Maturation of barley cysteine endopeptidase expressed in<i>Trichoderma reesei</i>is distorted by incomplete processing
Bibliographic record
Abstract
Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic in hibitors, 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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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".