In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes

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Abstract

In total, 37 080 protein sequences of the three trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi, were used to predict the trypanosomatid glycosomal proteome. All protein sequences were analyzed for the presence of either a C-terminal (PTS1) or an N-terminal (PTS2) peroxisomal targeting sequence. For L. major 191 potential PTS1-containing proteins and 68 potential PTS2-containing proteins with homologues in T. brucei and T. cruzi were identified. About 50% of them were hypothetical proteins to which no function was attributed. From those proteins with known function it appears that the predicted glycosomal proteome of L. major strongly resembles that of T. brucei and T. cruzi with respect to enzyme content. Glycosomes are not only involved in glycolysis, but are predicted to carry out also gluconeogenesis, reactions of the hexose-monophosphate pathway, purine salvage and pyrimidine biosynthesis, β-oxidation of fatty acids, fatty acid elongation and the biosynthesis of ether lipids. In addition, they seem to catalyze several reactions of isoprenoid synthesis and are involved in oxidant stress protection.

Introduction

Glycosomes of trypanosomatids belong to the microbody family of organelles. This family comprises also the glyoxysomes of plants and peroxisomes of all other eukaryotes. The organelles resemble each other with respect to morphology, metabolic functions, such as β-oxidation of fatty acids and biosynthesis of ether lipids, and their biogenesis. Specific to glycosomes of trypanosomatids is that they are involved in carbohydrate metabolism. In Trypanosoma brucei, seven enzymes of the glycolytic pathway, two enzymes of glycerol metabolism and various enzymes of purine salvage and pyrimidine metabolism have been identified inside glycosomes [1], [2]. In the case of other trypanosomatids, there is limited and highly scattered information about the enzymes or metabolic pathways present in their microbodies.

The biogenesis of microbodies is conserved between glycosomes and the other members of this family [3], [4]. Both the protein targeting of matrix enzymes and import mechanism into glycosomes and the components required for glycosomal biogenesis resemble those described for peroxisomes and glyoxysomes. Peroxisomal proteins are nuclear-encoded and synthesized in the cytosol on free polyribosomes [5]. The organelles acquire their matrix proteins by post-translational import from the cytosol via two pathways that rely on two kinds of conserved peroxisomal targeting signals (PTS). The majority of peroxisomal matrix proteins have a PTS1 at their extreme carboxyl terminus, consisting of just three amino acids – SKL – or a conservative variant thereof [6], [7]. A few enzymes (fructose-1,6-bisphosphate aldolase, hexokinase and 6-phosphogluconolactonase in glycosomes, and malate dehydrogenase, citrate synthase, acyl-CoA oxidase and 3-ketoacyl-CoA-thiolase in peroxisomes) are known to use a different targeting signal, the PTS2, located closely to the N-terminus, which is a bipartite signal with the consensus sequence [RK]-[LVI]-x5-[HQ]-[LA] [8]. Although most peroxisomal matrix proteins use a PTS for their targeting, there are a few proteins that lack a canonical targeting signal and that have been proposed to enter the peroxisomal matrix by “piggybacking” on other proteins bearing a PTS [3], [9]. The presence of such a targeting signal in protein sequences has often been a good indicator for a microbody location. Using the information about some 30 000 predicted protein sequences available through the L. major, T. brucei and T. cruzi genome projects [10] (http://www.genedb.org) we have attempted to predict the glycosomal proteomes of these three organisms. We have analyzed all predicted peptide sequences for the presence of either a C-terminal PTS1 or an N-terminally located PTS2. The results indicate that the glycosomes of L. major and T. cruzi strongly resemble those of T. brucei with respect to their enzyme content. In addition, this approach predicts many more proteins to be present in the glycosomes of both Leishmania and Trypanosoma. These proteins are not only involved in glycolysis, but also in the hexose-monophosphate pathway, in purine salvage and pyrimidine biosynthesis, the biosynthesis of ether lipids and possibly β-oxidation of fatty acids. In addition, glycosomes catalyze several reactions of the isoprenoid biosynthetic pathway and are involved in protection against oxidant stress.

Section snippets

Materials and methods

PTS1-containing proteins were identified using the Prosite pattern PS00342 [11]: [STAGCN]-[RKH]-[LIVMAFY], followed by an end-of-line anchor “$”. This resulted in an initial 190, 191 and 319 proteins when the L. major, T. brucei and T. cruzi databases were screened, respectively. 47–49% represented hypothetical proteins and 5–7 ribosomal proteins were found in each of the three datasets. In addition, the databases were searched for the presence of proteins with a C-terminal –SSL sequence and

Glycolysis, gluconeogenesis and glycerol metabolism

Seven proteins involved in glycolysis and two proteins in glycerol metabolism have been demonstrated to be present inside glycosomes, both of T. brucei [1], of T. cruzi [18] and of Leishmania spp. [19]. All of these enzymes, except triosephosphate isomerase (TPI) and phosphoglycerate kinase (PGK), were identified in the three trypanosomatid databases by the present search algorithms (Fig. 1). Hexokinase has a PTS2 in all three trypanosomatids. Glucose-6-phosphate isomerase and

Conclusion

With this analysis we have obtained a good impression of the various metabolic functions which are carried out by glycosomes. In addition to glycolysis, from which pathway they have derived their name, the organelles are involved in gluconeogenesis, in the hexose-monophosphate pathway, in purine salvage and pyrimidine biosynthesis, the biosynthesis of ether lipids and possibly the β-oxidation of fatty acids. They catalyze several reactions of the isoprenoid biosynthetic pathway and have their

Acknowledgments

The authors wish to thank Dr. Paul A.M. Michels for stimulating discussions and corrections on the manuscript. Sequence data used were produced by the Leishmania major Friedlin Genome project, the Trypanosoma brucei Genome Project at the Sanger Institute and the Trypanosoma cruzi genome project at The Institute of Genomic Research and the Karolinska Institute and can be obtained from http://www.genedb.org.

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