A0A0S4FKT4 · VSP1_CRODO
- ProteinThrombin-like enzyme collinein-1
- StatusUniProtKB reviewed (Swiss-Prot)
- Amino acids238 (go to sequence)
- Protein existenceEvidence at protein level
- Annotation score5/5
Function
function
Thrombin-like snake venom serine protease (PubMed:26227411, PubMed:31131001, PubMed:34506860).
Releases fibrinopeptide A and B in the conversion of fibrinogen to fibrin, with preferential activity on the alpha chain of fibrinogen (PubMed:26227411, PubMed:34506860).
Also hydrolyzes N-p-toluensulfonyl arginine ester (TAME) and chromogenic artificial substrates of the blood coagulation cascade: S-2222 for factor Xa, S-2302 for kallikrein and S-2238 for thrombin (PubMed:26227411).
When tested in vitro, the recombinant protein does not degrade blood clots, suggesting that this toxin lacks fibrinolytic activity (PubMed:31131001).
In addition, it moderately inhibits human Kv10.1/KCNH1/EAG1 currents, with a mechanism independent of its enzymatic activity. It selectively blocks Kv10.1/KCNH1/EAG1 in a time and dose-dependent manner (IC50=4.2 uM for native protein and IC50=2.5 uM for recombinant protein). It may have a preference in interacting with Kv10.1/KCNH1/EAG1 in its closed state, since the inhibitory effect of the toxin is decreased at more depolarized potentials. Corroboratively, it may have possible antitumor applications, since it reduces the viability of human breast cancer cell line MCF-7, which strongly expresses Kv10.1/KCNH1/EAG1, but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines, which weakly express Kv10.1/KCNH1/EAG1 (PubMed:32161292).
When tested on peripheral blood mononuclear cells (PBMC), the native protein shows mild cytotoxicity, whereas the recombinant protein does not show any cytotoxicity (PubMed:34506860).
Native form is not immununogenic, since it does not induce statistically significant antibody production in mice, whereas recombinant form shows an antibody titer slightly higher than the native form (PubMed:34506860).
In vivo, subplantar injection in mice paw induces a discreet paw edema (PubMed:31131001).
In addition, intraperitoneal injection of the recombinant protein into mice led to fibrinogen depletion, resulting in the blood incoagulability (PubMed:31131001).
Releases fibrinopeptide A and B in the conversion of fibrinogen to fibrin, with preferential activity on the alpha chain of fibrinogen (PubMed:26227411, PubMed:34506860).
Also hydrolyzes N-p-toluensulfonyl arginine ester (TAME) and chromogenic artificial substrates of the blood coagulation cascade: S-2222 for factor Xa, S-2302 for kallikrein and S-2238 for thrombin (PubMed:26227411).
When tested in vitro, the recombinant protein does not degrade blood clots, suggesting that this toxin lacks fibrinolytic activity (PubMed:31131001).
In addition, it moderately inhibits human Kv10.1/KCNH1/EAG1 currents, with a mechanism independent of its enzymatic activity. It selectively blocks Kv10.1/KCNH1/EAG1 in a time and dose-dependent manner (IC50=4.2 uM for native protein and IC50=2.5 uM for recombinant protein). It may have a preference in interacting with Kv10.1/KCNH1/EAG1 in its closed state, since the inhibitory effect of the toxin is decreased at more depolarized potentials. Corroboratively, it may have possible antitumor applications, since it reduces the viability of human breast cancer cell line MCF-7, which strongly expresses Kv10.1/KCNH1/EAG1, but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines, which weakly express Kv10.1/KCNH1/EAG1 (PubMed:32161292).
When tested on peripheral blood mononuclear cells (PBMC), the native protein shows mild cytotoxicity, whereas the recombinant protein does not show any cytotoxicity (PubMed:34506860).
Native form is not immununogenic, since it does not induce statistically significant antibody production in mice, whereas recombinant form shows an antibody titer slightly higher than the native form (PubMed:34506860).
In vivo, subplantar injection in mice paw induces a discreet paw edema (PubMed:31131001).
In addition, intraperitoneal injection of the recombinant protein into mice led to fibrinogen depletion, resulting in the blood incoagulability (PubMed:31131001).
Miscellaneous
Negative results: recombinant protein has no activity on many tested channels (Shaker IR, Kv1.4/KCNA4, Kv2.1/KCNB1, hKv11.1/KCNH2/ERG1, rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, rNav1.4/SCN4A, hNav1.5/SCN5A, mNav1.6/SCN8A, hNav1.8/SCN10A).
PEGylation of both native and recombinant forms increases 5-6-fold hERG1 inhibition (IC50=22.4 uM for PEG-collinein-1 and IC50=10.0 uM for PEGrCollinein-1). In contrast, PEGylated proteins displays a great loss in the capability of inhibiting Kv10.1/KCNH1/EAG1, compared to their non-PEGylated counterparts (PubMed:34506860).
It is noteworthy that PEGylated proteins have no activity towards all other channels tested (rKv1.1/KCNA1, rKv1.2/KCNA2, rKv1.4/KCNA4, rKv1.6/KCNA6, hKv2.1/KCNB1, hKv3.1/KCNC1, rKv4.2/KCND2, hKv7.2/7.3, hEAG1, Shaker, rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, rNav1.4/SCN4A, hNav1.5/SCN5A, mNav1.6/SCN8A, hNav1.8/SCN10A, rCav3.1/CACNA1G and rCav3.3/CACNA1I) (PubMed:34506860).
Both native and recombinant PEGylated forms are also not immununogenic, since they do not induce statistically significant antibody production in mice (PubMed:34506860).
It is noteworthy that PEGylated proteins have no activity towards all other channels tested (rKv1.1/KCNA1, rKv1.2/KCNA2, rKv1.4/KCNA4, rKv1.6/KCNA6, hKv2.1/KCNB1, hKv3.1/KCNC1, rKv4.2/KCND2, hKv7.2/7.3, hEAG1, Shaker, rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, rNav1.4/SCN4A, hNav1.5/SCN5A, mNav1.6/SCN8A, hNav1.8/SCN10A, rCav3.1/CACNA1G and rCav3.3/CACNA1I) (PubMed:34506860).
Both native and recombinant PEGylated forms are also not immununogenic, since they do not induce statistically significant antibody production in mice (PubMed:34506860).
Activity regulation
Inhibited by Cu2+ and, to a lesser extent, by Zn2+ and Ba2+. Not inhibited by Ca2+ and Mg2+.
Kinetics
KM | SUBSTRATE | pH | TEMPERATURE[C] | NOTES | EVIDENCE | |
---|---|---|---|---|---|---|
1.43 mM | TAME | |||||
0.92 mM | S-2302 |
Vmax | pH | TEMPERATURE[C] | NOTES | EVIDENCE | |
---|---|---|---|---|---|
0.06 mmol/min/ug | towards TAME |
pH Dependence
Activity is stable from pH 3.5 and 10.5.
Temperature Dependence
Thermostable. Activity is stable after incubation at 100 degrees Celsius for 30 minutes.
Features
Showing features for active site, site.
Type | ID | Position(s) | Description | |||
---|---|---|---|---|---|---|
Active site | 43 | Charge relay system | ||||
Sequence: H | ||||||
Site | 79 | Pharmacophore that directly interacts to Kv10.1/KCNH1/EAG1 channel selectivity filter | ||||
Sequence: R | ||||||
Active site | 88 | Charge relay system | ||||
Sequence: D | ||||||
Active site | 184 | Charge relay system | ||||
Sequence: S |
GO annotations
Aspect | Term | |
---|---|---|
Cellular Component | extracellular space | |
Molecular Function | potassium channel regulator activity | |
Molecular Function | serine-type endopeptidase activity | |
Molecular Function | toxin activity | |
Biological Process | proteolysis |
Keywords
- Molecular function
Enzyme and pathway databases
Names & Taxonomy
Protein names
- Recommended nameThrombin-like enzyme collinein-1
- EC number
- Short namesSVTLE collinein-1
- Alternative names
Organism names
- Taxonomic lineageEukaryota > Metazoa > Chordata > Craniata > Vertebrata > Euteleostomi > Lepidosauria > Squamata > Bifurcata > Unidentata > Episquamata > Toxicofera > Serpentes > Colubroidea > Viperidae > Crotalinae > Crotalus
Accessions
- Primary accessionA0A0S4FKT4
- Secondary accessions
Subcellular Location
Phenotypes & Variants
Features
Showing features for mutagenesis.
Type | ID | Position(s) | Description | |||
---|---|---|---|---|---|---|
Mutagenesis | 43 | No change in inhibition potency of Kv10.1/KCNH1/EAG1. | ||||
Sequence: H → R |
PTM/Processing
Features
Showing features for chain, disulfide bond.
Type | ID | Position(s) | Description | |||
---|---|---|---|---|---|---|
Chain | PRO_0000436478 | 1-238 | Thrombin-like enzyme collinein-1 | |||
Sequence: VIGGDECNINEHNFLVALYEYWSQSFLCGGTLINGEWVLTAAHCDRKHILIYVGVHDRSVQFDKEQRRFPKEKYFFNCRNNFTKWDKDIMLIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRVMGWGTIKSPQETLPDVPHCANINLLDYEVCRTAHPQFRLPATIRILCAGVLEGGIDTCHRDSGGPLICNGEFQGIVSWGDGSCAQPDKPALYSKVFDHLDWIQNIIAGSETVNCPS | ||||||
Disulfide bond | 7↔141 | |||||
Sequence: CNINEHNFLVALYEYWSQSFLCGGTLINGEWVLTAAHCDRKHILIYVGVHDRSVQFDKEQRRFPKEKYFFNCRNNFTKWDKDIMLIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRVMGWGTIKSPQETLPDVPHC | ||||||
Disulfide bond | 28↔44 | |||||
Sequence: CGGTLINGEWVLTAAHC | ||||||
Disulfide bond | 78↔236 | |||||
Sequence: CRNNFTKWDKDIMLIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRVMGWGTIKSPQETLPDVPHCANINLLDYEVCRTAHPQFRLPATIRILCAGVLEGGIDTCHRDSGGPLICNGEFQGIVSWGDGSCAQPDKPALYSKVFDHLDWIQNIIAGSETVNC | ||||||
Disulfide bond | 120↔190 | |||||
Sequence: CRVMGWGTIKSPQETLPDVPHCANINLLDYEVCRTAHPQFRLPATIRILCAGVLEGGIDTCHRDSGGPLIC | ||||||
Disulfide bond | 152↔169 | |||||
Sequence: CRTAHPQFRLPATIRILC | ||||||
Disulfide bond | 180↔205 | |||||
Sequence: CHRDSGGPLICNGEFQGIVSWGDGSC |
Keywords
- PTM
Expression
Tissue specificity
Expressed by the venom gland.
Interaction
Subunit
Monomer.
Structure
Family & Domains
Features
Showing features for domain.
Type | ID | Position(s) | Description | |||
---|---|---|---|---|---|---|
Domain | 1-229 | Peptidase S1 | ||||
Sequence: VIGGDECNINEHNFLVALYEYWSQSFLCGGTLINGEWVLTAAHCDRKHILIYVGVHDRSVQFDKEQRRFPKEKYFFNCRNNFTKWDKDIMLIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRVMGWGTIKSPQETLPDVPHCANINLLDYEVCRTAHPQFRLPATIRILCAGVLEGGIDTCHRDSGGPLICNGEFQGIVSWGDGSCAQPDKPALYSKVFDHLDWIQNIIA |
Sequence similarities
Belongs to the peptidase S1 family. Snake venom subfamily.
Family and domain databases
Sequence
- Sequence statusComplete
- Length238
- Mass (Da)26,664
- Last updated2016-02-17 v1
- ChecksumF0366AEBA3C760E3
Mass Spectrometry
Keywords
- Technical term