Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.
- MeSH
- Cysteine Endopeptidases metabolism MeSH
- Myelin Basic Protein metabolism MeSH
- Financing, Organized MeSH
- Transcription, Genetic MeSH
- Immunohistochemistry methods MeSH
- Trematode Infections metabolism MeSH
- Isomerism MeSH
- Cathepsin B analysis chemistry MeSH
- RNA, Messenger genetics MeSH
- Models, Molecular MeSH
- Molecular Sequence Data MeSH
- Enzyme Precursors analysis MeSH
- Recombinant Proteins analysis MeSH
- RNA, Helminth genetics MeSH
- Schistosomatidae genetics chemistry MeSH
- Base Sequence MeSH
- Sequence Alignment mortality MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Gelatinase B/MMP-9 is a member of matrix metalloproteinases with a major role in extracellular matrix degradation, cell proliferation and migration. Its proenzyme form has also been reported in pleural fluids as an inducible species, but its relation to pleural pathology has not yet been fully clarified. The primary goal of this study was to evaluate proMMP-9 as a potential marker for differentiating pleural effusions of both malignant and non-malignant origin. Pleural fluid samples were studied from 194 patients, including tumor etiology in 133 cases, inflammatory disorders in 33, transudates in 12, and unspecified disorders in 16 patients. The concentrations of proMMP-9 were estimated by means of immunoassays and/or by scanning zymography. Samples were also examined for C-reactive protein (CRP). The analysis of proMMP-9 showed significant differences among the etiological groups with the highest concentrations in para-inflammatory exudates, intermediate in para-neoplastic exudates, and the lowest in transudates. However, the analysis of the para-neoplastic group revealed a distinct heterogeneity with a minor portion of fluids reaching values typical for para-inflammatory effusions. A subsequent sorting based on tumor histology showed increased levels particularly in exudates associated with metastatic tumors. Interestingly, proMMP-9 values in general correlated with CRP, a systemic marker of inflammation. Thus, MMP-9 proenzyme appears to complement traditional markers distinguishing pleural fluids of different origin. Yet, the differentiation between paraneoplastic and para-inflammatory exudates must be regarded with caution due to the presence of a high-expressive paraneoplastic sub-population, including effusions associated with metastatic tumors.
- MeSH
- C-Reactive Protein analysis metabolism MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Financing, Organized MeSH
- Collagenases analysis metabolism MeSH
- Humans MeSH
- Matrix Metalloproteinase 9 MeSH
- Neoplasm Metastasis physiopathology MeSH
- Biomarkers, Tumor analysis MeSH
- Lung Neoplasms physiopathology MeSH
- Pleural Effusion MeSH
- Lung Diseases physiopathology MeSH
- Enzyme Precursors analysis metabolism MeSH
- Check Tag
- Humans MeSH
- MeSH
- Duodenal Ulcer enzymology physiopathology MeSH
- Electrophoresis methods MeSH
- Humans MeSH
- Stomach Neoplasms enzymology physiopathology MeSH
- Stomach Diseases enzymology physiopathology MeSH
- Enzyme Precursors analysis MeSH
- Stomach Ulcer enzymology physiopathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
