dolichyl Phosphate ◽  
Functional Homolog ◽  
Wide Range

In a wide range of organisms, dolichyl phosphate mannose (DPM) synthase is a complex of tree proteins Dpm1, Dpm2, and Dpm3. However, in the yeast Saccharomyces cerevisiae, it is believed to be a single Dpm1 protein. The function of Dpm3 is performed in S. cerevisiae by the C-terminal transmembrane domain of the catalytic subunit Dpm1. Until present, the regulatory Dpm2 protein has not been found in S. cerevisiae. In this study, we show that, in fact, the Yil102c-A protein interacts directly with Dpm1 in S. cerevisiae and influences its DPM synthase activity. Deletion of the YIL102c-A gene is lethal, and this phenotype is reversed by the dpm2 gene from Trichoderma reesei. Functional analysis of Yil102c-A revealed that it also interacts with glucosylphosphatidylinositol-N-acetylglucosaminyl transferase (GPI-GnT), similar to DPM2 in human cells. Taken together, these results show that Yil102c-A is a functional homolog of DPMII from T. reesei and DPM2 from humans.

scholarly journals Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae

2007 ◽  
Vol 7 (2) ◽  
pp. 401-414 ◽  
Author(s):  
Weihua Fei ◽  
Gabriel Alfaro ◽  
Baby-Periyanayaki Muthusamy ◽  
Zachary Klaassen ◽  
Todd R. Graham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Neutral Lipid ◽  
Lipid Synthesis ◽  
Secretory Protein ◽  
Lipid Storage ◽  
Protein Glycosylation ◽  
Particle Assembly ◽  
Genome Wide ◽  
And Storage

ABSTRACT The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.

O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei

1987 ◽  
Vol 33 (8) ◽  
pp. 698-703 ◽  
Author(s):  
C. P. Kubicek ◽  
T. Panda ◽  
G. Schreferl-kunar ◽  
F. Gruber ◽  
R. Messner
Keyword(s):  
Trichoderma Reesei ◽  
Protein Glycosylation ◽  
The Other ◽  
Detectable Effect ◽  
Secreted Protein ◽  
Intracellular Protein ◽  
Molecular Weights ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses

The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.

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