Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

https://doi.org/10.1016/j.parint.2016.06.008Get rights and content

Highlights

  • Two different Trypanosoma cruzi galactokinase genes were cloned and expressed in Escherichia coli.

  • Both galactokinase genes are expressed in Trypanosoma cruzi epimastigotes.

  • Both galactokinase enzymes are present inside glycosomes.

  • Trypanosoma cruzi may have an alternative pathway for UDP-galactose synthesis.

  • Epimastigotes can grow in a medium supplemented with galactose as carbon source.

Abstract

Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9 kDa and 51.3 kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108 mM (TcGALK-1) and 0.091 mM (TcGALK-2) and for ATP 0.36 mM (TcGALK-1) and 0.1 mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414 mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52–47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26–23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20 mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source.

Introduction

Trypanosoma cruzi, the causative agent of Chagas' disease, is a parasite belonging to the protistan Trypanosomatidae family. It undergoes multiple morphological and metabolic changes during its complex life cycle involving two hosts, a vertebrate and an insect [1]. The transition between the different developmental stages is accompanied by changes in the carbohydrate composition of the macromolecules on the cell surface, coated with a dense glycocalyx composed mainly of glycoconjugates, some of which play essential roles in parasite survival, infectivity and virulence [2].

The monosaccharides that make up these glycoconjugates are d-mannose (Man), d-N-acetylglucosamine (GlcNAc), d-glucosamine (GlcN), d-glucose (Glc), d-xylose (Xyl), l-rhamnopyranose (Rha), l-fucose (Fuc), d-galactose (d-Gal: d-galactopyranose and d-galactofuranose), with this latter sugar being particularly abundant in all trypanosomatids [3]. The glycoconjugates are synthesized by specific glycosyltransferases using nucleotide-sugars (NDP-sugars) as glycosyl donors [2].

Formation of nucleotide-sugars occurs via two distinct pathways, i) the de novo synthesis or interconversion of pre-existing NDP-sugars, and ii) a salvage pathway where free sugars are taken up from the environment by a specific sugar transporter or generated intracellularly upon the degradation of polysaccharides, glycoproteins and glycolipids. These free sugars can be phosphorylated at position C-1 by a specific kinase to form sugar 1-phosphates. Subsequently, a nucleotide diphosphate (NDP)-sugar pyrophosphorylase (USP) transfers a nucleotidyl residue to form the NDP-sugar [3]. It has been suggested that in trypanosomatids much of the synthesis of the nucleotide-sugars occurs inside glycosomes, based on the finding that some enzymes of these pathways contain a peroxisome-targeting signal (PTS-1 or PTS-2) [4], and for some of them localization in these organelles has been experimentally demonstrated [5], [6].

The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates that form the glycocalyx surface of T. cruzi. In this parasite the synthesis of UDP-Gal has been proposed to occur only by the direct conversion of UDP-Glc to UDP-Gal by UDP-glucose-4-epimerase (GALE) [7]. Galactose cannot be obtained from the environment as it is not recognized by the hexose transporter (TcrHT1) [8]. Moreover, it was found that, at least for the epimastigote stage, GALE is essential for the biosynthesis of UDP-Gal and derived glycoconjugates, and hence for the parasite's survival [9]. However, the genomes of T. cruzi[10] and Leishmania spp. [11], but not of Trypanosomabrucei [12] encode the entire enzymatic machinery for the salvage route, the Isselbacher pathway, to synthesize UDP-Gal [13]. In Leishmaniamajor both pathways are active and contribute to the UDP-Gal synthesis [14], [15]. In contrast, the T. brucei genome lacks a homologue of the USP gene and it has a pseudogene of GALK [12]. Indeed, in this parasite the UDP-Gal synthesis occurs only by the de novo pathway [16].

Isselbacher route enzymes encoded in the T. cruzi genome are: aldose-1-epimerase (TcCLB.509331.180), galactokinase (GALK) (two genes, TcCLB.507001.110 and TcCLB.510667.120), and UDP-sugar pyrophosphorylase (USP) (TcCLB.511761.10); this latter enzyme has been recently characterized in T. cruzi and Leishmania[3], [15]. In order to contribute to the elucidation of this pathway and its role in the UDP-Gal formation in T. cruzi, we aimed to study the enzyme galactokinase (EC 2.7.1.6), which catalyzes the ATP-dependent phosphorylation of α-d-galactose to produce α-d-galactose 1-phosphate (Gal-1-P). GALK plays an important role not only because it is involved in the synthesis of UDP-Gal, but the enzyme is also part of the Leloir pathway in species ranging from Escherichiacoli to human [17]. This latter pathway comprises galactose mutarotase, galactokinase (GALK), galactose-1-P uridyltransferase (GALT), and UDP-galactose 4′-epimerase (GALE), which together mediate the transformation of β-d-galactose into α-d-glucose-1-P [17], [18]. Finally, α-d-glucose-1-P is transformed into α-d-glucose-6-P by phosphoglucomutase (PGM). In this way the carbons from galactose can enter into the glycolytic pathway and be used as carbon and energy source.

The enzyme GALK has been isolated from different sources such as bacteria [19], yeast [20], mammalian liver [21], plants [22], and human [23]. In man, mutations in the GALK gene lead to the disease state referred as Type II galactosemia. Patients with this disorder exhibit neonatal cataracts that self-resolve upon dietary restriction of galactose [23]. On the basis of sequence comparisons, it was concluded that galactokinases belong to a unique class of ATP-dependent enzymes known as the GHMP superfamily (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) [24]. All these proteins contain three common structural/functional motifs, the second one being the highly conserved Pro-XXX-Gly-Leu-X-Ser-Ser-Ala motif that is involved in ATP binding [23], [24]. In this paper, we report the amplification and cloning of the two TcGALK genes of T. cruzi. The expressed proteins of the two genes TcCLB.507001.110 (TcGALK-1) and TcCLB.510667.120 (TcGALK-2) were soluble and able to phosphorylate α-d-galactose using ATP as phosphoryl donor, thus confirming them as GALKs. Both TcGALK genes are expressed in epimastigotes and trypomastigotes. Furthermore, galactokinase activity was detected in the epimastigote form of the parasite, and located within the glycosomes. In addition, we found that epimastigotes of T. cruzi are able to grow in LIT medium supplemented with galactose, supporting the notion that this sugar could be used as carbon and energy source.

Section snippets

Parasites and growth of T. cruzi epimastigotes and bloodstream trypomastigotes

Epimastigotes of T. cruzi strain EP were cultured axenically at 28 °C, with constant shaking, in liver infusion-tryptose (LIT) medium supplemented with 5% heat-inactivated fetal bovine serum [25]. Bloodstream trypomastigotes were obtained from infected Vero cells as described by Bertelli et al. [26].

Growth curves of T. cruzi epimastigotes were determined in three different conditions: i) low glucose, which is a partially depleted medium (1.5 mM d-glucose), supplemented with 5% heat-inactivated

Cloning and sequence analysis of the Trypanosoma cruzi galactokinase genes

Putative TcGALK genes of T. cruzi were identified in the GeneDB database (TcCLB.507001.110 and TcCLB.510667.120) and amplified by PCR using genomic DNA from the parasite (strain EP) and cloned. The putative TcGALK-1 gene is a 1407-bp long open-reading frame (ORF) for a polypeptide of 468 amino acids with a molecular mass of 51.9 kDa. The putative TcGALK-2 gene is a 1392-bp ORF that predicts a polypeptide of 463 amino acids with a molecular mass of 51.3 kDa. The predicted molecular weight of these

Discussion

In this paper we described the cloning and expression in E. coli of two predicted GALK genes identified in the T. cruzi genome. The recombinant TcGALK-1 and TcGALK-2 proteins were biochemically characterized confirming them as galactokinases. Their sequences contain all residues known to be involved in the substrates binding sites and the characteristic motif of the GHMP superfamily. These proteins are localized in glycosomes, routed by two different canonical PTS-1 motifs, SNL (TcGALK-1) and

Conclusions

Two different galactokinase genes are expressed in Trypanosoma cruzi, both enzymes are active, located in glycosomes, which suggests an alternative pathway to synthesize UDP-galactose.

Acknowledgements

We are grateful to Dr. Luisana Avilán, Dr. Melisa Gualdrón (ULA Mérida) and Dr. Paul Michels (University of Edinburgh) for their continuous support and helpful comments on the manuscript. We thank Betty Hernández and Silverio Díaz for their assistance with the parasite cultures. This research was supported by projects CDCHT-ULAC-1829-13-03-B and Misión Ciencia2007001425.

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