Onno Misset

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde‐phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody‐like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52‐57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1‐8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody‐like organelle, the glycosome, which they are believed to reach post‐translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde‐phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain. Modelling of the three‐dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two ‘hot spots’ about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three‐dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.

A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes.

Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.

Preliminary crystallographic studies of triosephosphate isomerase from the blood parasite Trypanosoma brucei brucei.

Crystals of triosephosphate isomerase (EC 5.3.1.1) from Trypanosoma brucei brucei have been grown. These crystals diffract to at least 2 A, even after 60 hours of exposure to X-rays. The space group is P212121, with cell dimensions a = 112.4 A, b = 97.8 A, c = 48.0 A. There is one dimer per asymmetric unit.