Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: An evolutionary scenario of subcellular compartmentation in Kinetoplastida

Abstract

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

“…Using digitonin titration, the subcellular localization of putG3PDH was experimentally addressed in cells expressing the V5-tagged version of the protein, as increasing concentrations of this nonionic detergent sequentially permeabilize cell membranes on the basis of their different levels of sterol content (32). Marker Abs for different T. brucei cell compartments were used as controls (Fig.…”

Trypanosoma cruzi -- the vector-parasite paradox

“…Phylogenetic analysis based on ssu rRNA sequence data indicate that the genus Trypanosoma is monophyletic (Stevens & Gibson 1999, Stevens et al 1999a,b, 2000 endorsed by studies based on the GAPDH gene (Wiemer et al 1995, Alvarez et al 1996, Hannaert et al 1998, and on 9S and 12S mitochondrial rRNA genes (Lake et al 1988), elongation factor 1α (Hashimoto et al 1995), trypanothione reductase and α-tubulin (Alvarez et al 1996) and phosphoglycerate kinase (Adjé et al 1998). All trypanosomes are parasitic, so the idea of of a monophyletic origin suggests an ancestral parasitic form that gave rise to the mammalian trypanosomes of Africa, America, and Australasia, as well as the various forms that parasitise fish.…”

Phylogeny of the bodonid flagellates (Kinetoplastida) based on small-subunit rRNA gene sequences.

“…( Wright et al, 1999), and the freeliving species Bodo caudatus (Fernandes et al, 1993), Dimastigella trypaniformis (Berchtold et al, 1994) and , 1997). By putting into phylogenetic context the data obtained by the analysis of RNA editing Lukes) et al, 1994 ;Blom et al, 1998Blom et al, , 2000, kDNA structure ( Yasuhira & Simpson, 1996 ;Lukes) et al, 1998), and glycosomes ( Wiemer et al, 1995) a better understanding of these processes and structures can be achieved. The aim of this work is to determine phylogenetic relationships within the suborder Bodonina, and to provide a framework for molecular and biochemical studies on bodonids.…”