RESUMEN
Proteinase-3 (PR-3) and neutrophil elastase (NE) are polymorphonuclear leukocyte serine proteinases that degrade extracellular matrix proteins including elastin and appear to be involved in the pathogenesis of several diseases characterized by tissue destruction most notably emphysema and Wegener's granulomatosis. In this report we characterize and compare the mouse PR-3 and NE genes and establish by FISH analysis a common location on mouse chromosome 10C2. Each gene consists of five exons and four introns conserving the typical granule-associated serine proteinase gene structure. The mouse PR-3 gene (Prtn3) is approximately 3.7 kb and is within 2.2 kb of the smaller (1.7 kb) NE gene (Ela2). The larger size of Prtn3 is accounted for by differences in intron sizes. A comparison between the mouse and human PR-3 cDNA reveals 73% homology, however, this drops to 60% when the amino acid sequences are compared. Homology between the mouse and human NE cDNA is 77% for both the cDNA and amino acid sequences. The catalytic triad and its placement are conserved among the four genes. The proximal promoter of mouse Prtn3 contains a TATA box, c-myb and an ets transcriptional site. As these are functional elements in the mouse Ela2 promoter they may also be important in the expression of Prtn3.
Asunto(s)
Mapeo Cromosómico , Elastasa de Leucocito/genética , Serina Endopeptidasas/genética , Animales , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Exones , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Mieloblastina , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
Human proteinase-3 is one of three serine proteinases present in the azurophil granules of polymorphonuclear leukocytes along with elastase and cathepsin G. Proteinase-3 gene expression is confined to the promyelocytic stage of polymorphonuclear leukocyte maturation. The present investigation identifies elements responsible for this highly controlled tissue- and developmental-specific expression of proteinase-3. Within the first 200 base pairs of the proteinase-3 promoter, two elements were identified as important for expression, these elements at -101 and -190 confer the majority of the activity. The element at -101 has a PU.1 consensus. It binds a myeloid nuclear protein of approximately 45 kDa that "supershifts" with PU.1 antibody and is competed by the CD11b PU.1 element. The element at -190 has a core sequence of CCCCGCCC (CG element). The cytidines but not the guanidine are essential for promoter activity. The CG element binds a second nuclear protein with a molecular mass of approximately 40 kDa that is found in cells of myeloid lineage as well as non-myeloid HeLa cells. However, the proteinase-3 promoter is not active in HeLa cells which suggests that the CG element alone is not sufficient for proteinase-3 gene expression. Maturation of promyelocytic cells results in an inhibition of proteinase-3 gene expression and a reduction in nuclear protein binding to the PU.1 and CG elements. Similar elements occur in the elastase and cathepsin G promoters. Using the elastase and cathepsin G PU.1 and CG-like elements as probes results in identical band-shift patterns to that obtained with proteinase-3 PU.1 and CG elements. These data suggest that there is cooperative interaction between a PU.1 and a CG element with a consensus of CCCCXCCC and that they are important control elements for tissue- and developmental-specific expression of azurophil serine proteinases of polymorphonuclear leukocytes.
Asunto(s)
Autoantígenos/genética , Citidina , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Granulomatosis con Poliangitis/genética , Proteínas Proto-Oncogénicas/farmacología , Serina Endopeptidasas/genética , Transactivadores , Secuencia de Bases , Catepsina G , Catepsinas/genética , Diferenciación Celular/efectos de los fármacos , Humanos , Elastasa de Leucocito/genética , Datos de Secuencia Molecular , Mieloblastina , Regiones Promotoras Genéticas , Eliminación de Secuencia , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Sporadic porphyria cutanea tarda (S-PCT) has been considered an acquired disease because of the generation of liver-specific inhibitors of uroporphyrinogen decarboxylase (URO-D) activity. Several families have been described with S-PCT in multiple generations, raising the possibility of an inherited basis for the disease. To determine if S-PCT is associated with mutant URO-Ds that might be sensitive to liver-specific inhibitors, a molecular analysis of genomic and hepatocellular URO-Ds was undertaken. METHODS: Total RNA from lymphoid cell lines from three unrelated patients with S-PCT and poly A+ RNA from liver biopsy samples from two additional patients was used as a template for single-stranded cDNA synthesis, and URO-D sequences were amplified and sequenced. DNA prepared from peripheral blood leukocytes was used as a template to polymerase chain reaction (PCR) amplify the promoter region of the URO-D gene. Sequencing of PCR products was performed completely in both directions by the chain termination method using a variety of custom oligonucleotide primers. RESULTS: Ten URO-D alleles were sequenced, and no mutations were found. The promoter region of the URO-D gene was also normal. CONCLUSIONS: It is concluded that S-PCT is not due to mutations at the URO-D locus. If inherited factors are involved, other loci must be affected.
Asunto(s)
ADN/genética , Porfiria Cutánea Tardía/enzimología , Uroporfirinógeno Descarboxilasa/genética , Adulto , Anciano , Secuencia de Bases , ADN/química , Femenino , Humanos , Hígado/enzimología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Porfiria Cutánea Tardía/genética , Uroporfirinógeno Descarboxilasa/metabolismoRESUMEN
Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It degrades a variety of extracellular matrix proteins including elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters. It is identical to the target autoantigen (c-ANCA) associated with Wegener's granulomatosis and to myeloblastin, a serine proteinase first identified in HL-60 leukemia cells. In this study, the gene encoding PR-3 was cloned and sequenced. The gene spans approximately 6.5 kilobase pairs and consists of five exons and four introns. The genomic organization of PR-3 is similar to that of the other serine proteinases expressed in hemopoietic cells. Each residue of the catalytic triad of PR-3 is located on a separate exon, and the positions of the residues within the exons are similar to those in human leukocyte elastase and cathepsin G. The phase and placement of the introns in the PR-3 gene are also similar to those in human leukocyte elastase and cathepsin G. The 400-base pair (bp) 5'-flanking sequence of the PR-3 gene contains a TATA box at position 379. There is no CAAT box promoter element. The 3'-untranslated region is 200 bp, extending from a TGA stop codon to the site of polyadenylation 10 bp after the canonical AATAAA signal. Amplification of PR-3 from a human/hamster hybrid cell line localizes the gene to human chromosome 19. Evidence from Northern analysis suggests that PR-3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development.
Asunto(s)
Autoantígenos/genética , Cromosomas Humanos Par 19 , Gránulos Citoplasmáticos/enzimología , Granulomatosis con Poliangitis/genética , Neutrófilos/enzimología , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Autoantígenos/sangre , Secuencia de Bases , Mapeo Cromosómico , Exones , Expresión Génica , Granulomatosis con Poliangitis/sangre , Granulomatosis con Poliangitis/enzimología , Humanos , Intrones , Datos de Secuencia Molecular , Mieloblastina , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Serina Endopeptidasas/inmunologíaRESUMEN
Uroporphyrinogen decarboxylase (URO-D) is a cytosolic heme-biosynthetic enzyme that converts uroporphyrinogen to coproporphyrinogen. Defects at the uroporphyrinogen decarboxylase locus cause the human genetic disease familial porphyria cutanea tarda. A splice site mutation has been found in a pedigree with familial porphyria cutanea tarda that causes exon 6 to be deleted from the mRNA. The intron/exon junctions on either side of exon 6 fall between codons, so the resulting protein is shorter than the normal protein, missing only the amino acids coded by exon 6. The shortened protein lacks catalytic activity, is rapidly degraded when exposed to human lymphocyte lysates, and is not detectable by Western blot analysis in lymphocyte lysates derived from affected individuals. The mutation was detected in five of 22 unrelated familial porphyria cutanea tarda pedigrees tested, so it appears to be common. This is the first splice site mutation to be found at the URO-D locus, and the first mutation that causes familial porphyria cutanea tarda to be found in more than one pedigree.