Stimulation of exoprotein secretion by choline and Tween 80 in Trichoderma reesei QM 9414 correlates with increased activities of dolichol phosphate mannose synthase

Abstract

~Addition of choline (20 mM) or Tween 80 (0.06 %) to the culture medium of Trichoderma reesei QM 9414 increased (a) the secretion of protein under both carbon-catabolite-repressed and -derepressed conditions, and (b) cellulase secretion under carbon-catabolite-derepressed conditions. In contrast, no stimulation by choline or Tween 80 was observed with the hypersecretory strain T. reesei RUT C-30. In view of the obligatory role of 0-glycosylation in protein secretion by this fungus, an investigation was made into the effects on this process of choline and Tween 80.A membrane preparation was isolated from both strains of T. reesei and used to assay enzymes involved in 0-glycosylation. Significant differences were observed with respect to the activity of dolichol phosphate mannose (Dol-P-Man) synthase only. Strain QM 9414, grown on media supplemented with choline or Tween 80 exhibited a two-to threefold higher activity of Dol-P-Man synthase compared to a control lacking these supplements. This stimulatory effect was observed during growth under both carbon-catabolite-repressed and -derepressed conditions. In contrast, strain RUT C-30 exhibited decreased activities of Dol-P-Man synthase when grown in media supplemented with choline. Choline had no effect on Dol-P-Man synthase in vifro, whereas Tween 80 decreased the activity. Thus the effect of Tween 80 or choline on protein secretion by T. reesei may be due to a stimulation of formation and/or activity of Dol-P-Man synthase, thereby elevating the level of 0-glycosylation and protein secretion.

Overexpression of the Gene Encoding GTP:Mannose-1-Phosphate Guanyltransferase, mpg1 , Increases Cellular GDP-Mannose Levels and Protein Mannosylation in Trichoderma reesei

“…Our earlier studies have shown a correlation between DPMS activity and protein secretion (14,17). mpg1 overexpression only slightly influenced the DPMS activity.…”

“…14 C]Man (specific activity, 288 Ci/mol [Amersham]) and 5 ng of dolichyl phosphate (DolP) according to the method of Kruszewska et al (14,15,18).…”

“…O mannosylation has been reported to be necessary for secretion of the proteins (19,24) and to be influenced in vivo by choline and Tween 80 added to the culture medium. The same compounds have been reported to affect the activity of dolichyl phosphate mannose synthase (DPMS) in this fungus (14), the key enzyme in the O glycosylation pathway (13). Overexpression of the Saccharomyces cerevisiae DPM1 gene encoding DPMS in T. reesei elevated the enzyme activity twofold and resulted in an increased level of protein secretion.…”

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

“…Many, if not all, of these extracellular proteins are glycosylated. Our previous study showed a close correlation between protein secretion and the activity of dolichylphosphate mannose (DPM)-synthase (EC 2.4.1.83), a key enzyme in O glycosylation in T. reesei (15,17). We have shown that in T. reesei, DPM, which is synthesized by DPM-synthase, donates the mannosyl residue that is transferred to the hydroxyl group of serine or threonine in protein O mannosylation (16).…”

Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation.

“…In our earlier study we identified some of the factors affecting protein secretion and activity of dolichylphosphate mannose synthase (DPMS), a key enzyme in O-glycosylation in T. reesei. Choline and Tween 80, when added to the cultivation medium, stimulated secretion and, at the same time, elevated DPMS activity (Kruszewska et al, 1990). Moreover, overexpression of the yeast DPM1 gene in T. reesei increased protein secretion of wild type glycosylated proteins up to seven-fold (Kruszewska et al, 1999).…”