Developmental and tissue-specific expression of 2-methyl branched-chain enoyl CoA reductase isoforms in the parasitic nematode, Ascaris suum.

The 2-methyl branched-chain enoyl CoA reductase (ECR) plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode, Ascaris suum. Two-dimensional gel electrophoresis of the purified ECR yielded multiple spots, with two distinct but overlapping N-terminal sequences. These multiple isoforms were not the result of population effects, as the pattern observed on 2-D gels of the purified ECR was identical to those on immunoblots of muscle homogenates isolated from individual worms. A full-length cDNA coding for the major ECR isoform (ECRI) has been cloned and sequenced and compared with that of the minor isoform (ECRII) which has been described previously (Duran et al. J Biol Chem 1993;268:22391-22396). ECRI contained the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, a 5' untranslated region (UTR) of 13 nucleotides, an open reading frame (ORF) of 1257 nucleotides, a 3'-UTR of 110 nucleotides that included the polyadenylation signal AATAAA downstream of the termination codon and a short poly(A) tail. The ORF predicted a 16 amino acid leader sequence not found in the native protein and a mature protein of 403 amino acids with a molecular weight of 43 698 and a predicted pI of 6.2. ECRI and ECRII were 73% identical at the predicted amino acid level and their mRNAs exhibited significant structural similarity even though they were products of separate genes. Comparison of ECRI and ECRII with the sequences of acyl CoA dehydrogenases from a variety of different sources revealed a high degree of interspecies sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene. This result is surprising since the ascarid enzymes function as reductases, not as dehydrogenases. Both ECRs were tissue-specific and developmentally regulated and were found in transitional third-stage larvae (L3) and adult muscle, but not in early, aerobic larval stages or adult testis, ovary, or intestine. The ratio of ECRII to ECRI was greater in L3 than in adult muscle. Interestingly, both ECRs also appeared to be expressed in pharyngeal muscle, suggesting that branched-chain fatty acid synthesis may not be confined exclusively to body wall muscle.

Pyruvate dehydrogenase complexes from the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum, helminths exhibiting anaerobic mitochondrial metabolism.

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from 2 parasitic helminths exhibiting anaerobic mitochondrial metabolism, the equine nematode, Parascaris equorum, and the canine cestode, Dipylidium caninum. The P. equorum PDC yielded 7 major bands when separated by SDS-PAGE. The bands of 72, 55-53.5, 41 and 36 kDa corresponded to E2, E3, E1 alpha and E1 beta, respectively. The complex also contained additional unidentified proteins of 43 and 45 kDa. Incubation of the complex with [2-14C]pyruvate resulted in the acetylation of only E2. These results suggest that the P. equorum PDC lacks protein X and exhibits an altered subunit composition, as has been described previously for the PDC of the related nematode, Ascaris suum. In contrast, the D. caninum PDC yielded only four major bands after SDS-PAGE of 59, 58, 39 and 34 kDa, which corresponded to E3, E2, E1 alpha and E1 beta, respectively. Incubation of the D. caninum complex with [2-14C]pyruvate resulted in the acetylation of E2 and a second protein which comigrated with E3, suggesting that the D. caninum complex contained protein X and had a subunit composition similar to PDCs from other eukaryotic organisms. Both helminth complexes appeared less sensitive to inhibition by elevated NADH/NAD+ ratios than complexes isolated from aerobic organisms, as would be predicted for PDCs from organisms exploiting microaerobic habitats. These results suggest that although these helminths have similar anaerobic mitochondrial pathways, they contain significantly different PDCs.

Characterization of cDNA clones for the 2-methyl branched-chain enoyl-CoA reductase. An enzyme involved in branched-chain fatty acid synthesis in anaerobic mitochondria of the parasitic nematode Ascaris suum.

The 2-methyl branched-chain enoyl-CoA reductase plays a pivotal role in the reversal of beta-oxidation operating in anaerobic mitochondria of the parasitic nematode Ascaris suum. An affinity-purified polyclonal anti-serum against the reductase was used to screen a cDNA library constructed in lambda gt11 with poly(A)+ RNA from adult A. suum muscle. A 1.2-kilobase partial cDNA clone was isolated, subcloned, and sequenced in both directions. Additional sequence at the 5' end of the mRNA was determined by the RACE (rapid amplification of cDNA ends) procedure. Nucleotide sequence analysis of the cDNAs revealed the 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1236 nucleotides and a 3'-untranslated sequence of 109 nucleotides including a short poly(A) tail 14 nucleotides from a polyadenylation signal (AATAAA). The open reading frame encoded a 396-amino acid sequence (M(r) 43,046) including a 16-amino acid leader peptide. Two-dimensional gel electrophoresis of the purified reductase yielded multiple spots with two distinct but overlapping amino-terminal amino acid sequences. Both sequences overlapped with the sequence predicted from the mRNA, and one of the sequences was identical to the predicted sequence. Comparison of the ascarid sequence with that of mammalian acyl-CoA dehydrogenases revealed a high degree of sequence identity, suggesting that these enzymes may have evolved from a common ancestral gene even though the ascarid enzyme functions as a reductase, not as a dehydrogenase. Immunoblotting of A. suum larval stages and adult tissues with antisera that cross-reacted with each of the spots separated on two-dimensional gels suggested that the reductase was only found in adult muscle. Northern blotting using the partial cDNA revealed a hybridization band of about 1.5 kilobases and also suggested that the enzyme was tissue-specific and developmentally regulated in agreement with the results of the immunoblotting.

Enzymes of galactose utilization in the rat tapeworm, Hymenolepis diminuta.

1. Crude enzyme preparations from Hymenolepis diminuta contained galactokinase, galactose 1-phosphate uridyl transferase and UDPgalactose 4-epimerase activity, although their specific activities were low. 2. Galactose 1-phosphate non-competitively inhibited galactose phosphorylation. This inhibition, together with the low specific activities of the enzymes in the pathway of galactose utilization, probably accounts for the inadequacy of galactose as a main nutritive carbohydrate for development of the worm.