ID VSP1_CRODO Reviewed; 238 AA. AC A0A0S4FKT4; C0HJR5; DT 08-JUN-2016, integrated into UniProtKB/Swiss-Prot. DT 17-FEB-2016, sequence version 1. DT 22-FEB-2023, entry version 21. DE RecName: Full=Thrombin-like enzyme collinein-1 {ECO:0000303|PubMed:26227411}; DE Short=SVTLE collinein-1 {ECO:0000303|PubMed:26227411}; DE EC=3.4.21.- {ECO:0000255|PROSITE-ProRule:PRU00274, ECO:0000269|PubMed:26227411}; DE AltName: Full=Fibrinogen-clotting enzyme {ECO:0000250|UniProtKB:Q9PSN3}; DE AltName: Full=Snake venom serine protease {ECO:0000303|PubMed:26227411}; DE Short=SVSP {ECO:0000303|PubMed:26227411}; OS Crotalus durissus collilineatus (Brazilian rattlesnake). OC Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; OC Lepidosauria; Squamata; Bifurcata; Unidentata; Episquamata; Toxicofera; OC Serpentes; Colubroidea; Viperidae; Crotalinae; Crotalus. OX NCBI_TaxID=221569 {ECO:0000312|EMBL:CEJ25501.1}; RN [1] RP NUCLEOTIDE SEQUENCE [MRNA], PARTIAL PROTEIN SEQUENCE, FUNCTION, ACTIVITY RP REGULATION, BIOPHYSICOCHEMICAL PROPERTIES, SUBCELLULAR LOCATION, MASS RP SPECTROMETRY, AND IDENTIFICATION BY MASS SPECTROMETRY. RC TISSUE=Venom, and Venom gland; RX PubMed=26227411; DOI=10.1007/s00253-015-6836-2; RA Boldrini-Franca J., Santos Rodrigues R., Santos-Silva L.K., de Souza D.L., RA Gomes M.S., Cologna C.T., de Pauw E., Quinton L., Henrique-Silva F., RA de Melo Rodrigues V., Arantes E.C.; RT "Expression of a new serine protease from Crotalus durissus collilineatus RT venom in Pichia pastoris and functional comparison with the native RT enzyme."; RL Appl. Microbiol. Biotechnol. 99:9971-9986(2015). RN [2] RP FUNCTION, AND RECOMBINANT EXPRESSION. RX PubMed=31131001; DOI=10.1590/1678-9199-jvatitd-1471-18; RA Boldrini-Franca J., Pinheiro-Junior E.L., Arantes E.C.; RT "Functional and biological insights of rCollinein-1, a recombinant serine RT protease from Crotalus durissus collilineatus."; RL J. Venom. Anim. Toxins Incl. Trop. Dis. 25:e147118-e147118(2019). RN [3] RP FUNCTION, MUTAGENESIS OF HIS-43, RECOMBINANT EXPRESSION, IN SILICO RP MOLECULAR DOCKING, AND 3D-STRUCTURE MODELING. RC TISSUE=Venom; RX PubMed=32161292; DOI=10.1038/s41598-020-61258-x; RA Boldrini-Franca J., Pinheiro-Junior E.L., Peigneur S., Pucca M.B., RA Cerni F.A., Borges R.J., Costa T.R., Carone S.E.I., Fontes M.R.M., RA Sampaio S.V., Arantes E.C., Tytgat J.; RT "Beyond hemostasis: a snake venom serine protease with potassium channel RT blocking and potential antitumor activities."; RL Sci. Rep. 10:4476-4476(2020). RN [4] RP FUNCTION, MASS SPECTROMETRY, RECOMBINANT EXPRESSION, PEGYLATION OF NATIVE RP AND RECOMBINANT FORMS, AND 3D-STRUCTURE MODELING. RC TISSUE=Venom; RX PubMed=34506860; DOI=10.1016/j.ijbiomac.2021.09.004; RA Pinheiro-Junior E.L., Boldrini-Franca J., Takeda A.A.S., Costa T.R., RA Peigneur S., Cardoso I.A., Oliveira I.S., Sampaio S.V., RA de Mattos Fontes M.R., Tytgat J., Arantes E.C.; RT "Towards toxin PEGylation: the example of rCollinein-1, a snake venom RT thrombin-like enzyme, as a PEGylated biopharmaceutical prototype."; RL Int. J. Biol. Macromol. 190:564-573(2021). CC -!- FUNCTION: Thrombin-like snake venom serine protease (PubMed:26227411, CC PubMed:31131001, PubMed:34506860). Releases fibrinopeptide A and B in CC the conversion of fibrinogen to fibrin, with preferential activity on CC the alpha chain of fibrinogen (PubMed:26227411, PubMed:34506860). Also CC hydrolyzes N-p-toluensulfonyl arginine ester (TAME) and chromogenic CC artificial substrates of the blood coagulation cascade: S-2222 for CC factor Xa, S-2302 for kallikrein and S-2238 for thrombin CC (PubMed:26227411). When tested in vitro, the recombinant protein does CC not degrade blood clots, suggesting that this toxin lacks fibrinolytic CC activity (PubMed:31131001). In addition, it moderately inhibits human CC Kv10.1/KCNH1/EAG1 currents, with a mechanism independent of its CC enzymatic activity. It selectively blocks Kv10.1/KCNH1/EAG1 in a time CC and dose-dependent manner (IC(50)=4.2 uM for native protein and CC IC(50)=2.5 uM for recombinant protein). It may have a preference in CC interacting with Kv10.1/KCNH1/EAG1 in its closed state, since the CC inhibitory effect of the toxin is decreased at more depolarized CC potentials. Corroboratively, it may have possible antitumor CC applications, since it reduces the viability of human breast cancer CC cell line MCF-7, which strongly expresses Kv10.1/KCNH1/EAG1, but does CC not affect the liver carcinoma and the non-tumorigenic epithelial CC breast cell lines, which weakly express Kv10.1/KCNH1/EAG1 CC (PubMed:32161292). When tested on peripheral blood mononuclear cells CC (PBMC), the native protein shows mild cytotoxicity, whereas the CC recombinant protein does not show any cytotoxicity (PubMed:34506860). CC Native form is not immununogenic, since it does not induce CC statistically significant antibody production in mice, whereas CC recombinant form shows an antibody titer slightly higher than the CC native form (PubMed:34506860). In vivo, subplantar injection in mice CC paw induces a discreet paw edema (PubMed:31131001). In addition, CC intraperitoneal injection of the recombinant protein into mice led to CC fibrinogen depletion, resulting in the blood incoagulability CC (PubMed:31131001). {ECO:0000269|PubMed:26227411, CC ECO:0000269|PubMed:31131001, ECO:0000269|PubMed:32161292, CC ECO:0000269|PubMed:34506860}. CC -!- ACTIVITY REGULATION: Inhibited by Cu(2+) and, to a lesser extent, by CC Zn(2+) and Ba(2+). Not inhibited by Ca(2+) and Mg(2+). CC {ECO:0000269|PubMed:26227411}. CC -!- BIOPHYSICOCHEMICAL PROPERTIES: CC Kinetic parameters: CC KM=1.43 mM for TAME {ECO:0000269|PubMed:26227411}; CC KM=0.92 mM for S-2302 {ECO:0000269|PubMed:34506860}; CC Vmax=0.06 mmol/min/ug enzyme towards TAME CC {ECO:0000269|PubMed:26227411}; CC pH dependence: CC Activity is stable from pH 3.5 and 10.5. CC {ECO:0000269|PubMed:26227411}; CC Temperature dependence: CC Thermostable. Activity is stable after incubation at 100 degrees CC Celsius for 30 min. {ECO:0000269|PubMed:26227411}; CC -!- SUBUNIT: Monomer. {ECO:0000305|PubMed:26227411}. CC -!- SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:26227411}. CC -!- TISSUE SPECIFICITY: Expressed by the venom gland. CC {ECO:0000305|PubMed:26227411}. CC -!- MASS SPECTROMETRY: Mass=29474; Method=Electrospray; CC Evidence={ECO:0000269|PubMed:26227411, ECO:0000269|PubMed:34506860}; CC -!- MISCELLANEOUS: Negative results: recombinant protein has no activity on CC many tested channels (Shaker IR, Kv1.4/KCNA4, Kv2.1/KCNB1, CC hKv11.1/KCNH2/ERG1, rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, CC rNav1.4/SCN4A, hNav1.5/SCN5A, mNav1.6/SCN8A, hNav1.8/SCN10A). CC {ECO:0000269|PubMed:32161292}. CC -!- MISCELLANEOUS: PEGylation of both native and recombinant forms CC increases 5-6-fold hERG1 inhibition (IC(50)=22.4 uM for PEG-collinein-1 CC and IC(50)=10.0 uM for PEGrCollinein-1). In contrast, PEGylated CC proteins displays a great loss in the capability of inhibiting CC Kv10.1/KCNH1/EAG1, compared to their non-PEGylated counterparts CC (PubMed:34506860). It is noteworthy that PEGylated proteins have no CC activity towards all other channels tested (rKv1.1/KCNA1, rKv1.2/KCNA2, CC rKv1.4/KCNA4, rKv1.6/KCNA6, hKv2.1/KCNB1, hKv3.1/KCNC1, rKv4.2/KCND2, CC hKv7.2/7.3, hEAG1, Shaker, rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, CC rNav1.4/SCN4A, hNav1.5/SCN5A, mNav1.6/SCN8A, hNav1.8/SCN10A, CC rCav3.1/CACNA1G and rCav3.3/CACNA1I) (PubMed:34506860). Both native and CC recombinant PEGylated forms are also not immununogenic, since they do CC not induce statistically significant antibody production in mice CC (PubMed:34506860). {ECO:0000269|PubMed:34506860}. CC -!- SIMILARITY: Belongs to the peptidase S1 family. Snake venom subfamily. CC {ECO:0000255}. CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- DR EMBL; LN651356; CEJ25501.1; -; mRNA. DR AlphaFoldDB; A0A0S4FKT4; -. DR SMR; A0A0S4FKT4; -. DR SABIO-RK; A0A0S4FKT4; -. DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell. DR GO; GO:0015459; F:potassium channel regulator activity; IEA:UniProtKB-KW. DR GO; GO:0004252; F:serine-type endopeptidase activity; IEA:InterPro. DR GO; GO:0090729; F:toxin activity; IEA:UniProtKB-KW. DR GO; GO:0006508; P:proteolysis; IEA:UniProtKB-KW. DR CDD; cd00190; Tryp_SPc; 1. DR Gene3D; 2.40.10.10; Trypsin-like serine proteases; 2. DR InterPro; IPR009003; Peptidase_S1_PA. DR InterPro; IPR043504; Peptidase_S1_PA_chymotrypsin. DR InterPro; IPR001314; Peptidase_S1A. DR InterPro; IPR001254; Trypsin_dom. DR InterPro; IPR018114; TRYPSIN_HIS. DR PANTHER; PTHR24271:SF89; KALLIKREIN-2; 1. DR PANTHER; PTHR24271; KALLIKREIN-RELATED; 1. DR Pfam; PF00089; Trypsin; 1. DR PRINTS; PR00722; CHYMOTRYPSIN. DR SMART; SM00020; Tryp_SPc; 1. DR SUPFAM; SSF50494; Trypsin-like serine proteases; 1. DR PROSITE; PS50240; TRYPSIN_DOM; 1. DR PROSITE; PS00134; TRYPSIN_HIS; 1. PE 1: Evidence at protein level; KW Blood coagulation cascade activating toxin; Direct protein sequencing; KW Disulfide bond; Hemostasis impairing toxin; Hydrolase; KW Ion channel impairing toxin; Potassium channel impairing toxin; Protease; KW Secreted; Serine protease; Toxin; KW Voltage-gated potassium channel impairing toxin. FT CHAIN 1..238 FT /note="Thrombin-like enzyme collinein-1" FT /evidence="ECO:0000305" FT /id="PRO_0000436478" FT DOMAIN 1..229 FT /note="Peptidase S1" FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00274" FT ACT_SITE 43 FT /note="Charge relay system" FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00274" FT ACT_SITE 88 FT /note="Charge relay system" FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00274" FT ACT_SITE 184 FT /note="Charge relay system" FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00274" FT SITE 79 FT /note="Pharmacophore that directly interacts to FT Kv10.1/KCNH1/EAG1 channel selectivity filter" FT /evidence="ECO:0000305|PubMed:32161292" FT DISULFID 7..141 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3" FT DISULFID 28..44 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3, FT ECO:0000255|PROSITE-ProRule:PRU00274" FT DISULFID 78..236 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3" FT DISULFID 120..190 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3, FT ECO:0000255|PROSITE-ProRule:PRU00274" FT DISULFID 152..169 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3, FT ECO:0000255|PROSITE-ProRule:PRU00274" FT DISULFID 180..205 FT /evidence="ECO:0000250|UniProtKB:Q9PSN3, FT ECO:0000255|PROSITE-ProRule:PRU00274" FT MUTAGEN 43 FT /note="H->R: No change in inhibition potency of FT Kv10.1/KCNH1/EAG1." FT /evidence="ECO:0000269|PubMed:32161292" SQ SEQUENCE 238 AA; 26664 MW; F0366AEBA3C760E3 CRC64; VIGGDECNIN EHNFLVALYE YWSQSFLCGG TLINGEWVLT AAHCDRKHIL IYVGVHDRSV QFDKEQRRFP KEKYFFNCRN NFTKWDKDIM LIRLNKPVSY SEHIAPLSLP SSPPIVGSVC RVMGWGTIKS PQETLPDVPH CANINLLDYE VCRTAHPQFR LPATIRILCA GVLEGGIDTC HRDSGGPLIC NGEFQGIVSW GDGSCAQPDK PALYSKVFDH LDWIQNIIAG SETVNCPS //