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Robert E Mackenzie - One of the best experts on this subject based on the ideXlab platform.
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the molecular basis of glutamate Formiminotransferase deficiency
Human Mutation, 2003Co-Authors: John Hilton, Karen E Christensen, David Watkins, Benjamin A Raby, Yannick Renaud, Susanna De La Luna, Xavier Estivill, Robert E MackenzieAbstract:Glutamate Formiminotransferase deficiency, an autosomal recessive disorder and the second most common inborn error of folate metabolism, is presumed to be due to defects in the bifunctional enzyme glutamate Formiminotransferase-Cyclodeaminase (FTCD). Features of a severe phenotype, first identified in patients of Japanese descent, include elevated levels of formiminoglutamate (FIGLU) in the urine in response to histidine administration, megaloblastic anemia, and mental retardation. Features of a mild phenotype include high urinary excretion of FIGLU in the absence of histidine administration, mild developmental delay, and no hematological abnormalities. We found mutations in the human FTCD gene in three patients with putative glutamate Formiminotransferase deficiency. Two siblings were heterozygous for missense mutations, c.457C>T (R135C) and c.940G>C (R299P). Mutagenesis of porcine FTCD and expression in E. coli showed that the R135C mutation reduced Formiminotransferase activity to 61% of wild-type, whereas the R299P mutation reduced this activity to 57% of wild-type. The third patient was hemizygous for c.1033insG, with quantitative PCR indicating that the other allele contained a deletion. These mutations are the first identified in glutamate Formiminotransferase deficiency and demonstrate that mutations in FTCD represent the molecular basis for the mild phenotype of this disease. Hum Mutat 22:67–73, 2003. © 2003 Wiley-Liss, Inc.
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the crystal structure of the Formiminotransferase domain of Formiminotransferase Cyclodeaminase implications for substrate channeling in a bifunctional enzyme
Structure, 2000Co-Authors: Darcy Kohls, Robert E Mackenzie, Traian Sulea, Enrico O Purisima, Alice VrielinkAbstract:Abstract Background: The bifunctional enzyme Formiminotransferase-Cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a γ-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the Cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. Results : The crystal structure of the Formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel α/β fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6 R )-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. Conclusions : The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the Formiminotransferase mechanism.
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crystallization and preliminary x ray analysis of the Formiminotransferase domain from the bifunctional enzyme Formiminotransferase Cyclodeaminase
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Darcy Kohls, Robert E Mackenzie, Nathalie Croteau, Narciso R Mejia, Alice VrielinkAbstract:Formiminotransferase–Cyclodeaminase (E.C. 2.1.2.5–E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The Formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.
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monofunctional domains of Formiminotransferase Cyclodeaminase retain similar conformational stabilities outside the bifunctional octamer
Biochimica et Biophysica Acta, 1997Co-Authors: Laura Lea Murley, Robert E MackenzieAbstract:Each identical subunit of octameric Formiminotransferase Cyclodeaminase consists of a transferase and a deaminase domain connected by a short linker sequence. Both domains can be independently expressed in Escherichia coli as monofunctional dimers and show no indication of associating, suggesting that the linker mediates the only substantial interaction between the transferase and deaminase domains. To better understand the benefits arising from octamer formation, we have used equilibrium unfolding methods to examine the properties of the transferase and deaminase domains independently and within the octamer. Each isolated dimeric domain undergoes an apparent change in tertiary structure at low concentrations of urea (<2 mol/l) which results in the concurrent loss of intrinsic fluorescence and catalytic activity. The full length octameric enzyme also undergoes inactivation and a loss of intrinsic fluorescence over this concentration range, without apparent change in secondary or quaternary structure. Between 2 and 2.5 M urea the isolated transferase and deaminase domains dissociate to monomers. However, only one of the subunit interfaces in the octamer is disrupted at this urea concentration and dissociation of the second interface occurs between 3.5 and 5 M urea. While each domain shows similar stability to denaturation within and outside of the octamer, one type of subunit interface achieves increased stability within the full length enzyme.
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the two monofunctional domains of octameric Formiminotransferase Cyclodeaminase exist as dimers
Biochemistry, 1995Co-Authors: Laura Lea Murley, Robert E MackenzieAbstract:Formiminotransferase-Cyclodeaminase is a bifunctional enzyme arranged as a circular tetramer of dimers that exhibits the ability to efficiently channel polyglutamylated folate between catalytic sites. Through deletion mutagenesis we demonstrate that each subunit consists of an N-terminal transferase active domain and a C-terminal deaminase active domain separated by a linker sequence of minimally eight residues. The full-length enzyme and both isolated domains have been expressed as C-terminally histidine-tagged proteins. Both domains self-dimerize, providing direct evidence for the existence of two types of subunit interfaces. The results suggest that both the transferase and the deaminase activities are dependent on the formation of specific subunit interfaces. Because channeling is not observed between isolated domains, only the octamer appears able to directly transfer pentaglutamylated intermediate between active sites.
Fernando Alvarez - One of the best experts on this subject based on the ideXlab platform.
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characterization of the antigenicity of the Formiminotransferase Cyclodeaminase in type 2 autoimmune hepatitis
Experimental Cell Research, 2004Co-Authors: Reginald Renous, Pascal Lapierre, Idriss Djilalisaiah, Susana Vitozzi, Fernando AlvarezAbstract:Human Formiminotransferase-Cyclodeaminase (hFTCD) is the autoantigen recognized by anti-liver cytosol type 1 (LC1) autoantibodies in type 2 autoimmune hepatitis (AIH) patients. In rats, this octameric protein is localized on the Golgi apparatus and binds brain microtubules (MTs) and vimentin. Subcellular localization of human Formiminotransferase-Cyclodeaminase and its implication in the pathogenesis of autoimmune hepatitis are unknown. Localization of the human Formiminotransferase-Cyclodeaminase in human hepatocytes was done using indirect immunofluorescence and subcellular fractionations followed by in vitro binding techniques. The Formiminotransferase-Cyclodeaminase antigen at two distinct locations in hepatocytes, free in the cytosol and associated with the Golgi membranes are recognized by anti-liver cytosol type 1 autoantibodies. The human Formiminotransferase-Cyclodeaminase binds reversibly to the Golgi membranes and this complex formation is increased by anti-liver cytosol type 1 autoantibodies. Finally, human Formiminotransferase-Cyclodeaminase does not interact with liver-specific cytoskeleton proteins. Anti-liver cytosol type 1 autoantibodies are directed against the mature high molecular form of human Formiminotransferase-Cyclodeaminase. Therefore, the subcellular location of the protein may influence the production of autoantibodies and their role in the pathogenesis of type 2 autoimmune hepatitis. This antigen-driven response does not appear to be facilitated or enhanced by a possible interaction between human Formiminotransferase-Cyclodeaminase and hepatocyte cytoskeleton proteins.
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characterization of the b cell response of patients with anti liver cytosol autoantibodies in type 2 autoimmune hepatitis
European Journal of Immunology, 2003Co-Authors: Pascal Lapierre, Catherine Johanet, Fernando AlvarezAbstract:Anti-liver cytosol type 1 (LC1) autoantibody is detected in 30% of sera from patients with type 2 autoimmune hepatitis (AIH), and is the only circulating autoantibody in 10% of cases. Human Formiminotransferase Cyclodeaminase (FTCD) has been shown to be the specific liver antigen recognized by anti-LC1 autoantibodies. The aim of this study was to identify the dominant epitope on human FTCD and to analyze antigenic-site sequences for clues on the development of AIH. Recombinant proteins and peptides covering the entire cDNA of human FTCD were tested against anti-LC1 autoantibodies. Conformational epitopes were found throughout the protein but linear epitopes were found exclusively in the C-terminal 146 amino acids. Two groups of sera with different reactivities were found: 69%of the sera recognized two specific linear epitopes at positions 428–434 (NTPEEKD) and 440–447 (LQEGLRRA) of human FTCD; others reacted only with a discontinuous epitope between the amino acids at position 395 and 528. FTCD autoantibody production is thus a polyclonal-antigen-driven B cell response. Autoantibodies against conformational or discontinuous epitopes were found in all patients and two-thirds also recognized linear epitopes on human FTCD.
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Formiminotransferase Cyclodeaminase is an organ specific autoantigen recognized by sera of patients with autoimmune hepatitis
Gastroenterology, 1999Co-Authors: Pascal Lapierre, Oumnia Hajoui, Jeanclaude Homberg, Fernando AlvarezAbstract:Abstract Background & Aims: Anti–liver cytosol type 1 autoantibodies have been reported in association with anti–liver-kidney microsome type 1 autoantibodies in 30% of patients with autoimmune hepatitis type II. In 10% of cases, anti–liver cytosol type 1 antibodies are the only liver-related circulating autoantibodies. The liver cytosol antigen is a liver-specific 62-kilodalton protein present in the cell as an oligomer of ~240 kilodaltons. The aim of this study was to identify the antigen recognized by anti–liver cytosol antibody. Methods: To identify the liver cytosol antigen, an anti–liver cytosol type 1–positive serum was used for the screening of a complementary DNA library from HepG2 cells. Double immunodiffusion method was used to show the identity between the cytosolic and the cloned protein. Results: The sequence of two isolated clones showed 85.2% homology with the Formiminotransferase Cyclodeaminase (FTCD) enzyme from pig liver. Antibodies purified by affinity with the recombinant protein and sera from mice immunized with FTCD recognized a 62-kilodalton human cytosolic protein when tested by immunoblot. The identity of precipitation lines was found between the cytosolic antigen and FTCD. Conclusions: This enzyme is a liver-specific antigen recognized by the sera of patients with autoimmune hepatitis. GASTROENTEROLOGY 1999;116:643-649
Alice Vrielink - One of the best experts on this subject based on the ideXlab platform.
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the crystal structure of the Formiminotransferase domain of Formiminotransferase Cyclodeaminase implications for substrate channeling in a bifunctional enzyme
Structure, 2000Co-Authors: Darcy Kohls, Robert E Mackenzie, Traian Sulea, Enrico O Purisima, Alice VrielinkAbstract:Abstract Background: The bifunctional enzyme Formiminotransferase-Cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a γ-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the Cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. Results : The crystal structure of the Formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel α/β fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6 R )-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. Conclusions : The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the Formiminotransferase mechanism.
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crystallization and preliminary x ray analysis of the Formiminotransferase domain from the bifunctional enzyme Formiminotransferase Cyclodeaminase
Acta Crystallographica Section D-biological Crystallography, 1999Co-Authors: Darcy Kohls, Robert E Mackenzie, Nathalie Croteau, Narciso R Mejia, Alice VrielinkAbstract:Formiminotransferase–Cyclodeaminase (E.C. 2.1.2.5–E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The Formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.
Laura Lea Murley - One of the best experts on this subject based on the ideXlab platform.
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a Formiminotransferase Cyclodeaminase isoform is localized to the golgi complex and can mediate interaction of trans golgi network derived vesicles with microtubules
Journal of Biological Chemistry, 1998Co-Authors: Dagmar Hennig, Laura Lea Murley, Suzie J Scales, Anne Moreau, Jan R De Mey, Thomas E KreisAbstract:A protein of 60 kDa (p60) has been identified using a quantitative in vitro vesicle-microtubule binding assay. Purified p60 induces co-sedimentation with microtubules of trans-Golgi network-derived vesicles isolated from polarized, perforated Madin-Darby canine kidney cells. Sequencing of the cDNA coding for this protein revealed that it is the chicken homologue of Formiminotransferase Cyclodeaminase (FTCD), a liver-specific enzyme involved in the histidine degradation pathway. Purified p60 from chicken liver has Formiminotransferase activity, confirming that it is FTCD or an isoform of this enzyme. Isoforms of FTCD were identified in chicken hepatoma and HeLa cells, and immunolocalize to the region of the Golgi complex and vesicular structures in its vicinity. Furthermore, 58K, a previously identified microtubule-binding Golgi protein from rat liver (Bloom, G. S., and Brashear, T. A. (1989) J. Biol. Chem. 264, 16083–16092), is identical to FTCD. Both proteins co-purify with microtubules and co-localize with membranes of the Golgi complex. The capacity of FTCD to bind both to microtubules and Golgi-derived membranes may suggest that this protein, or one of its isoforms, might have in addition to its enzymatic activity, a second physiological function in mediating interaction of Golgi-derived membranes with microtubules.
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monofunctional domains of Formiminotransferase Cyclodeaminase retain similar conformational stabilities outside the bifunctional octamer
Biochimica et Biophysica Acta, 1997Co-Authors: Laura Lea Murley, Robert E MackenzieAbstract:Each identical subunit of octameric Formiminotransferase Cyclodeaminase consists of a transferase and a deaminase domain connected by a short linker sequence. Both domains can be independently expressed in Escherichia coli as monofunctional dimers and show no indication of associating, suggesting that the linker mediates the only substantial interaction between the transferase and deaminase domains. To better understand the benefits arising from octamer formation, we have used equilibrium unfolding methods to examine the properties of the transferase and deaminase domains independently and within the octamer. Each isolated dimeric domain undergoes an apparent change in tertiary structure at low concentrations of urea (<2 mol/l) which results in the concurrent loss of intrinsic fluorescence and catalytic activity. The full length octameric enzyme also undergoes inactivation and a loss of intrinsic fluorescence over this concentration range, without apparent change in secondary or quaternary structure. Between 2 and 2.5 M urea the isolated transferase and deaminase domains dissociate to monomers. However, only one of the subunit interfaces in the octamer is disrupted at this urea concentration and dissociation of the second interface occurs between 3.5 and 5 M urea. While each domain shows similar stability to denaturation within and outside of the octamer, one type of subunit interface achieves increased stability within the full length enzyme.
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the two monofunctional domains of octameric Formiminotransferase Cyclodeaminase exist as dimers
Biochemistry, 1995Co-Authors: Laura Lea Murley, Robert E MackenzieAbstract:Formiminotransferase-Cyclodeaminase is a bifunctional enzyme arranged as a circular tetramer of dimers that exhibits the ability to efficiently channel polyglutamylated folate between catalytic sites. Through deletion mutagenesis we demonstrate that each subunit consists of an N-terminal transferase active domain and a C-terminal deaminase active domain separated by a linker sequence of minimally eight residues. The full-length enzyme and both isolated domains have been expressed as C-terminally histidine-tagged proteins. Both domains self-dimerize, providing direct evidence for the existence of two types of subunit interfaces. The results suggest that both the transferase and the deaminase activities are dependent on the formation of specific subunit interfaces. Because channeling is not observed between isolated domains, only the octamer appears able to directly transfer pentaglutamylated intermediate between active sites.
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the nucleotide sequence of porcine Formiminotransferase Cyclodeaminase expression and purification from escherichia coli
Journal of Biological Chemistry, 1993Co-Authors: Laura Lea Murley, Narciso R Mejia, Robert E MackenzieAbstract:We have isolated and characterized cDNA clones encoding the porcine liver octameric enzyme, 5-formiminotetrahydrofolate:L-glutamate N-Formiminotransferase (EC 2.1.2.5)-formiminotetrahydrofolate Cyclodeaminase (EC 4.3.1.4). The cDNA encodes a novel amino acid sequence of 541 residues which contains exact matches to two sequences derived by automated sequence analysis of CNBr cleavage fragments isolated from the porcine enzyme. The recombinant enzyme has been expressed as a soluble protein in Escherichia coli at levels 4-fold higher than those observed in liver, and is bifunctional, displaying both transferase and deaminase activities. With a calculated subunit molecular mass of 58,926 Da, it is similar in size to the enzyme isolated from porcine liver. Purification of the enzyme from E. coli involves chromatography on a novel polyglutamate column which might interact with the folylpolyglutamate binding site of the protein. The purified recombinant enzyme has a transferase specific activity of 39-41 units/mg/min.
Michael P Manns - One of the best experts on this subject based on the ideXlab platform.
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the influence of genetic predisposition and autoimmune hepatitis inducing antigens in disease development
Journal of Autoimmunity, 2017Co-Authors: Matthias Hardtkewolenski, Janine Dywicki, Katja Fischer, Martin Hapke, Maren Sievers, Jerome Schlue, Mark S Anderson, Richard Taubert, Fatih Noyan, Michael P MannsAbstract:Abstract Autoimmune hepatitis (AIH) is defined as a chronic liver inflammation with loss of tolerance against hepatocytes. The etiology and pathophysiology of AIH are still poorly understood because reliable animal models are limited. Therefore, we recently introduced a model of experimental murine AIH by a self-limited adenoviral infection with the AIH type 2 antigen Formiminotransferase Cyclodeaminase (FTCD). We could demonstrate that break of humoral tolerance towards liver specific autoantigens like FTCD and cytochrome P450 2D6 (CYP2D6) is not dependent on the genetic background. However, the development of AIH in autoantibody positive animals is determined by genetic background genes. We could also show that the break of humoral tolerance is necessary but not sufficient for the development of AIH. In contrast the break of tolerance against the ubiquitously expressed nuclear antigens (ANAs) is strictly dependent on genetic predisposition. Priming with the UGA suppressor tRNA-associated protein (soluble liver antigen; SLA) is a strong inducer of ANA reactivity, but not sufficient to cause AIH development thereby questioning the importance of anti-SLA immune response as an important driver in AIH. Monogenetic mutations such as Aire-deficiency can cause AIH in otherwise genetically resistant strains. Conclusion The results have important implications for our understanding of the pathophysiology of AIH development and for the interpretation of humoral antibody responses in AIH.
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advances in the diagnosis pathogenesis and management of autoimmune hepatitis
Gastroenterology, 2010Co-Authors: Albert J Czaja, Michael P MannsAbstract:Autoimmune hepatitis (AIH) is characterized by chronic inflammation of the liver, interface hepatitis (based on histologic examination), hypergammaglobulinemia, and production of autoantibodies. Many clinical and basic science studies have provided important insights into the pathogenesis and treatment of AIH. Transgenic mice that express human antigens and develop autoantibodies, liver-infiltrating CD4 + T cells, liver inflammation, and fibrosis have been developed as models of AIH. AIH has been associated with autoantibodies against members of the cytochrome P450 superfamily of enzymes, transfer RNA selenocysteine synthase, Formiminotransferase Cyclodeaminase, and the uridine diphosphate glucuronosyltransferases, whereas alleles such as DRB1*0301 and DRB1*0401 are genetic risk factors in white North American and northern European populations. Deficiencies in the number and function of CD4 + CD25 + (regulatory) T cells disrupt immune homeostasis and might be corrected as a therapeutic strategy. Treatment can be improved by continuing corticosteroid therapy until normal liver test results and normal liver tissue are within normal limits, instituting ancillary therapies to prevent drug-related side effects, identifying problematic patients early, and providing long-term maintenance therapy after patients experience a first relapse. Calcineurin inhibitors and mycophenolate mofetil are potential salvage therapies, and reagents such as recombinant interleukin-10, abatacept, and CD3-specific antibodies are feasible as therapeutics. Liver transplantation is an effective salvage therapy, even in the elderly, and AIH must be considered in all patients with graft dysfunction after liver transplantation. Identification of the key defects in immune homeostasis and antigen targets will direct new therapies.
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58 liver cytosol antigen type 1 autoantibodies liver kidney microsomal autoantibodies and liver microsomal autoantibodies
Autoantibodies (Second Edition), 2007Co-Authors: C P Strassburg, Michael P MannsAbstract:Reactivity against targets of the endoplasmic reticulum designated liver kidney microsomal autoantibodies (LKM) was first detected by Rizzetto in 1973. Since then a considerable body of analyses has led to the subclassification of these autoantibodies into LKM1, LKM2, LKM3, liver microsomal (LM), as well as to the identification of liver cytosolic (LC-1) autoantibodies. Disease associations are remarkably heterogeneous and include the detection of LKM/LM autoantibodies in autoimmune hepatitis, viral hepatitis C and D, the genetically determined autoimmune polyglandular syndrome type 1 (APECED), as well as drug reactions with anticonvulsants, dihydralazine and ticrynafen. Anti-microsomal reactivity converges on two groups of drug metabolizing enzymes resident in the membranes of the endoplasmatic reticulum: cytochrome P450s and UDP-glucuronosyltransferases (UGTs). Present day diagnostic methods as well as the still incomplete understanding of the mechanisms involved in autoimmunity result from the analysis of recombinant target proteins and their epitope recognition patterns. In clinical practice the detection of LKM/LM immunofluorescence patterns requires the application of specific tests which can contribute to achieving a scientifically founded subclassification of autoantibodies and thereby their association with autoimmune disease or simple serological autoimmunity. LC-1 autoanbodies in contrast are reactive with cytosolic proteins of the liver. Initial screening by indirect immunofluorescence is not sensitive and therefore requires immunodiffusion analysis or the demonstration of reactivity with Formiminotransferase Cyclodeaminase. Microsomal autoantibodies provide one of the most interesting models to study autoimmunity since they span mechanisms of drug metabolism-induced immunology, virus-induced autoimmunity, mimicry and genetic predisposition, all converging on a focused array of target proteins.