VSP1_CRODO
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 03-AUG-2022, entry version 19.
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 MCF7, 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: Recombinant protein has no activity on many tested
CC channels (Shaker IR, Kv1.4/KCNA4, Kv2.1/KCNB1, hKv11.1/KCNH2/ERG1,
CC rNav1.1/SCN1A, rNav1.2/SCN2A, rNav1.3/SCN3A, rNav1.4/SCN4A,
CC 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; -; 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 Pfam; PF00089; Trypsin; 1.
DR PRINTS; PR00722; CHYMOTRYPSIN.
DR SMART; SM00020; Tryp_SPc; 1.
DR SUPFAM; SSF50494; SSF50494; 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
