Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites

Nene, Vishvanath; Iams, Keith P.; Gobright, Elke; Musoke, A.J.

doi:10.1016/0166-6851(92)90196-q

Cited by 82 publications

(64 citation statements)

References 25 publications

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“…Their enzymic characteristics and amino acid sequences (where known) are similar to those of papain and cathepsins L and B (Barrett and Kirschke, 1981 ;Mottram et al, 1989;Eakin et al, 1990;Nene et al, 1990). We have shown that cysteine protcase activity in T. congolense, Trypanosoma brucei and Trypanosoma vivux changes from one life-cycle stage to another (Lonsdale-Eccles and Mpimbaza, 1986;Mbawa et al, 1991a).…”

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Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Mbawa¹,

Gumm²,

Shaw

et al. 1992

European Journal of Biochemistry

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A cysteine protease (trypanopain-Tc) with cathepsin-L-like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31 -3 2 kDa by SDS/ PAGE and 66 kDa by gel chromatography. It has a PI 7.4 and a high affinity for concanavalin A, Trypanopain-Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant-surface glycoprotein. It has minimal or no activity against casein or elastin.A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain-Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain-Tc was Z-Phe-Arg-NHMec (Z, benzyloxycarbonyl ; NHMec, 7-amido-4-methylcoumarin). The kinetic constants for the hydrolysis, of ZPhe-Arg-NHMec were k,,, = 17.4 SKI, K, = 4.4 pM, kcal/Km = 4.0 pM-' ' s-', which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z-ArgArg-NHMec) and cathepsin H (Arg-NHMec) were not hydrolysed by trypanopain-Tc under the conditions tested. The pH optimum of trypanopain-Tc against Z-Phe-Arg-NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low-molecular-mass thiol compounds and inhibited by cystatin, ~-trans-epoxysucciny1-4-guanidinobutane (E-64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z-Leu-Leu-Met-CHN2, Z-Leu-Met-CHN2 and Z-Leu-Lys-CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vituo after 24 -48 h incubation in 2 20 pM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.Cathepsins L and B play important roles in intracellular protein turnover (for review see Ballard, 1987;Marzella and Glaumann, 1987). They are also implicated in the degradation of extracellular matrix in diseases such as muscular dystrophy, emphysema and arthritis, and in association with tumour metastasis (Rochefort ct al., 1987;Kominami et al., 1987; Burnett and Stockley, 1985;Etherington et al., 1988). Recently, interest has been shown in the role of cysteine proteases in the pathology of parasitic infections. These proteases can be released from the parasites (Blum, 1976;Keene, 1986; Lockwood et al., 1988; Garber and Lemchuk-Favel, 1989;North et al., 1989; Boutignon et al., 1990) andmay beinvolved in the pathology of the diseases and the survival of the parasites within the host (Bremmer et al

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confidence: 95%

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Mbawa¹,

Gumm²,

Shaw

et al. 1992

European Journal of Biochemistry

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“…Washing after hybridization was done at 65˚C with 0.2X SSC containing 0.1% SDS for 30 min and the blot was exposed to X-ray film at 80˚C overnight. parva and T. annulata [1,7]. Our present result suggests that cysteine proteinase may also be expressed in intraerythrocytic stages in Theileria parasites.…”

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confidence: 56%

“…The cysteine proteinase of P. falciparum has been characterized and the interruption of parasite development by inhibition of its enzyme activity was demonstrated [10,12]. The growth of T. parva schizont-infected bovine lymphocytes was also impaired by addition of Z-Leu-Val-Gly-CHN 2 , a diazomethel ketone inhibitor of cysteine proteinases [7].…”

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confidence: 99%

“…1). Comparison of the translated amino acid sequence of the T. sergenti protein with those of T. parva [7] and T. annulata [1] resulted in 29.3% and 35.1% amino acid identity, respectively. Southern blot analysis with this cDNA clone as a probe detected only a single hybridization band with T. sergenti genomic DNA digested several enzymes (Fig.…”

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confidence: 99%

“…From the conserved regions of T. parva (Genbank accession No: M37791) and T. annulata (Genbank accession No: M86659) cysteine proteinases [1,7], we designed a set of primers, CyP-1: 5'-TGTGGTTCTTGTTGGGCATT-3' (forward) and CyP-2: 5'-CAATCTTCTCCCCATGAGTT-3' (reverse). Polymerase chain reaction (PCR) was performed in a 100 µl reaction mixture containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1 µM of each primer, 100 ng of genomic DNA extracted from piroplasms (Shintoku stock), 1.5 mM MgCl 2 , 0.2 mM of each dNTP and 0.5 units of Taq DNA polymerase (Pharmacia).…”

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Cloning of a Cysteine Proteinase Gene of Theileria sergenti.

Sako

Sugimoto

Onuma

1999

The Journal of Veterinary Medical Science

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ABSTRACT. A cDNA encoding cysteine proteinase of Theileria sergenti was isolated from a piroplasm cDNA library and its nucleotide sequence was determined. The gene encodes a polypeptide of 402 amino acids with predicted molecular mass of 46.4 kDa. Analysis of the predicted amino acid sequence revealed a number of features common to known cysteine proteinases. Southern blot analysis showed that the cysteine proteinase gene was likely to be a single copy per genome.-KEY WORDS: cysteine proteinase, piroplasm, Theileria sergenti.

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Theileria

and

Baumgärtner

2009

Intracellular Niches of Microbes

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Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites

Cited by 82 publications

References 25 publications

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense

Cloning of a Cysteine Proteinase Gene of Theileria sergenti.

Theileria

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