RESUMO
Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A- and C-type pepsins appear to be conserved in mammals and amphibians.
Assuntos
Ácido Aspártico Proteases/genética , Mucosa Gástrica/metabolismo , Salamandridae/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico Proteases/classificação , Ácido Aspártico Proteases/isolamento & purificação , Catepsina E/classificação , Catepsina E/genética , Catepsina E/isolamento & purificação , Clonagem Molecular , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Precursores Enzimáticos/classificação , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Pepsina A/classificação , Pepsina A/genética , Pepsina A/isolamento & purificação , Pepsinogênios/classificação , Pepsinogênios/genética , Pepsinogênios/isolamento & purificação , Pepstatinas/farmacologia , Filogenia , Inibidores de Proteases/farmacologiaRESUMO
The full-length cDNAs of three pepsinogens (Pgs) were cloned from the stomach of newt, Cynops pyrrhogaster, and nucleotide sequences of the full-length cDNAs were determined. Molecular phylogenetic analysis showed that two Pgs, named PgC1 and PgC2, belong to the pepsinogen C group, and one Pg, named PgA, belongs to the pepsinogen A group. The sequences contain an open reading frame (ORF) encoding 385 amino acid residues for PgC1, 383 amino acid residues for PgC2 and 377 amino acid residues for PgA. In addition, all of the three amino acid sequences conserve some unique characteristics such as six cysteine residues and putative active site two aspartic acid residues. All of the pepsinogen mRNAs were detected in the stomach by RT-PCR but not in other organs. Although a slight difference at the time of the start of expression was seen among the three pepsinogen genes, all of them were expressed in the larval stage after hatching. This is the first report on cloning of pepsinogens from urodele stomach.
Assuntos
Mucosa Gástrica/metabolismo , Pepsinogênio A/genética , Pepsinogênio C/genética , Salamandridae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/genética , DNA Complementar/genética , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Pepsinogênio A/metabolismo , Pepsinogênio C/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salamandridae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Through subtractive hybridization, H+/K+-ATPase beta subunit mRNA, highly expressed in the larval stomach of Xenopus laevis, was isolated. In situ hybridization demonstrated that the H+/K+-ATPase beta subunit mRNA was exclusively expressed in manicotto gland cells of the larval stomach, not in any other cell. Northern blot analysis showed that metamorphosis-associated changes of the H+/K+-ATPase beta subunit mRNA expression in the stomach were characterized by high expression in tadpoles, a considerably lower expression in metamorphosing tadpoles, and a re-increase of expression in froglets. Further in situ hybridization showed that the decrease of expression correlated with the degeneration of larval type epithelium in the manicotto gland, while the re-increase correlated with the differentiation of oxynticopeptic cells of the adult type stomach. Moreover, the H+/K+-ATPase beta subunit mRNA was expressed in adult epithelial primordia. Such changes were found in thyroid hormone-induced precocious metamorphosis. Based on studies using this ATPase as well as xP1 and PgC (pepsinogen C) as molecular markers, this study discusses a probable cell lineage involved in metamorphosis-associated stomach remodeling. The pH of luminal contents of the larval stomach was found to be lower than 2. In addition, the pH of an isolated stomach changed from 7.2 to lower than 4 after incubation in Ringer's solution, suggesting acid production from the larval stomach. This is the first demonstration of the H+/K+-ATPase-mediated acid production and secretion in the larval stomach of Xenopus laevis.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Metamorfose Biológica/fisiologia , Estômago/enzimologia , Xenopus laevis/fisiologia , Animais , Diferenciação Celular , Ácido Gástrico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , ATPase Trocadora de Hidrogênio-Potássio/genética , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Metamorfose Biológica/genética , Especificidade de Órgãos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/análise , Estômago/citologia , Estômago/crescimento & desenvolvimento , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Cathepsin E (CE) was purified from the foregut of Xenopus laevis tadpoles as a mature dimeric form. The purified enzyme was a typical CE among aspartic proteinases with respect to pH dependence of proteolytic activity, susceptibility to pepstatin, and having N-linked high-mannose type oligosaccharide chains. We isolated two cDNAs for the CE (CE1 and CE2) from adult stomach. The amino acid sequence of the N-terminal region of the purified CE coincided with the corresponding sequence predicted from CE1. Northern blot analysis and in situ hybridization were performed. The CE1 mRNA was highly expressed in surface mucous cells and gland cells constituting the larval epithelium of the foregut of pro-metamorphic tadpoles. As metamorphosis began and progressed, CE1 mRNA drastically decreased in amount, and subsequently both CE1 and CE2 mRNAs gradually increased. The increase in CE2 mRNA was detected shortly after the increase in CE1 mRNA. The decrease in CE1 expression correlated with degeneration of the larval type epithelium, while the increases in both CE1 and CE2 expression correlated with formation of the adult type epithelium. Thus, cathepsin E gene expression was differentially regulated during metamorphosis-associated remodeling of the larval to adult type epithelium in stomach.
Assuntos
Catepsina E/metabolismo , Epitélio/crescimento & desenvolvimento , Metamorfose Biológica , Estômago/enzimologia , Xenopus laevis/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/isolamento & purificação , Epitélio/metabolismo , Concentração de Íons de Hidrogênio , Larva/fisiologia , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Xenopus laevis/metabolismoRESUMO
A cDNA library was constructed from a poly(A)(+) RNA fraction of the gastric mucosa of bullfrog Rana catesbeiana. We cloned a cDNA encoding preprocathepsin E (Pre-Pro-CE) from the library. The present study is the first demonstration of the Pre-Pro-CE cDNA of lower vertebrate such as amphibian. Amino acid sequence deduced from the cDNA was compared with partial amino acid sequence determined by Edman degradation, suggesting that the cDNA comprises an open reading frame encoding a signal peptide (16 amino acids), a pro-sequence (33 amino acids) and a mature protein region (348 amino acids). Two consensus tri-peptide sequences (FDT and VDT) as active site and positions of seven cysteine residues were conserved in this amphibian CE. Although the bullfrog CE was deduced to contain one potential N-linked glycosylation site, its position (Asn(139)-Leu(140)-Thr(141)) was different from that of mammalian CEs. Molecular phylogenetic analysis showed that the bullfrog Pro-CE belongs to the typical Pro-CE group among various aspartic proteinases.
