Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Shiga toxin has a single A subunit non-covalently associated with a pentamer of B subunits. The toxin has a trypsin-sensitive region near the COOH-terminal end of the A-chain, and upon cleavage, two disulfide bonded fragments, A1 and A2, are generated. These fragments are also formed upon incubation with cells. The disulfide loop contains the sequence (Arg-X-X-Arg), which is a consensus motif for cleavage by the membrane-anchored protease furin. We found that a soluble form of furin cleaves intact A-chain producing A1 and A2 fragments, and furin also seems to be responsible for rapid cellular cleavage of Shiga toxin. LoVo cells, which normally do not produce functional furin, cleave intact A-chain very efficiently when transfected with furin (LoVo/fur), whereas a control cell (LoVo/neo) cleaves the toxin very slowly. To investigate the role of this cleavage for intoxication of cells, we studied the ability of unnicked and furin-nicked toxin to inhibit protein synthesis in LoVo/fur and LoVo/neo cells. LoVo/fur cells were intoxicated equally well with unnicked and nicked toxin, whereas in LoVo/neo cells nicked toxin was about 20 times more active than unnicked toxin. The results suggest that cleavage of Shiga toxin is important for intoxication of cells, and they indicate that furin can cleave and thereby activate Shiga toxin in cells.
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PMID:Furin-induced cleavage and activation of Shiga toxin. 773 18

The role for proteolytic activation of Shiga-like toxin type II variant (SLT-IIv) A subunit was examined using site-directed mutagenesis. Processing of the enzymatically active A subunit by trypsin results in cleavage at an arginine residue(s) (Arg247 and/or Arg250) located between two cysteines. After reduction of the disulfide bond, the processed A subunit separates into an enzymatically active A1 and an A2 peptide. Substitution mutations were created in SLT-IIv that replaced each or both of the two arginines with either glutamic acid (R247E, R250E, or R247E/R250E) or histidine (R247H, R250H, or R247H/R250H). The products of all glutamic acid substitution mutations were immunoreactive but were not cytotoxic due to an inability to assemble into holotoxin. The products of all histidine substitution mutations had cytotoxic activities, enzymatic activities, and a lethal dose for mice similar to that of native toxin. R247H and R250H were susceptible to proteolytic cleavage while R247H/R250H was resistant to processing by exogenously added trypsin. Native toxin incubated with Vero cells was completely cleaved while only a fraction of R247H/R250H was cleaved. These results demonstrate that cleavage of SLT-IIv can be mediated by proteases with different specificities and suggest efficient cleavage is not required for toxicity.
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PMID:Evidence that proteolytic separation of Shiga-like toxin type IIv A subunit into A1 and A2 subunits is not required for toxin activity. 790 71

Escherichia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids. The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues. By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments. Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol. In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur. However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells. We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alternative accessible sites to compensate for loss of target sites within the loop.
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PMID:Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity. 796 13

The enterotoxic moiety present in the cell-free culture supernatants of Salmonella typhiurium strains (p/536 and p/603) was purified to apparent homogeneity by salt precipitation with ammonium sulphate and successive chromatography through Sephadex G-100 and G-200 columns. It was non-dialysable, heat labile at 90 degrees C and active within pH 6-8. Its activity was completely lost on treatment with trypsin, protease and papain. The enterotoxin appeared to be of high molecular weight (100 kDa) and was highly immunogenic in rabbit. Antigenically, it was not related to cholera toxin, Shiga toxin or the heat labile enterotoxin of Escherichia coli. It did not bind to the GM1 ganglioside. The enterotoxic, delayed permeability and CHO cell elongation activities were attributed to a single protein moiety.
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PMID:Purification and characterization of enterotoxic moiety present in cell-free culture supernatant of Salmonella typhimurium. 804 72

The effect of Shiga toxin with mutations in the A fragment has been tested on cells in order to get more information about the processing of the A fragment during entry into the cytosol. A mutant with a deletion between the A1 and A2 domain in the A fragment is resistant to cleavage by trypsin and is less toxic than wild type toxin on both Vero and A431 cells. The results support the view that processing of the A fragment is important for the high toxicity of the wild type toxin. A number of cell lines are resistant to Shiga toxin although they bind the toxin. However, A431 cells can be sensitized by butyric acid treatment, and transport of Shiga toxin to the Golgi apparatus seems to be required for the intoxication in the sensitized cells. The role of retrograde transport through the Golgi apparatus to the endoplasmic reticulum (ER) will be discussed.
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PMID:Entry of Shiga toxin into cells. 834 33

The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from mouse small intestine and colon and that growth in small-intestine mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce SLT-II or SLT-IIc, were approximately 35- to 350-fold more toxic for Vero cells than supernatants from B2F1 grown in LB broth. This increased toxicity was not reflected by a concomitant increase in SLT antigen content. Furthermore, when culture supernatants from B2F1 or K-12 strains carrying plasmids encoding SLTs cloned from H414-36/89 or purified SLT-IIvhb from B2F1 were incubated with mouse intestinal mucus, the samples exhibited greater cytotoxicity than when they were incubated with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. These toxin preparations also showed increased cytotoxicity after incubation with human colonic mucus. In contrast, culture supernatants from LB-grown EHEC isolates that produced SLT-I, SLT-II, SLT-IIc or SLT-IIe did not show increased cytotoxicity after incubation with mouse or human intestinal mucus. The A subunits of purified SLT-II and SLT-IIvhb that had been treated with mouse intestinal mucus or trypsin were cleaved to A1 fragments by the mucus, but trypsin-mediated cleavage, unlike treatment with mouse intestinal mucus, did not result in increased Vero cell cytotoxicity activity. This finding implies that the increased cytotoxicity of SLT-IIvhb detected after incubation with mucus is probably not due to cleavage of the A subunit into the A1 and A2 fragments. Taken together, these results indicate that mouse or human intestinal mucus directly activates SLT-II-related toxins from B2F1 and H414-36/89 and suggest that toxin activation may explain the low 50% lethal doses of B2F1 and H414-36/89 in streptomycin-treated mice.
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PMID:Activation of Shiga-like toxins by mouse and human intestinal mucus correlates with virulence of enterohemorrhagic Escherichia coli O91:H21 isolates in orally infected, streptomycin-treated mice. 861 62

Some clinical strains of Vibrio cholerae non-O1 produce an extracellular factor that evokes a rapid and dramatic cytotoxic response which manifests as cell rounding of Chinese hamster ovary (CHO) and HeLa cells without accompanying membrane damage. This study was performed to establish the identity of the non-membrane-damaging cytotoxin (NMDCY), which was not inhibited by antitoxins against cholera toxin, heat-labile toxin of enterotoxigenic Escherichia coli, El Tor hemolysin, Shiga-like toxin I, and Shiga-like toxin II, indicating that NMDCY did not bear an apparent immunological relationship with the above toxins and hemolysin. Brain heart infusion broth and AKI medium supported the maximal production of NMDCY; culture supernatant of AKI medium was found to be free of hemolysin activity, whereas in brain heart infusion broth hemolysin was coproduced with NMDCY. Maximal production of NMDCY in AKI medium was observed at 37 degrees C under shaking conditions with the pH of the medium adjusted to 8.5. NMDCY was purified to homogeneity by a three-step purification procedure which increased the specific activity of the cytotoxin by 1.7 X 10(5)-fold. The denatured molecular weight of the purified toxin was 35,000, and the cytotoxin was heat labile and sensitive to trypsin. Purification of the cytotoxin revealed an enterotoxic activity as reflected by its ability to accumulate fluid in the rabbit ileal loop. Both the cytotoxic and enterotoxic activities of NMDCY could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified NMDCY and antiserum gave a single well-defined precipitin band which showed reactions of complete identity, while, in an immunoblot assay, a well-defined single band was observed in the 35-kDa region. Our results indicate that the cytotoxic and enterotoxic activities expressed by NMDCY appear to contribute to the pathogenesis of the disease associated with V. cholerae non-O1 strains which produce this cytotoxin.
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PMID:Purification and characterization of an extracellular secretogenic non-membrane-damaging cytotoxin produced by clinical strains of Vibrio cholerae non-O1. 875 40

Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.
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PMID:Role of the disulfide bond in Shiga toxin A-chain for toxin entry into cells. 911 Oct 51

Shiga toxin and Shiga-like toxins are ribosome-inactivating proteins with RNA-N-glycosidase activity which remove a specific adenine from 28S RNA. The toxins are composed of an A subunit non-covalently associated to a multimer of receptor-binding B subunits. Near the COOH-terminus of the A subunit, a disulfide-bonded loop contains two trypsin-sensitive arginine residues. Proteolytic nicking at these sites, followed by reduction, removes from the A subunit the C-terminal end together with the associated B subunits. The requirement of such cleavage for biological activity of Shiga toxin and Shiga-like toxins has been recently questioned. The present paper reports the kinetic constants of the adenine release from highly purified Artemia salina ribosomes catalysed by Shiga-like toxin I and by its A subunit before and after treatment with trypsin, urea and dithiothreitol or urea and dithiothreitol alone. All reactions had approximately the same Km (1 microM). The Kcat was 0.6 min-1 for the untreated holotoxin and 6 min-1 for the isolated A subunit, respectively. The trypsin treatment increased 1000-fold the Kcat of the holotoxin (770 min-1) and 100-fold the Kcat of the A subunit (640 min-1). The same Kcat (693 min -1) was also observed when the A subunit was treated only with urea and dithiothreitol. Thus the full activity of Shiga-like toxin I required not only removal of the B subunits but also activation of the A subunit itself. Such activation could be largely induced in vitro by drastic loosening of the molecule induced by urea and dithiothreitol, but in vivo would probably require a proteolytic cleavage of the toxin. Inactivation of ribosomes by Shiga-like toxin I did not require sensitization of ribosomes by ATP and macromolecular cofactors present in postribosomal supernatants.
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PMID:The RNA-N-glycosidase activity of Shiga-like toxin I: kinetic parameters of the native and activated toxin. 940 66

Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.
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PMID:Disruption of an internal membrane-spanning region in Shiga toxin 1 reduces cytotoxicity. 978 30


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