Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complementary DNA cloning of the 130-kD pemphigus vulgaris (PV) autoantigen (PVA) has indicated that it is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules. By homology with typical cadherins, PVA has five extracellular domains (EC1 through EC5). To localize immunogenic domains and to determine whether antibodies against them might be pathogenic, we produced beta-galactosidase fusion proteins with cDNA encoding different portions of the extracellular domains of PVA (EC1-2, EC3-5, and each individual domain). Immunoblot analysis of these fusion proteins with 23 PV patients' sera demonstrated that major immunogenic regions of PVA are located on the EC1, EC2, and EC4 domains. IgG was affinity-purified from PV sera on fusion proteins representing the amino (EC1-2) and carboxy (EC3-5) terminus of the extracellular PVA, and injected into neonatal mice. PV IgG affinity-purified on the EC1-2 fusion protein caused suprabasilar acantholysis, the typical histological finding of PV, but IgG affinity-purified on the EC3-5 fusion protein or beta-galactosidase alone did not. These results indicate that at least one pathogenic epitope, which is sufficient to cause suprabasilar acantholysis in neonatal mice, is located on the amino-terminal region of PVA, an area thought to be important in cadherin homophilic adhesion.
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PMID:Autoantibodies against the amino-terminal cadherin-like binding domain of pemphigus vulgaris antigen are pathogenic. 152 42

Antibodies to ribosomal P proteins are markers for systemic lupus erythematosus (SLE) but are often missed by assays utilizing routine immunofluorescence or immunoprecipitation. Expression of autoantigenic sites encoded by complementary DNA (cDNA) clones offers an inexpensive source of antigen for use in quantitative immunoassays. Using a monospecific ribosomal P positive serum to screen a human placental lambda gt11 expression library, a cDNA clone was isolated which reacted with all anti-P human sera tested. Autoantibodies which were affinity purified from the expressed cDNA reacted with the 38 kDa ribosomal Po protein and reactivity was blocked by absorption with recombinant fusion protein. A stable beta-galactosidase fusion protein of 150 kDa was partially purified by a simple differential centrifugation method and used in an enzyme-linked immunoabsorbent assay to reliably quantitate anti-P responses, with a sensitivity and specificity equal to Western blotting. A survey of 84 normals and 151 patients with autoimmune diseases confirmed the high specificity of anti-P antibodies for SLE. The availability of cloned autoantigen will facilitate more detailed clinico-serologic correlations for anti-P antibodies.
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PMID:Detection of autoantibodies to ribosomal P protein using recombinant autoantigen in a quantitative immunoassay. 239 41

The cDNA encoding the ribosomal protein P2 antigen was cloned from a human cDNA library constructed in the lambda gt11 expression vector. A beta-galactosidase-P2 fusion protein was purified to near homogeneity and used to develop an ELISA which was highly specific for anti-P antibodies produced in murine and human SLE. The median concentration of human IgG anti-P antibodies in serum was estimated to be 100 micrograms/ml (range 6-450 micrograms/ml). Pre-incubation of human anti-P sera with a synthetic peptide, corresponding to the C-terminal 22 amino acids of P2, completely inhibited reactivity with the fusion protein in the ELISA. These findings confirm that lupus anti-P sera show a striking restriction in epitope specificity and indicate that the P2 fusion protein is a useful alternative to the synthetic peptide antigen for detection and quantification of anti-P antibodies. To investigate the possibility that anti-P antibodies were induced by 'altered-self', cDNA encoding P2 were also cloned from lupus patients and control mononuclear cells. The predicted amino acid sequences of the patients' P2 were identical to that of the normal controls indicating that a primary structural abnormality of the P2 autoantigen was unlikely.
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PMID:Quantification of lupus anti-ribosome P antibodies using a recombinant P2 fusion protein and determination of the predicted amino acid sequence of the autoantigen in patients' mononuclear cells. 275 93

A human insulinoma cDNA library was constructed in the expression plasmid vector pUEX1. The clone pUEX1Ins12 was selected by means of hybridization with an insulin probe. It codes for full size amino acid sequence preproinsulin. The bacterial strain pUEX3Ins8 producing proinsulin as beta-galactosidase fusion protein was obtained for the use of recombinant protein as an antigen in an ELISA to detect serum antibodies in subjects with IDDM. Recombinant clones containing the middle, N- and C-terminal domains of the GAD65, the major autoantigen in IDDM, were constructed in pVEX1. These clones may become important tools to study the nature of GAD autoreactivity in IDDM. The clone pHICEO.9 was selected from the human insulinoma cDNA library by immunoscreening with total human insulinoma protein antibodies. This clone expresses the C-terminal fragment of human cholesterol esterase/lipase containing its antigenic determinant and can be used for blood lipase determination. Four clones containing cDNA inserts (0.47-1.42 kb) without any significant homologies to the known sequences in the Gene Bank were obtained by means of statistic selection.
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PMID:[Study on structural gene expression in human insulinoma]. 774 51

The mechanisms involved in the targeting of proteins to different cytosolic compartments are still largely unknown. In this study we have investigated the targeting signal of the 65-kD isoform of glutamic acid decarboxylase (GAD65), a major autoantigen in two autoimmune diseases: Stiff-Man syndrome and insulin-dependent diabetes mellitus. GAD65 is expressed in neurons and in pancreatic beta-cells, where it is concentrated in the Golgi complex region and in proximity to GABA-containing vesicles. GAD65, but not the similar isoform GAD67 which has a more diffuse cytosolic distribution, is palmitoylated within its first 100 amino acids (a.a.). We have previously demonstrated that the domain corresponding to a.a. 1-83 of GAD65 is required for the targeting of GAD65 to the Golgi complex region. Here we show that this domain is sufficient to target an unrelated protein, beta-galactosidase, to the same region. Site-directed mutagenesis of all the putative acceptor sites for thiopalmitoylation within this domain did not abolish targeting of GAD65 to the Golgi complex region. The replacement of a.a. 1-29 of GAD67 with the corresponding a.a. 1-27 of GAD65 was sufficient to target the otherwise soluble GAD67 to the Golgi complex region. Conversely, the replacement of a.a. 1-27 of GAD65 with a.a. 1-29 of GAD67 resulted in a GAD65 protein that had a diffuse cytosolic distribution and was primarily hydrophilic, suggesting that targeting to the Golgi complex region is required for palmitoylation of GAD65. We propose that the domain corresponding to a.a. 1-27 of GAD65, contains a signal required for the targeting of GAD65 to the Golgi complex region.
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PMID:A signal located within amino acids 1-27 of GAD65 is required for its targeting to the Golgi complex region. 803 38

Recent studies have indicated that GM2-1, a pancreatic islet monosialo-ganglioside, is an islet-specific component whose expression is metabolically regulable and represents one of the target antigens of cytoplasmic islet cell antibodies. In the present study we aimed to biochemically characterize this molecule using a panel of biochemical techniques including gas chromatography, thin layer chromatography, enzymatic digestion and mass spectrometry. GM2-1 ganglioside was extracted from human pancreas and purified by thin-layer chromatography. Fatty acids in the ceramide (the hydrophobic portion of the molecule), identified by gas chromatography ranged from C16:1 to C24:1. The oligosaccharide chain was enzymatically digested by the sequential application of various exoglycosidases (neuraminidase followed by beta-galactosidase, followed by beta-hexosaminidase) and characterized by gas chromatography identification of the liberated sugars. The following structure was deducted from enzymatic studies and confirmed by mass spectrometry analysis: N-acetyl neuraminic acid-galactose-galactosamine-galactosamine-glucose-ceramide. This is a novel ganglioside structure, not yet described, which shares characteristics with a neuronal glycolipid autoantigen: the LM1 ganglioside. Both GM2-1 and LM1 have a single sialic acid residue in the terminal position, the same migration position on thin layer chromatography and the same number of carbohydrate moieties. In conclusion, we have characterized a novel islet-specific ganglioside molecule with unusual characteristics, such as the terminal sialic acid and the galactosamine residues, which may facilitate both its antigenicity and its involvement in beta-cell autoimmunity.
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PMID:GM2-1 pancreatic islet ganglioside: identification and characterization of a novel islet-specific molecule. 859 28

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance.
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PMID:An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis. 961 5

Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction. Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library. Surprisingly, both clones encoded the heavy chain of human IgG1. The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo. In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific. Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM. Ro52 could also precipitate IgG directly from serum. The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity. We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region. In conclusion, our data support an unusual interaction between native Ro52 and IgG. The potential biological significance of this unusual protein-protein interaction is discussed.
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PMID:Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain. 1035 73

Recent studies revealing active mechanisms of immune privilege in neural tissues have diminished the putative role of passive tolerance. To examine the significance of Ag localization in the retina on immune privilege, the immune responses of transgenic mice expressing high and low levels of beta-galactosidase (beta-gal) in the photoreceptor cells of the retina were compared with those of normal mice and those of mice expressing moderate levels of beta-gal systemically. Immunization with beta-gal induced experimental autoimmune uveoretinitis indistinguishable from that induced by known photoreceptor cell autoantigens, including destruction of photoreceptor cells, in transgenic mice with high level retinal expression. Retinal expression had no apparent effect on the immune responses to beta-gal, showing that tolerance was not elicited by levels of retinal beta-gal sufficient to serve as a target for autoimmune disease. Mice with systemic expression exhibited reduced lymphoproliferative responses following immunization with beta-gal and did not develop autoimmune disease. T cells prepared from normal mice immunized with beta-gal transferred experimental autoimmune uveoretinitis to the transgenic mice with high level retinal beta-gal expression, but no disease was found in mice with systemic transgene expression under these conditions. The results of our experiments are most consistent with sequestration being the primary mechanism of retinal immune privilege. The results also show that beta-gal can serve as an immunopathogenic neural autoantigen, and that T cells raised by immunization of normal mice with a foreign Ag can be immunopathogenic in certain transgenic recipients.
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PMID:Retinal expression of a neo-self antigen, beta-galactosidase, is not tolerogenic and creates a target for autoimmune uveoretinitis. 1039 7

Autoimmune gastritis, in which the H+/K(+)-ATPase of parietal cells is the major antigen, is one of the most common autoimmune diseases. Here we examined if specific properties of the H+/K(+)-ATPase or parietal cells are involved in rendering them autoimmune targets. The model antigens beta-galactosidase and ovalbumin (OVA) were expressed in parietal cells of transgenic mice. On experimental induction of autoimmune gastritis by neonatal thymectomy, autoantibodies to beta-galactosidase developed in mice expressing beta-galactosidase in parietal cells, a response that was independent of either the response to the gastric H+/K(+)-ATPase or gastric inflammation. In contrast, mice that expressed OVA in parietal cells did not exhibit an antibody response to OVA after thymectomy. However, increasing the frequency of anti-OVA T lymphocytes in OVA-expressing mice resulted in autoantibodies to OVA and gastritis. These studies indicate that parietal cells can present a variety of antigens to the immune system. Factors such as the identity and expression level of the autoantigen and the frequency of autoreactive T cells play a role in determining the prevalence and outcome of the particular immune response. In addition, as not all mice of a particular genotype displayed autoimmunity, random events are involved in determining the target of autoimmune recognition.
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PMID:Requirements for autoimmune responses to mouse gastric autoantigens. 1189 24


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