Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains

Díaz, Francisca; Komuniecki, Richard

doi:10.1016/0166-6851(95)02498-0

Cited by 8 publications

(7 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant ABA-1 (rABA-1) allergen from A. lumbricoides was produced by similar methods, as will be described in detail elsewhere. 2 Antibody Generation and Immunoblotting-Purified rAs-p18 was separated on 12% SDS-polyacrylamide gels and used to immunize rabbits as described previously (15). Antibodies against rAs-p18 were affinity-purified using protein bound to CNBr-activated Sepharose 4B (Sigma) according to Kent (16).…”

Section: Methodsmentioning

confidence: 99%

Secretion of a Novel, Developmentally Regulated Fatty Acid-binding Protein into the Perivitelline Fluid of the Parasitic Nematode, Ascaris suum

Mei

Kennedy²,

Beauchamp

et al. 1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Early development of the parasitic nematode, Ascaris suum, occurs inside a highly resistant eggshell, and the developing larva is bathed in perivitelline fluid. Twodimensional gel analysis of perivitelline fluid from infective larvae reveals seven major proteins; a cDNA encoding one of these, As-p18, has been cloned, sequenced, and protein expressed in Escherichia coli. The predicted amino acid sequence of As-p18 exhibits similarities to the intracellular lipid-binding protein (iLBP) family including retinoid-and fatty acid-binding proteins (FABP). As-p18 is unusual in that it possesses a hydrophobic leader that is not present in the mature protein, the developmental regulation of its expression, and in terms of its predicted structure.Recombinant As-p18 is a functional FABP with a high affinity for both a fluorescent fatty acid analog (11-(((5-(dimethylamino)-1-naphthalenyl )sulfonyl ) amino ) undecanoic acid) and oleic acid, but not retinol. Circular dichroism of rAs-p18 reveals a high ␤-sheet content (62%), which is consistent with secondary structure for the protein predicted from sequence algorithms, and the structure of iLBPs. Unusual features are apparent in a structural model of As-p18 generated from existing crystal structures of iLBPs.As-p18 is not found in unembryonated eggs, begins to be synthesized at about day 3 of development, reaches a maximal concentration with the formation of the firststage larva and remains abundant in the perivitelline fluid of the second-stage larva. Since As-p18 is not present in the post-infective third-stage larva or adult worm tissues, it appears to be exclusive to the egg. Surprisingly, however, Northern blot analysis yields mRNA for As-p18 not only in the early larval stages, but also the unembryonated egg, third-stage larvae, and ovaries of adult worms, even though the protein is not detectable from any of those sources. As-p18 may play a role in sequestering potentially toxic fatty acids and their peroxidation products, or it may be involved in the maintenance of the impermeable lipid layer of the eggshell.Parasitic nematodes cause medical and economic damage on a global scale, in part the result of the longevity and environmental robustness of their dispersal stages, their eggs. Arguably the most successful and widely distributed nematode parasite of humans is Ascaris lumbricoides, which infects over 1 billion people. A morphologically indistinguishable species, Ascaris suum, occurs in pigs and has become the best understood nematode in biochemical terms. The eggs contaminate soil, and the embryos develop to infective, second-stage larvae (L2s)

show abstract

Section: Methodsmentioning

confidence: 99%

Secretion of a Novel, Developmentally Regulated Fatty Acid-binding Protein into the Perivitelline Fluid of the Parasitic Nematode, Ascaris suum

Mei

Kennedy²,

Beauchamp

et al. 1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, its occurence in anaerobic organisms (Dietrichs and Andreesen, 1990) and in bloodstream African trypanosomes, which lack a functional mitochondrion (Danson et al, 1987), led to the proposal that the function of LipDH is not restricted to its roles within the multienzyme complexes. From the insect parasite Crithidia fasciculata the enzyme has recently been characterized as part of the pyruvate dehydrogenase complex (Diaz and Komuniecki, 1995). Structural and functional studies on pyruvate dehydrogenase from Escherichia coli have shown that the complex can generate free SH groups (Frey et al, 1978;Bunik et al, 1995).…”

mentioning

confidence: 99%

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Schöneck

Billaut-Mulot

Numrich

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50066. Comparison of the deduced amino acid sequence of LipDH from 7: crzizi with that of Trypanosoma brucei and man shows identities of 81 % and 50%, respectively.An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpdl of 7: cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for AsnlO which represents the N-terminus of the mature protei-n. This system allowed the synthesis of 1000 U 7: cruzi LipDH/l bacterial cell culture. The recombinant protein was purified to homogeneity by (NH,),SO,-precipitation and affinity chromatography on 5' AMP-Sepharose. The K,,, values for NAD', NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite.LipDH is present in all major developmental stages of 7: cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite.In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason 7: cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of 7: cruzi LipDH in drug activation and the crystallization of the protein.

show abstract

“…Whether this reduced specific activity is related to the absence of a lipoyl domain on E3BP is not clear. Recently, an E3BP in the PDC of the insect trypanosomatid, Crithidia fasciculata, has been identified which appears to contain, not one, but multiple lipoyl domains (40). A similar situation has been described previously for E2s, where 1, 2, or 3 lipoyl domains have been identified, depending on the species, and there appears to be no physiological significance to the number of lipoyl domains in E2 (41).…”

Section: Discussionmentioning

confidence: 63%

Identification of a Novel Dihydrolipoyl Dehydrogenase-binding Protein in the Pyruvate Dehydrogenase Complex of the Anaerobic Parasitic Nematode, Ascaris suum

Klingbeil

Walker

Arnette

et al. 1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which lacks an amino-terminal lipoyl domain, p45, has been identified in the pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited significant similarity with internal E3-binding domains of dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution of a pyruvate dehydrogenase-depleted adult A. suum PDC in guanidine hydrochloride resulted in two E3-depleted E2 core preparations which were either enriched or substantially depleted of p45. Following reconstitution, the p45-enriched E2 core exhibited enhanced E3 binding, whereas, the p45-depleted E2 core exhibited dramatically reduced E3 binding. Reconstitution of either the bovine kidney or A. suum PDCs with the A. suum E3 suggested that the ascarid E3 was more sensitive to NADH inhibition when bound to the bovine kidney core. The expression of p45 was developmentally regulated and p45 was most abundant in anaerobic muscle. In contrast, E3s isolated from anaerobic muscle or aerobic second-stage larvae were identical. These results suggest that during the transition to anaerobic metabolism, E3 remains unchanged, but it appears that a novel E3BP, p45, is expressed which may help to maintain the activity of the PDC in the face of the elevated intramitochondrial NADH/NAD ؉ ratios associated with anaerobiosis.The pyruvate dehydrogenase complex (PDC) 1 plays a key role in the unique mitochondrial metabolism of the parasitic nematode, Ascaris suum (1, 2). During larval development, A. suum exhibits a marked aerobic-anaerobic transition in energy metabolism. Early larval stages are aerobic, and the PDC functions to supply acetyl-CoA for tricarboxylic acid cycle oxidation (3). In contrast, energy metabolism in adult ascarid muscle is anaerobic. Adult muscle mitochondria lack both a functional tricarboxylic acid cycle and cytochrome oxidase activity and use unsaturated organic acids, such as fumarate and 2-methyl branched-chain enoyl-CoAs, instead of oxygen, as terminal electron acceptors (4 -6). The PDC is overexpressed in adult muscle and is designed to function under the elevated NADH/ NAD ϩ and acyl-CoA/CoA ratios present in the microaerobic environment of the host gut (7,8).The subunit composition of the adult ascarid muscle PDC differs significantly from the PDCs isolated from other eukaryotic organisms (7, 9, 10). In both yeast and mammalian PDCs, incubation of the complex with [2-14 C]pyruvate in the absence of CoA results in the acetylation of two proteins, dihydrolipoyl transacetylase (E2) and dihydrolipoyl dehydrogenase (E3)-binding protein (BP) (11, 12). E2 and E3BP are present in a ratio of about 5:1, and both contain similar multidomain structures consisting of an amino-terminal lipoyl domain, a subunit binding domain, and an inner domain (13-15). E2 preferentially binds pyruvate dehydrogenase (E1) and exhibits transacetylase activity, whereas E3BP contains a high-affinity binding site for E3 (16). In contrast, ...

show abstract

Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains

Cited by 8 publications

References 34 publications

Secretion of a Novel, Developmentally Regulated Fatty Acid-binding Protein into the Perivitelline Fluid of the Parasitic Nematode, Ascaris suum

Secretion of a Novel, Developmentally Regulated Fatty Acid-binding Protein into the Perivitelline Fluid of the Parasitic Nematode, Ascaris suum

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Identification of a Novel Dihydrolipoyl Dehydrogenase-binding Protein in the Pyruvate Dehydrogenase Complex of the Anaerobic Parasitic Nematode, Ascaris suum

Contact Info

Product

Resources

About