Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain alpha-ketoacid dehydrogenase complex

Antimitochondrial antibodies (AMA) recognizing the acetyltransferase (E2) of the pyruvate dehydrogenase (PDH) complex have been previously well-documented and the immunodominant epitope mapped. In this study, we demonstrate that sera from patients with primary biliary cirrhosis (PBC) react with another lipoic acid containing acyltransferase enzyme, namely the E2 of the branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Indeed, 85/120 (71%) sera from patients with PBC reacted with BCKD-E2 by immunoblotting against purified BCKD complex. In contrast, sera from patients with chronic active hepatitis or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with any component enzymes of the BCKD complex. More importantly, BCKD enzyme activity was inhibited after incubation of the BCKD complex with either PBC sera against BCKD-E2 or with affinity purified antisera to BCKD-E2. Enzyme activity was unaltered by control sera or with PBC sera that reacted with PDH-E2 but not BCKD-E2. Furthermore, immunoblots of purified mitochondria probed with PBC sera absorbed with BCKD-E2 demonstrated that BCKD-E2 and PDH-E2 are each recognized by distinct AMA populations which do not cross-react. In addition, affinity purified PBC sera against BCKD-E2 did not react with PDH-E2 nor inhibit PDH enzyme activity, thus providing further evidence that BCKD-E2 and PDH-E2 are recognized by separate AMA. These data further suggest that the BCKD-E2 epitope recognized by AMA contains, or is close to, a functional domain of this enzyme. The availability of cDNA clones encoding BCKD-E2 and PDH-E2 will allow the study of how key metabolic enzymes may be involved in the immunology and pathology of PBC.     

Comparative epitope mapping of murine monoclonal and human autoantibodies to human PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis.

Immunization with recombinant human pyruvate dehydrogenase (PDH)-E2, the major autoantigen of primary biliary cirrhosis, readily induces a vigorous murine antibody response but does not generate hepatic disease. To determine the fine specificity of this response, 18 mAb were generated from three strains of mice and the reactive epitopes mapped. An initial examination of mAb suggested that they behaved similarly to the antimitochondrial autoantibodies in primary biliary cirrhosis (PBC) because i) all polyclonal antisera and 2 of 18 mAb reacted with all species of mammalian PDH-E2 examined including mouse PDH-E2, ii) 15 of 18 mAb inhibited PDH enzyme function, and iii) the reactivity of mAb toward rPDH-E2 were blocked by PBC sera. However, fine examination of the reactive sequences of the PDH-E2 complex revealed that antibodies identical to those in PBC patients were not produced by experimental immunization. In contrast to PBC, none of the mAb or murine polyclonal sera were able to react with protein X, a lipoic acid-containing component of the PDH complex previously shown to cross-react with PDH-E2 when probed with PBC sera. Although the epitopes for 12 mAb were localized within the inner lipoyl domain, none reacted with mouse PDH-E2 or cross-reacted with the outer lipoyl domain as observed in PBC. In addition, the epitopes of the two mAb which did react with all mammalian species of mitochondria were not localized within the PBC epitope. These findings indicate the highly immunogenic nature of the inner lipoyl domain of PDH-E2. The inability to elicit antibodies of the same specificity in mice, considered together with the highly localized autoantibody response in humans, suggests that antimitochondrial autoantibodies are most likely the result of specific breakdown of tolerance to a unique autoepitope.     

Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope.

The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC). Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site. Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites. By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region. The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area. By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition. Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized. In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag. The possible implications for such results are discussed.     

Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease that includes the presence of lymphoid infiltrates in portal tracts, high titer autoantibodies against pyruvate dehydrogenase-E2 (PDH-E2) and branched chain ketoacid dehydrogenase-E2 (BCKD-E2), and biliary tract destruction. The mechanism by which the autoimmune response is induced, the specificity of damage to the biliary epithelium, and the role of T cells in PBC are still unknown. To address these issues, we have taken advantage of a mouse mAb, coined C355.1, and studied its reactivity against a panel of liver tissue from normal subjects as well as a panel of liver specimens from patients with PBC, progressive sclerosing cholangitis, and chronic active hepatitis (CAH). C355.1, much like human autoantibodies to PDH-E2, reacts exclusively by immunoblotting with PDH-E2, binds to the inner lipoyl domain of the protein, and inhibits PDH-E2 activity in vitro. In addition, we have also attempted to develop cloned T cell lines that react with PDH-E2 and/or BCKD-E2 using liver biopsies from patients with PBC, compared with CAH. Although monoclonal C355.1 produced typical mitochondrial fluorescence on sections of normal liver, pancreas, lung, heart, thyroid, and kidney, it produced a distinct and intense reactivity when used to stain the bile ducts of patients with PBC. Nine of 13 PBC liver biopsies studied herein contained bile ducts on light microscopy, all of which reacted intensely at a 1:100 culture supernatant dilution of monoclonal C355.1. In contrast, although bile ducts of liver specimens from normals, CAH, and progressive sclerosing cholangitis also reacted with C355.1, such reactivity was exclusively mitochondrial and readily detectable only at a dilution of 1:2. More importantly, we generated CD4+, CD8-, alpha beta TCR+ cloned T cell lines from patients with PBC, but not from CAH, that produced IL-2 specifically in response to PDH-E2 or BCKD-E2.     

Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos

We describe a new strategy for producing tissue-specific cDNA libraries and subsequently identifying tissue-specific clones. This method was used to screen for cDNA clones corresponding to RNAs expressed in the Drosophila head that cannot be detected in the early embryo. RNA blots were used to assess the spatial and temporal patterns of expression of these RNAs. The ensemble of 436 head-not-embryo clones identified roughly 700 distinct RNAs that are differentially expressed in the Drosophila head. The RNA expression patterns can be classified into five major categories. it is argued that this ensemble of clones represents a large fraction of all genes differentially expressed in the adult head, but not detected in the early embryo. Many of these genes are likely to encode eye- and nervous system-specific products.     

Regulation of PDH in human arm and leg muscles at rest and during intense exercise

To test the hypothesis that pyruvate dehydrogenase (PDH) is differentially regulated in specific human muscles, regulation of PDH was examined in triceps, deltoid, and vastus lateralis at rest and during intense exercise. To elicit considerable glycogen use, subjects performed 30 min of exhaustive arm cycling on two occasions and leg cycling exercise on a third day. Muscle biopsies were obtained from deltoid or triceps on the arm exercise days and from vastus lateralis on the leg cycling day. Resting PDH protein content and phosphorylation on PDH-E1 alpha sites 1 and 2 were higher (P < or = 0.05) in vastus lateralis than in triceps and deltoid as was the activity of oxidative enzymes. Net muscle glycogen utilization was similar in vastus lateralis and triceps ( approximately 50%) but less in deltoid (likely reflecting less recruitment of deltoid), while muscle lactate accumulation was approximately 55% higher (P < or = 0.05) in triceps than vastus lateralis. Exercise induced (P < or = 0.05) dephosphorylation of both PDH-E1 alpha site 1 and site 2 in all three muscles, but it was more pronounced at PDH-E1 alpha site 1 in triceps than in vastus lateralis (P < or = 0.05). The increase in activity of the active form of PDH (PDHa) after 10 min of exercise was more marked in vastus lateralis ( approximately 246%) than in triceps ( approximately 160%), but when it was related to total PDH-E1 alpha protein content, no difference was evident. In conclusion, PDH protein content seems to be related to metabolic enzyme profile, rather than myosin heavy chain composition, and less PDH capacity in triceps is a likely contributing factor to higher lactate accumulation in triceps than in vastus lateralis.     

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Antigen-specific CD8⁺ T cells play a key role in the host’s antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8⁺ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8⁺ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8⁺ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8⁺ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.     

Three genes for enzymes of the pyruvate dehydrogenase complex map to human chromosomes 3, 7, and X.

The genes for three proteins of the pyruvate dehydrogenase (PDH) complex have been assigned to human chromosomes by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these genes. PDH-E1 alpha has been localized on human chromosome 3p13-q23. The assignments of lipoamide dehydrogenase(E3) and PDH-E1 alpha [corrected] to chromosomes 7 and Xp, respectively, have been confirmed. Restrictive-fragment-length polymorphisms have been identified with E3, which will permit further localization of this gene by genetic linkage analysis.     

Gene trap and gene inversion methods for conditional gene inactivation in the mouse

Conditional inactivation of individual genes in mice using site-specific recombinases is an extremely powerful method for determining the complex roles of mammalian genes in developmental and tissue-specific contexts, a major goal of post-genomic research. However, the process of generating mice with recombinase recognition sequences placed at specific locations within a gene, while maintaining a functional allele, is time consuming, expensive and technically challenging. We describe a system that combines gene trap and site-specific DNA inversion to generate mouse embryonic stem (ES) cell clones for the rapid production of conditional knockout mice, and the use of this system in an initial gene trap screen. Gene trapping should allow the selection of thousands of ES cell clones with defined insertions that can be used to generate conditional knockout mice, thereby providing extensive parallelism that eliminates the time-consuming steps of targeting vector construction and homologous recombination for each gene.     

The ornithine aminotransferase-encoding gene family of rat: cloning, characterization, and evolutionary relationships between a single expressed gene and three pseudogenes

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.     

Heterogeneity and tissue-specific expression of eukaryotic transcription factor S-II-related protein mRNA

Two new cDNA clones for S-II-related proteins were isolated from a mouse liver cDNA library. Analyses of these clones suggested that S-II proteins are a family of proteins with relatively conserved C-terminal regions but variable N-terminal regions, and that some members of this family are expressed in a tissue-specific manner.     

Activity of β-glucuronidase in Root Tips of Different Types of Transgenic Sugar Beet Plants

Expression of the -glucuronidase (GUS) reporter gene driven by the CaMV 35S, rolC, nos and mas promoters was assessed in the tips of 12 independent clones of transgenic sugar beet (Beta vulgaris) roots. Three questions were addressed: 1) expression pattern specific for a given promoter, 2) expression pattern variability, and 3) relationship between gene expression and cell differentiation. Characteristic patterns of tissue-specific expression were distinguished for each promoter. Striking differences, however, were found between some clones, bearing the same construct. Statistical analysis of the pattern variability proved that the variability is significantly lower within the construct than between constructs. rolC-GUS clones exhibited the lowest and CaMV 35S clones the highest pattern variability. Comparisons between the four promoters showed consistent GUS activity in areas playing a key role in tissue determination (the elongation zone) where cells switch from frequent mitosis and mostly isodiametrical growth, typical for the promeristem, to rapid elongation and differentiation. All of the clones were highly GUS-positive in the elongation zone of stele. Activity was commonly localised in the stele of the maturation zone for CaMV 35S, rolC and mas-GUS clones. CaMV 35S-GUS clones were highly active in the promeristem.     

The L-type calcium channel alpha 1C subunit gene undergoes extensive, uncoordinated alternative splicing

The alpha1C subunit is the pore-forming protein for the L-type calcium channel. Previous studies indicate that there is possible tissue-specific alternative splicing of this gene. In this study we cloned the entire open reading frame of the alpha1C subunit cDNA from adult rat cardiac myocytes in a single piece (6.64 kb). Using 75 positive clones that were identified by restriction enzyme mapping, we tested the alternative splicing patterns of the Ca(v) 1.2 gene that encodes the alpha1C subunit protein and focused on five loci: IS6, post-IS6, IIIS2, IVS3, and the c-terminus. The results indicate that: (1) alternative splicing occurs in most of the loci, giving rise to two or three different isoforms at those sites; (2) there is a predominant form for each splicing site, (3) there does not appear to be consistent coordination of splicing at multiple loci of this gene. Alternative splicing is not tissue-specific in most regions.