Trypanosoma cruzi Arginine Kinase Characterization and Cloning

Abstract

This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates l-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mm l-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease. This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates l-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mm l-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease. polymerase chain reaction base pair(s) kilobases kilobase pair(s) N-Phosphorylated guanidino compounds, commonly referred to as phosphagens, play a critical role as an energy reserve because of the high energy phosphate that can be transferred when the renewal of ATP is needed. It has also been proposed that these compounds function in spatial buffering of cellular energy production sites. So, phosphagens act as reserves not only of ATP but also of inorganic phosphate, which is mostly returned to the medium by metabolic consumption of ATP. Phosphoarginine is the main reserve of high energy phosphate compounds in a wide variety of invertebrates. In addition phosphocreatine, phosphoglycocyamine, phosphotaurocyamine, phosphohypotaurocyamine, phosphoopheline, and phospholombricine are also found, whereas in vertebrates the only one present is phosphocreatine (1.Morrison J.F. Boyer P.D. The Enzymes. 8. Academic Press, New York1973: 457-486Google Scholar, 2.Huennekens F.M. Whiteley H.R. Florkin M. Mason H.S. Comparative Biochemistry. 1. Academic Press, New York1960: 107-180Crossref Google Scholar). Arginine kinase (EC 2.7.3.3) is a member of a conserved family of phosphotransferases which also includes creatine kinase. These enzymes catalyze the reversible transfer of a phosphoryl group from ATP to a guanidino acceptor, which can be either an amino acid (e.g.lombricine or arginine) or a carboxylate (e.g. creatine or glycocyamine; Reaction 1).MgATP+guanidino acceptor⇄P­guanidino acceptor+MgADP+H+REACTION 1Arginine kinase is present in Annelida, Celenterata, Platyhelmintes, Nemertea, Mollusca, Phoronida, Arthropoda, Equinodermata, Hemichordata, and Chordata, where, like other phosphagen kinases, it maintains ATP homeostasis during muscle contraction (1.Morrison J.F. Boyer P.D. The Enzymes. 8. Academic Press, New York1973: 457-486Google Scholar,2.Huennekens F.M. Whiteley H.R. Florkin M. Mason H.S. Comparative Biochemistry. 1. Academic Press, New York1960: 107-180Crossref Google Scholar). Common in most of these phosphotransferases is the presence in the binding sites of substrates of five arginine residues interacting with ATP, two carboxylate amino acids, and one cysteine residue interacting with the guanidino acceptor group (3.Zhou G. Smasundaram T. Blank E. Parthasarathy G. Ellington W.R. Chapman M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). In addition, some arginine kinases contain a domain for interaction with actin (4.Reddy R.R. Houmeida A. Benyamin Y. Roustan C. Eur. J. Biochem. 1992; 206: 251-257Crossref PubMed Scopus (21) Google Scholar). Most of the enzymatic reactions studied in trypanosomatids, which involves l-arginine, are related to the ornithine-arginine pathway. Distinct genus of trypanosomatids utilizes different enzymes in arginine catabolism. Members of the Trypanosoma genus include T. cruzi, the causative agent of Chagas' disease, devoid of ornithine decarboxylase, arginine decarboxylase, and arginase (5.Camargo E.P. Coelho J.A. Moraes G. Figueiredo E.N. Exp. Parasitol. 1978; 46: 141-144Crossref PubMed Scopus (57) Google Scholar, 6.Yoshida N. Camargo E.P. J. Bacteriol. 1978; 136: 1184-1186Crossref PubMed Google Scholar). In addition, the existence of the nitric oxide pathway inT. cruzi was demonstrated recently by this laboratory. This signaling pathway involves a putativel-glutamate/N-methyl-d-aspartate receptor, a nitric oxide synthase, and a guanylyl cyclase and seems to be a control step in epimastigote flagellar motility (7.Paveto C. Pereira C. Espinosa J. Montagna A.E. Farber M. Esteva M. Flawiá M.M. Torres H.N. J. Biol. Chem. 1995; 270: 16576-16579Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 8.Pereira C. Paveto C. Espinosa J. Alonso G. Flawiá M.M. Torres H.N. J. Eukar. Microbiol. 1997; 44: 155-156Crossref PubMed Scopus (25) Google Scholar). The existence of a high affinity and very specific l-arginine transporter was also demonstrated in T. cruzi epimastigotes. One of the major products of l-arginine uptake was characterized as phosphoarginine (9.Pereira C.A. Alonso G.D. Paveto M.C. Flawiá M.M. Torres H.N. J. Eukar. Microbiol. 1999; 46: 566-570Crossref PubMed Scopus (65) Google Scholar). The present article provides wide information on the enzymatic and genetic characterization of T. cruzi arginine kinase. Enzymes and PCR1 reagents were provided by Promega Corporation (Madison, WI). Oligonucleotides were from Biosynthesis, Inc. (Lewisville, TX), and other reagents were from Sigma Chemical Co. (St. Louis, MO). Unless otherwise indicated, T. cruziepimastigote forms of the Tulahuen 2 strain were cultured at 28 °C in a medium containing 5 g/liter liver infusion, 5 g/liter Bacto-tryptose (Difco Laboratories, Detroit, MI), 68 mmNaCl, 5.3 mm KCl, 22 mmNa2HPO4, 0.2% glucose, 0.002% hemin supplemented with 10% fetal calf serum, 10 units/ml penicillin, and 10 mg/liter streptomycin. Cell viability was assessed by direct microscopic examination. For arginine kinase purification, epimastigotes from 7-day cultures (late logarithmic phase) were collected by centrifugation at 1,000 × g, washed three times with 25 mm Hepes adjusted to pH 7.3 with Tris and containing 1.8 mmCaCl2, 0.8 mm MgSO4, 2.7 mm KCl, 1.2 mm KH2PO4, 154 mm NaCl, and 5.5 mm d-glucose (buffer A). The pellet was then resuspended (1 ml/each 30 ml of culture) in 25 mm Hepes buffer, pH 7.3, containing 0.01 mg/ml leupeptin, 2 mg/ml soybean trypsin inhibitor, 1 mmbenzamidine, 25 units/ml Trasylol, 0.1 mmphenylmethylsulfonyl fluoride, and 0.5 mm tosyl-lysine chloromethyl ketone, and lysed by six cycles of freezing in liquid N2 and thawing at 4 °C. The extract was then centrifuged 5 min at 8,000 × g. The supernatant thus obtained was used as a source of epimastigote proteins for the arginine kinase purification. The supernatant fluid (5.0 ml) was applied to a 5 × 1-cm Whatman DE-52 column equilibrated with 25 mm Hepes buffer, pH 7.6, and washed with 25 ml of the same buffer solution. The enzyme activity was recovered in the percolate. After a concentration step using Amicon ultracentrifugation cells, the sample (0.5 ml) was applied to a Superose-6 HR10/30 column (Amersham Pharmacia Biotech) equilibrated as above. The effluent, obtained at a rate of 0.3 ml/min, was collected in 0.5-ml fractions. The peak activity fractions were pooled and used as a source of kinase activity. These fractions were enriched 80-fold in enzyme activity with a recovery of about 55% (specific activity 0.8–1.2 μmol·min−1·mg of protein−1; see Fig. 3 B). Purification of recombinant arginine kinase was performed on a Ni-NTA agarose column (QIAGEN, Valencia, CA) according to the manufacturer's instructions for denatured polypeptides. The eluate was then dialyzed successively for 24 h against decreasing urea concentrations (from 7.0 m to none) in 0.1 m phosphate buffer, pH 7.2, containing 7 mm 2-mercaptoethanol, 10% glycerol, and 0.15 m NaCl, to obtain recombinant arginine kinase with a specific activity of 10–16 μmol·min−1·mg of protein−1 and a yield of 10–20 mg of protein/liter of culture (see Fig. 3 A). The incubation mixture contained 25 mm Hepes buffer, pH 7.3, 2 mm ATP, 5 mm magnesium acetate, 10 mm 2-mercaptoethanol, 1 mm l-[2,3-3H]arginine (NEN Life Scienc