Proteolytic Enzymes: Serine and Cysteine Peptidases
Proteases mechanism of action classifies them as either serine, cysteine or threonine proteases amino-terminal nucleophile hydrolases , or as aspartic, metallo and glutamic proteases with glutamic proteases being the only subtype not found in mammals so far. Proteases are involved in many aspects of human biology. For example, in the small intestine, proteases digest dietary proteins to allow absorption of amino acids.
Other processes mediated by proteases include blood coagulation, immune function, maturation of prohormones, bone formation, programmed cell death and the recycling of cellular proteins that are no longer needed. Proteases also offer a valuable target in many therapeutic settings, including Alzheimer's, cancer, and viral infection. MMP-9 , a matrix metallopeptidase, plays a role in angiogenesis and is a therapeutic target for cancer. Because of their significance in the pathology of disease, proteases are a relevant drug target class. Proteases activity are central to diverse physiological cascades throughout biology.
Some are essential for coagulation, while others contribute to cancer pathology. When the protein material is passed to the small intestine , proteins, which are only partially digested in the stomach, are further attacked by proteolytic enzymes secreted by the pancreas.
These enzymes are liberated in the small intestine from inactive precursors produced by the acinar cells in the pancreas. The precursors are called trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase. Trypsinogen is transformed to an endopeptidase called trypsin by an enzyme enterokinase secreted from the walls of the small intestine.
Trypsin then activates the precursors of chymotrypsin, elastase, and carboxypeptidase. When the pancreatic enzymes become activated in the intestine, they convert proteins into free amino acids, which are easily absorbed by the cells of the intestinal wall. The pancreas also produces a protein that inhibits trypsin. It is thought that in this manner the pancreas protects itself from autodigestion.
Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets
Proteolytic enzyme. Info Print Cite. Submit Feedback. Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored.
The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I.
It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms.
The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the partially extracellular?
Peptidases and proteinases | Enzymes | IUPHAR/BPS Guide to PHARMACOLOGY
High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis. Blood-feeding monogeneans of the family Diplozoidae Heteronchoinea are ectoparasites that live on the gills of cyprinid fishes. One member of the family, Eudiplozoon nipponicum , is an important invasive species, first recorded in Europe in on farmed carp in France [ 1 ].
It is currently found throughout Europe and is a widespread representative of the helminth fauna of the common carp Cyprinus carpio in the Czech Republic [ 2 ]. The common carp is a fish of high economic importance in many Asian and European countries, with global aquaculture production yielding over 4 million tons in [ 3 ]. Since the parasite affects the health of farmed fish, although precise estimates are unknown, it is assumed it causes economic losses.
Pathogenic effects of E. Monogenea are a rather neglected group of Neodermata and only a handful of papers on their biochemistry and bioactive molecules have ever been published. Based on previous ultrastructural studies, it has been assumed that in diplozoid monogeneans, the digestion of blood, gathered by combined action of their powerful buccal suckers and muscular pharynx, takes place within the lysosomal cycle in the specialised cells of the intestinal epidermis [ 5 , 6 , 7 , 8 , 9 ], similarly as in blood-sucking mites such as ticks [ 10 , 11 ].
Our previous study [ 12 ] has shown that the processing of blood in E. Among the endopeptidases of E. In helminths, cathepsins L and B play various roles. Due to their histolytic potency, they are involved in host invasion and tissue migration, but they also play a role in various pathological processes, immune evasion, and other parasite-host interactions, as well as in helminth reproduction, nutrient digestion, etc.
The use of different peptidases with overlapping substrate specificities helps heteroxenous and tissue-migrating parasites to adapt to various environments and sources of nutrition within the hosts. On the other hand, little is known about the complex functioning of cysteine peptidases in monoxenous blood-feeding monogeneans that spend most of their life attached to a single type of host tissue, such as the gills. In the present study, we focused on clan CA cysteine peptidases, namely cathepsins L and B, selected due to their abundance in the transcriptome of adult E. We have employed phylogenetic and bioinformatic analysis to investigate their relationship to other helminth peptidases.
We have selected two of the most abundant cathepsins L and the only expressed cathepsin B, and produced these as functionally active recombinant forms using the Pichia pastoris expression system. A biochemical and functional characterisation of the recombinant enzymes was performed in order to understand their specificity and substrate preference. Our work thus presents the first detailed functional characterisation of monogenean peptidases. Adult worms of E.
Transcripts from the transcriptome of adult E. They were annotated by searching for the closest homologues. Sequences encoding cathepsin L-like and B-like peptidases of E. The relative abundance of cathepsin L transcripts, which reflects the rate of transcription of the corresponding genes, was predicted by back mapping of raw Illumina reads to the assembled transcripts using Kallisto v. The presence of a signal sequence in amino acid sequences of the enzymes was predicted by SignalP 4. Potential N-glycosylation and O-glycosylation sites were identified using an online tool at NetNGlyc 1.
Divergent and incomplete sequences were manually removed, which resulted in a dataset of sequences. Bayesian inference of phylogeny was run under the C60 site-heterogeneous mixture model using Phylobayes. The amplified products were ligated into a P. Protein expression in P. The yeast medium was centrifuged and the supernatant filtered 0. The amplified product was ligated into the E. EnCL1 was expressed in E. The solubilised mixture was filtered 0. Chromatographic fractions were analysed by SDS-PAGE and the identity of suspected protein bands was verified by mass spectrometry as described in [ 12 ].
Processing of the enzyme to its mature form was monitored by SDS-PAGE, Western blotting and Edman degradation of N-terminus and subsequently confirmed by fluorometry with the corresponding synthetic peptide substrates see below. Peptidase activities of recombinant cathepsins were measured with fluorogenic aminomethylcoumarin peptide substrates Bachem as described previously [ 12 ].
The constructs were linearised and used as a template for RNA probes. In situ hybridisation was performed using a modified protocol [ 44 ]. Detection was achieved with alkaline phosphatase-conjugated anti-digoxigenin antibodies , Roche and Fast Red TR substrate Sigma-Aldrich.
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Negative controls were incubated under the same conditions but with an anti-sense probe or without a probe. Specific strand RT-PCR was used for the detection of natural anti-sense transcripts occurring in the monogenean cells [ 45 ]. Anti-EnCL1 antibodies were produced in mice injected intraperitoneally with c. Control sera were taken from the same mice prior to immunisation. The experiment was performed according to a modified protocol [ 40 ].
Subsequently, the sections were blocked and immunostained according to a modified Immunocytochemistry and Immunofluorescence Protocol Abcam. Immunofluorescence was observed and photographed under fluorescence or confocal microscope.
Ten unique transcripts encoding different cathepsin L-like sequences were discovered in the transcriptome of adult E. Of the predicted CL amino acid sequences, seven represented complete sequences of mature processed enzymes, with six encoding the complete sequences of zymogens including the pro-sequences.
The third group contains only EnCL2 , in which the S2 pocket differs substantially from that of the other EnCLs : Ala is replaced by the more hydrophobic Val at the bottom, and moreover, the two leucines are replaced by Trp67 and Phe, which could make the S2 pocket highly hydrophobic.
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In the case of CL2, residue is a positively charged Arg, whereas in CL6e, it is the negatively charged aspartate. The rate of transcription of the particular EnCLs was predicted by a reverse mapping of raw reads towards the selected transcripts. Relative transcription rate of E. The relative transcription rate predicted by a back mapping of raw Illumina reads to the assembled transcripts was calculated as the percentage of all cathepsin L transcripts.
Phylogenetic analysis has shown that E. The two clusters belong to separate clades, whereby each of the clusters also includes different cathepsins L of free-living rhabditophorans and CLs of digeneans and cestodes Fig.
It can thus be concluded that E. Collapsed phylogram showing relationships between E. Collapsed unrooted maximum-likelihood tree of selected E. Leaves of related organisms are collapsed. The cluster containing EnCL1 and EnCL3 , for which the function in blood digestion was confirmed in this study, occurs within the same clade as the CL of another haematophagous monogenean, Protopolystoma xenopodis. The various CLs of the mucophagous Gyrodactylus salaris fall into both clades where the two clusters of EnCLs are located. Its primary structure contains the typical motif of an occluding loop, which is responsible for peptidyl dipeptidase activity of cathepsins B.