Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded "echA12_2" in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2-7months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.

Enoyl-coenzyme A hydratase and antigen 85B of Mycobacterium habana are specifically recognized by antibodies in sera from leprosy patients.

Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico.

[Preparation and identification of monoclonal antibody against enoyl-CoA hydratase 1].

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against enoyl-CoA hydratase 1 (ECH1). METHODS: Normal human liver tissues were homogenized, and the mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine hybridoma technique. The mAbs were characterized by ELISA, Western blotting and immunohistochemistry. The specificity of the antibody was identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening. RESULTS: One clone of the hybridoma BGB095 secreting specific mAb against ECH1 was obtained. The mAb was identified to belong to Ig subclass IgG1 and could be used in ELISA, Western blotting, immunohistochemistry, and immunoprecipitation. CONCLUSION: A hybridoma cell line stably secreting specific mAb against ECH1 has been established. The specific mAb against ECH1 can be of great value for functional and distribution studies of ECH1.

A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency.

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.

Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli.

Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains. CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays. The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds. In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine. The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB. When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).

Different recognition by peroxisome proliferator structures in rat peroxisomal induction: application of sandwich ELISA using monoclonal antibody against rat peroxisomes.

A novel assay for a peroxisomal beta-oxidation enzyme by sandwich ELISA using a monoclonal antibody (RPX-5) against purified rat liver peroxisomes was developed. Immunoblot analysis revealed that RPX-5 recognized a 78 Kd protein, which is a peroxisomal bifunctional enzyme (PBE) in the beta-oxidation pathway. Immunoprecipitation by RPX-5 and the resulting reduction of PBE activity were dependent on RPX-5 concentrations. Sandwich ELISA using RPX-5 could be used to assay PBE in the range of 30 to 2000 ng protein/ml. In rat hepatocyte cultures, the PBE amount by this assay correlated well with PBE activity, with correlation coefficients of 0.965. Studying the mechanisms of peroxisomal induction, patterns of peroxisomal induction were examined by co-treatment of rat hepatocytes with various peroxisome proliferators (PxPs). Treatment with clofibrate and bezafibrate resulted in neither an additive nor synergistic effect on PBE level. On the other hand, co-treatment with either bezafibrate-Wy-14,643 or clofibrate-MEHP(mono(2-ethylhexyl)phthalate) both resulted in an additive effect. From these results, it is suggested that PxPs of the fibrate group may exert their functions via a common process, and non-fibrate PxPs via a different process in hepatocytes. The cognition site for peroxisome proliferators, therefore, might not involve a single site for inducing peroxisomal enzymes.

Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

Biochemical and immunological identity of the hepatic peroxisomal and microsomal trans-2-enoyl CoA hydratase bifunctional protein.

In the present study, the hepatic microsomal and peroxisomal bifunctional trans-2-enoyl CoA hydratases were isolated and purified from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These two enzymes (microsomal and peroxisomal) were purified with the identical purification procedures and had identical molecular masses of 76 kDa. A single band was observed on an electrophoretic gel of an equimixture of the two proteins. Both preparations had identical pI's of 8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5 for the hydratase activity. Two-dimensional gel analysis of an equimixture of the two preparations showed only one band. Ouchterlony double-diffusion analysis showed that an antibody raised against the purified microsomal enzyme interacted at a point with the peroxisomal enzyme, indicating immunologic identity. Western blot analysis demonstrated that the antibody formed a single band with total microsomal and peroxisomal fractions. The antibody inhibited the enzymatic activities of both preparations in a similar manner. Interestingly, the antibody had a markedly greater inhibitory effect on the reductase activity of the two enzyme preparations, and a much less inhibitory effect on the hydratase activity, suggesting that the antigenic determinants reside at or near the catalytic site of the reductase portion of the protein. These results suggest that the microsomal and peroxisomal bifunctional proteins are identical.

Induction, identification, and cell-free translation of mRNAs coding for peroxisomal proteins in Candida tropicalis.

Peroxisomes have been purified from Candida tropicalis grown on oleic acid and shown to be nearly pure by marker enzyme analysis, electron microscopy, and comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They contain approximately 20 polypeptides, among which acyl-CoA oxidase, trifunctional hydratase-dehydrogenase-epimerase, and catalase have been identified. Rabbit antisera have been elicited that react with these three proteins. When C. tropicalis is grown on alkanes, a dozen mRNAs are strikingly induced. Nine of the 12 induced mRNAs code for polypeptides that comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with peroxisomal proteins, among which three have been identified immunochemically as the acyl-CoA oxidase, the trifunctional protein, and catalase. These results indicate that some genes coding for peroxisomal proteins are strongly expressed during growth of C. tropicalis on alkanes. The data are consistent with evidence in other species that peroxisomes form by the post-translational incorporation of newly made proteins into pre-existing peroxisomes, generally without proteolytic processing, followed by peroxisome division.