Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

Osinga, K.A.; Swinkels, B.W.; Gibson, Wendy; Borst, Piet; Veeneman, G. H.; Boom, J. H. van; Michels, P.A.M.; Opperdoes, Frederik

doi:10.1002/j.1460-2075.1985.tb04152.x

Cited by 171 publications

(97 citation statements)

References 41 publications

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“…A similar result has been found for the glycosomal phosphoglycerate kinase which has a predicted subunit mass of 46.890 kDa [27], in agreement with the value on SDS gels for the purified enzyme: 47.0 f 0.7 kDa. The sequence of the cytosolic isoenzyme (44.914 kDa) is 20 amino acids shorter than the glycosomal isoenzyme [27] which we have also confirmed on SDS gels.…”

Section: Do Glycosomal Polypeptides Have Signals For Import?supporting

confidence: 89%

“…The sequence of the cytosolic isoenzyme (44.914 kDa) is 20 amino acids shorter than the glycosomal isoenzyme [27] which we have also confirmed on SDS gels. Also the yeast enzyme with 25 amino acids less [27] than the glycosomal isoenzyme behaved on SDS gels as a polypeptide which was 3 kDa smaller than the glycosomal one.…”

Section: Do Glycosomal Polypeptides Have Signals For Import?supporting

confidence: 77%

“…In this respect it is highly relevant that the glycosomal enzymes are basic proteins and have PI values which are 1 -6 higher than those found for other organisms. This results in highly positively charged proteins (for instance, a net charge of + 13 for glycosomal phosphoglycerate kinase [27] and + 10 for aldolase [29]) and such a positive charge or charges could play an important role in the recognition of the enzyme by, or its entry into, the organelle. Although we cannot exclude that the above-described unique properties of the glycosomal enzymes play a role in their proper functioning inside the glycosome, we now strongly believe that we are dealing here with signals for import.…”

Section: Do Glycosomal Polypeptides Have Signals For Import?mentioning

confidence: 99%

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Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

1986

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We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-l,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8 -55%.Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM-and DEAE-cellulose) which resulted in the purification of all nine enzymes.The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80 -90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1 -5 kDa larger in size.Together these nine enzymes contribute 3.3 f 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants.A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with PI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3 -6 higher than in the case of the various unicellular organisms.It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).

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Section: Do Glycosomal Polypeptides Have Signals For Import?supporting

confidence: 89%

Section: Do Glycosomal Polypeptides Have Signals For Import?supporting

confidence: 77%

Section: Do Glycosomal Polypeptides Have Signals For Import?mentioning

confidence: 99%

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Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

1986

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“…Osinga et al [17] have recently brought forward the hypothesis that the positively charged amino acids, present in N-terminally located insertions of glycosomal enzymes, constitute part of a topogenic signal required for import of glycosomal enzymes into the organelle. This hypothesis was based on an extensive comparison of properties [3], sequences [2, [17][18][19] and structures (Wierenga, R. K. and Hol, W. G. In short most glycosomal enzymes (a) carry a high positive charge and (b) contain unique N-terminal insertions rich in lysine and/or arginine residues as well as C-terminal unique amino acid substitutions rich in the same residues; in addition (c) at least three enzymes tend to cluster these additional positive charges into two surface areas some 4nm apart.…”

Section: Y D S V H G K F K H S V S T T K S K P S V a K D D T L V~' N mentioning

confidence: 99%

“…This hypothesis was based on an extensive comparison of properties [3], sequences [2, [17][18][19] and structures (Wierenga, R. K. and Hol, W. G. In short most glycosomal enzymes (a) carry a high positive charge and (b) contain unique N-terminal insertions rich in lysine and/or arginine residues as well as C-terminal unique amino acid substitutions rich in the same residues; in addition (c) at least three enzymes tend to cluster these additional positive charges into two surface areas some 4nm apart. The glycosomal GAPDH differs from all other GAPDHs described to date in that its amino acid sequence contains four unique insertions and a short C-terminal extension [2], while the cytosolic enzyme much more closely resembles the other GAPDHs, in that it lacks at least two and probably the other insertions present in the glycosomal enzyme as well.…”

Section: Y D S V H G K F K H S V S T T K S K P S V a K D D T L V~' N mentioning

confidence: 99%

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Misset

Beeumen²,

Lambeir

et al. 1987

European Journal of Biochemistry

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Trypanosoma bruceicontains two glyceraldehyde-phosphate (GAPDH; EC 1.2.1.12) isoenzymes; one is located in glycosomes and represents 80% of the total activity, whereas the other is present in the cytosol. The purification of the cytosolic GAPDH, which is identical in both bloodstream-form and insect-stage trypanosomes, is described, and the enzyme compared with its glycosomal counterpart. Cytosolic GAPDH is specific for NAD. It is a tetrameric enzyme with subunits of 33.5 kDa, 5 kDa smaller than those of the glycosomal GAPDH. The native enzyme has a pl of 7.9, which is 1.5 pH units less basic than the glycosobal enzyme. Both enzymes display maximal activity at pH 8 but the cytosolic enzyme has a much broader activity profile especially towards lower pH values.Sequence comparison of the first 85 amino acids reveals that the N-terminal parts of both isoenzymes differ by 52%. The N terminus of the cytosolic isoenzyme resembles the corresponding N termini of ten other known GAPDH sequences in that they all lack three amino-acid insertions, which so far only have been found in the glycosomal isoenzyme of T. brucei. This observation explains in part the great difference in subunit size between the two T. brucei isoenzymes and suggests that at least one of these insertions is responsible for import of the glycosomal isoenzyme into the organelle.In Trypanosoma brucei, glyceraldehyde-phosphate dehydrogenase (GAPDH) has been found in two separate cellular compartments: the cytosol and the glycosomes [l]. However, thus far no direct proof has been brought forward for the existence of two separate isoenzymes in this organism.Recently two tandemly linked identical genes coding for GAPDH have been identified in the genome of T. brucei [2]. From sequence data it was inferred that both genes code for the glycosomal enzyme; subunit molecular mass, isoelectric point 131, amino-acid composition and the N-terminal sequence of the isolated glycosomal protein [2] were all in excellent agreement with the properties predicted from the amino acid sequence as deduced from the gene. A gene coding for a possible cytosolic isoenzyme has not yet been identified.In this paper we now present evidence for the existence of a separate cytosolic isoenzyme of GAPDH in cultured procyclic (insect-stage) as well as in bloodstream-form trypanosomes. We describe its purification, characterization and N-terminal amino acid sequence and compare the properties of both the glycosomal and the cytosolic counterparts.

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Antiprotozoal Agents

Friès¹,

Fairlamb²

2003

Burger's Medicinal Chemistry and Drug Discovery

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The chapter provides coverage of all drugs currently used for the treatment of human African trypanosomiasis (African sleeping sickness), American trypanosomiases (Chagas' disease), and leishmaniasis. Collectively, this group of diseases is caused by flagellated protozoan of the order Kinetoplastida. The drugs currently used to treat these diseases include melarsoprol, suramin, pentamidine, organic antimonials, and several nitroheterocyclic agents. The agents have all been in use for 25–75 years. They have a high degree of toxicity and the first four in the preceding list must be given by parenteral injection over a 2‐ to 6‐week time period. Tolerance has developed to most of the drugs in at least some geographical areas. Eflornithine is the only agent approved in the last 50 years for the treatment of African trypanosomiasis, although it is expensive, requires prolonged systemic dosage, and is effective only against T. brucei gambiense . Megazol, trybazine, DB289, and CGP 40215A have all yielded encouraging results in in vivo preclinical studies against African trypanosomiasis. All four of these agents have entered or are scheduled to enter clinical trials. Nifurtimox and benznidazole are the only agents approved to treat Chagas' disease. The agents are toxic, rarely produce long‐term cures, and resistance to them has developed. The third‐generation triazole antifungal agents posaconazole and UR‐9825 have given encouraging preclinical results against Chagas' disease. Meltefosine has been found to be orally active and to produce 98% cure rates of visceral leishmaniasis. New classes of experimental antitrypanosomal agents include the bisphosphonates, which disrupt lipid metabolism, and cysteine protease inhibitors. Agents from both of these mechanistic classes are being advanced as potential antitrypanosomal agents. Kinetoplastid biochemistry offers numerous opportunities for the development of chemotherapeutic agents having a high degree of selective toxicity. Trypanothione, the N 1 , N 8 ‐bis(glutathionyl)spermidine metabolite that replaces glutathione in redox protection reactions in kinetoplastids, is one hopeful target for drug design. A second possible target is the kinetoplastid organelle named the glycosome. African trypanosomes and perhaps other kinetoplastids are highly dependent on glycolysis for energy production. Enclosing the enzymes for glycolysis in glycosomes increases the efficiency of glycolysis compared to its rate in mammalian cells. Any potent inhibitor of an enzyme contained in the glycosome, or of any of the biochemical processes associated with glycosomal function, might be an effective drug against these parasites.

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Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

Cited by 171 publications

References 41 publications

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei

Antiprotozoal Agents

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