RESUMEN

BACKGROUND: It is well known that pepsinogen (PGs), as an important precursor of pepsin performing digestive function, has a good correlation with the occurrence and development of gastric cancer and it is also known that ectopic PGs expression is related to the prognosis of some cancers. However, the panoramic picture of pepsinogen gene family in human cancer is not clear. This study focused on elucidating the expression profile, activated pathway, immune cells infiltration, mutation, and copy number variation of PGs and their potential role in human cancer. METHOD: Based on the next generation sequence data from TCGA, Oncomine, and CCLE, the molecular changes and clinical correlation of PGs in 33 tumor types were analyzed systematically by R language, including the expression, mutation, and copy number variation of PGs and their correlation with cancer-related signal transduction pathway, immune cell infiltration, and prognostic potential in different cancers. RESULTS: PGs expression profiles appear different in 33 tumors. The transcriptional expression of PGs was detected in 16 of all 33 tumors. PGC was highly expressed in cholangiocarcinoma, colon adenocarcinoma, rectum adenocarcinoma, uterine corpus endometrial carcinoma, bladder urothelial carcinoma and breast cancer, while decreased in stomach adenocarcinoma, kidney renal clear cell carcinoma, prostate adenocarcinoma, lung squamous cell carcinoma, and esophageal carcinoma. PGA3, PGA4, and PGA5 were expressed in most normal tissues, but decreased in cancer tissues. PGs expression was significantly related to the activation or inhibition of many signal transduction pathways, in which PGC and PGA5 are more likely to be associated with cancer-related pathways. PGC participated in 33 regulatory network pathways in pan-cancer, mainly distributed in stomach adenocarcinoma, esophageal carcinoma, and lung squamous cell carcinoma, respectively. PGC and PGA3 expression were significantly correlated with immune cell infiltration. The results of survival analysis showed that different PGs expression play significantly different prognostic roles in different cancers. PGC was correlated with poor survival in brain lower grade glioma, skin cutaneous melanoma, and higher survival in kidney renal clear cell carcinoma, acute myeloid leukemia, mesothelioma, and uveal melanoma. PGA4 was only associated with higher survival in kidney renal clear cell carcinoma. Genetic variation analysis showed that PGC gene often mutated in uterine corpus endometrial carcinoma and stomach adenocarcinoma had extensive copy number amplification in various tumor types. PGC expression was upregulated with the increase of copy number in cholangiocarcinoma, esophageal carcinoma, and kidney renal papillary cell carcinoma, while in stomach adenocarcinoma, PGC was upregulated regardless of whether the copy number was increased or decreased. CONCLUSIONS: PGs was expressed unevenly in a variety of cancer tissues and was related to many carcinogenic pathways and involved in the immune regulation. PGC participated in 33 regulatory pathways in human cancer. Different PGs expression play significantly different prognostic roles in different cancers. The variation of copy number of PGC gene could affect the PGC expression. These findings suggested that PGs, especially PGC have characteristic of broad-spectrum expression in multiple cancers rather than being confined to the gastric mucosa, which may made PGs be useful biomarkers for prediction/diagnosis/prognosis and effective targets for treatment in human cancer.

Asunto(s)

Neoplasias/genética , Pepsinógenos/genética , Transcriptoma , Biología Computacional , Variaciones en el Número de Copia de ADN , Bases de Datos Genéticas , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Mutación , Neoplasias/enzimología , Neoplasias/mortalidad , Neoplasias/terapia , Pepsinógenos/metabolismo , Pronóstico , Mapas de Interacción de Proteínas , Transducción de Señal

RESUMEN

BACKGROUND: Persistent infections that induce prolonged inflammation might negatively affect the leukocyte telomere length (LTL); however, the role in LTL of Helicobacter pylori (H. pylori) infection, which persistently colonizes the stomach, remains unknown. The study objective was to examine associations of sero-prevalence of H. pylori immunoglobulin G (IgG) antibody and serum pepsinogens (PGs), as markers of atrophic gastritis, with LTL. A cross-sectional study was performed among 934 Arab residents of East Jerusalem, aged 27-78 years, randomly selected from Israel's national population registry. Sera were tested for H. pylori IgG and PG levels by ELISA. LTL was measured by southern blots. Multiple linear regression models were fitted to adjust for sociodemographic and lifestyle factors. RESULTS: LTL decreased significantly with age (p < 0.001) and was shorter in men than women (p = 0.032). The mean LTL was longer in H. pylori sero-positive persons than negative ones: mean difference 0.13 kb (95% CI 0.02, 0.24), p = 0.016. Participants with atrophic gastritis (PGI < 30 µg/L or a PGI: PGII < 3.0) had shorter LTL than did those without: mean difference - 0.18 (95% CI - 0.32, - 0.04). The difference was of larger magnitude between persons who had past H. pylori infection (sero-negative to H. pylori IgG antibody) and atrophic gastritis, compared to those who were H. pylori sero-negative and did not have atrophic gastritis: mean difference - 0.32 kb (95% CI - 0.55, - 0.10). This association remained significant after adjustment for age, sex, and religiosity: beta coefficient - 0.21 kb (95% CI - 0.41, - 0.001), p = 0.049. The results were similar after further adjustment for lifestyle factors. In bivariate analysis, mean LTL was longer in physically active persons than non-active ones, and shorter in persons with than without obesity; however, these differences were diminished and were not significant in the multivariable model. CONCLUSIONS: H. pylori IgG sero-positivity per se was not related to reduced LTL. However, persons with past H. pylori infection (i.e., lacking H. pylori IgG serum antibody) and with serological evidence of atrophic gastritis, had a significantly shorter LTL than did those without atrophic gastritis.

Asunto(s)

Gastritis Atrófica/sangre , Infecciones por Helicobacter/sangre , Inmunoglobulina G/sangre , Pepsinógenos/sangre , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Árabes/genética , Biomarcadores/sangre , Femenino , Gastritis Atrófica/genética , Gastritis Atrófica/microbiología , Gastritis Atrófica/patología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Humanos , Israel/epidemiología , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Persona de Mediana Edad , Pepsinógenos/genética , Telómero/genética , Telómero/microbiología

RESUMEN

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.

Asunto(s)

Proteasas de Ácido Aspártico/genética , Mucosa Gástrica/metabolismo , Salamandridae/metabolismo , Secuencia de Aminoácidos , Animales , Proteasas de Ácido Aspártico/clasificación , Proteasas de Ácido Aspártico/aislamiento & purificación , Catepsina E/clasificación , Catepsina E/genética , Catepsina E/aislamiento & purificación , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas , Precursores Enzimáticos/clasificación , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Pepsina A/clasificación , Pepsina A/genética , Pepsina A/aislamiento & purificación , Pepsinógenos/clasificación , Pepsinógenos/genética , Pepsinógenos/aislamiento & purificación , Pepstatinas/farmacología , Filogenia , Inhibidores de Proteasas/farmacología

RESUMEN

The nucleotide sequences of largemouth bass pepsinogens (PG1, 2 and 3) were determined after molecular cloning of the respective cDNAs. Encoded PG1, 2 and 3 were classified as fish pepsinogens A1, A2 and C, respectively. Molecular evolutionary analyses show that vertebrate pepsinogens are classified into seven monophyletic groups, i.e. pepsinogens A, F, Y (prochymosins), C, B, and fish pepsinogens A and C. Regarding the primary structures, extensive deletion was obvious in S'1 loop residues in fish pepsin A as well as tetrapod pepsin Y. This deletion resulted in a decrease in hydrophobic residues in the S'1 site. Hydrolytic specificities of bass pepsins A1 and A2 were investigated with a pepsin substrate and its variants. Bass pepsins preferred both hydrophobic/aromatic residues and charged residues at the P'1 sites of substrates, showing the dual character of S'1 sites. Thermodynamic analyses of bass pepsin A2 showed that its activation Gibbs energy change (∆G(‡)) was lower than that of porcine pepsin A. Several sites of bass pepsin A2 moiety were found to be under positive selection, and most of them are located on the surface of the molecule, where they are involved in conformational flexibility. The broad S'1 specificity and flexible structure of bass pepsin A2 are thought to cause its high proteolytic activity.

Asunto(s)

Lubina/genética , ADN Complementario/genética , Evolución Molecular , Proteínas de Peces/genética , Pepsinógenos/genética , Secuencia de Aminoácidos , Animales , Lubina/clasificación , Lubina/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Expresión Génica , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Pepsina A/química , Pepsina A/genética , Pepsina A/metabolismo , Pepsinógenos/química , Pepsinógenos/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Porcinos , Termodinámica

RESUMEN

The native folding of certain zymogen-derived enzymes is completely dependent upon a prosegment domain to stabilize the folding transition state, thereby catalyzing the folding reaction. Generally little is known about how the prosegment accomplishes this task. It was previously shown that the prosegment catalyzes a late-stage folding transition between a stable misfolded state and the native state of pepsin. In this study, the contributions of specific prosegment residues to catalyzing pepsin folding were investigated by introducing individual Ala substitutions and measuring the effects on the bimolecular folding reaction between the prosegment peptide and pepsin. The effects of mutations on the free energies of the individual misfolded and native ground states and the transition state were compared using measurements of prosegment-pepsin binding and folding kinetics. Five out of the seven prosegment residues examined yielded relatively large kinetic effects and minimal ground state perturbations upon mutation, findings which indicate that these residues form strengthened and/or non-native contacts in the transition state. These five residues are semi- to strictly conserved, while only a non-conserved residue had no kinetic effect. One conserved residue was shown to form native structure in the transition state. These results indicated that the prosegment, which is only 44 residues long, has evolved a high density of contacts that preferentially stabilize the folding transition state over the ground states. It is postulated that the prosegment forms extensive non-native contacts during the process of catalyzing correct inter- and intra-domain contacts during the final stages of folding. These results have implications for understanding the folding of multi-domain proteins and for the evolution of prosegment-catalyzed folding.

Asunto(s)

Pepsina A/química , Pepsinógenos/química , Pliegue de Proteína , Secuencias de Aminoácidos , Animales , Humanos , Cinética , Mutación , Pepsina A/genética , Pepsina A/metabolismo , Pepsinógenos/genética , Pepsinógenos/metabolismo , Estructura Terciaria de Proteína , Porcinos

RESUMEN

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish rice field eel (Monopterus albus Zuiew) by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephacryl S-200 HR. The molecular masses of the three purified PGs were all estimated as 36 kDa using SDS-PAGE. Two-dimensional gel electrophoresis (2D-PAGE) showed that pI values of the three PGs were 5.1, 4.8, and 4.6, respectively. All the PGs converted into corresponding pepsins quickly at pH 2.0, and their activities could be specifically inhibited by aspartic proteinase inhibitor pepstatin A. Optimum pH and temperature of the enzymes for hydrolyzing hemoglobin were 3.0-3.5 and 40-45 °C. The K (m) values of them were 1.2 × 10⁻4 M, 8.7 × 10⁻5 M, and 6.9 × 10⁻5 M, respectively. The turnover numbers (k(cat)) of them were 23.2, 24.0, and 42.6 s⁻¹. Purified pepsins were effective in the degradation of fish muscular proteins, suggesting their digestive functions physiologically.

Asunto(s)

Anguilas/fisiología , Mucosa Gástrica/metabolismo , Pepsina A/metabolismo , Pepsinógenos/metabolismo , Animales , Regulación Enzimológica de la Expresión Génica/fisiología , Concentración de Iones de Hidrógeno , Pepsina A/química , Pepsina A/genética , Pepsinógenos/química , Pepsinógenos/genética , Temperatura

RESUMEN

The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1'-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology.

Asunto(s)

Evolución Molecular , Pepsinógenos/química , Pepsinógenos/genética , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Datos de Secuencia Molecular , Señales de Clasificación de Proteína , Homología Estructural de Proteína , Atún

RESUMEN

Two major pepsinogens, PG1 and PG2, and one minor pepsinogen, PG3, were purified from the gastric mucosa of African coelacanth, Latimeria chalumnae (Actinistia). PG1 and PG2 were much less acidic than PG3. Their molecular masses were estimated by SDS-PAGE to be 37.0, 37.0 and 39.3 kD, respectively. When incubated at pH 2.0, PG1 and PG2 were converted autocatalytically to the mature pepsins through an intermediate form, whereas PG3 was converted to an intermediate form, but not to the mature pepsin autocatalytically. The N-terminal sequencing indicated that the 42 residue sequences of the propeptides of PG1 and PG2 were essentially identical with each other, but different from that of PG3. A phylogenetic tree based on the N-terminal propeptide sequences indicates that PG1 and PG2 belong to the pepsinogen A group, and PG3 to the pepsinogen C group. From the phylogenetic comparison, coelacanth PG1 and PG2 appear to be evolutionally closer to tetrapod pepsinogens A than ray-finned fish pepsinogens A, consistent with the traditional systematics. Pepsins 1 and 2 were essentially identical with each other and rather similar to mammalian pepsins A in the pH optimum toward hemoglobin (pH 2-2.5), the cleavage specificity toward oxidized insulin B chain and strong inhibition by pepstatin, except that they possessed a significant level of activity in the higher pH range unlike mammalian pepsins A.

Asunto(s)

Peces/genética , Mucosa Gástrica/enzimología , Pepsina A/genética , Pepsinógenos/genética , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Activación Enzimática , Datos de Secuencia Molecular , Pepsina A/química , Pepsina A/metabolismo , Pepsinógenos/aislamiento & purificación , Pepsinógenos/metabolismo , Filogenia , Homología de Secuencia de Aminoácido

RESUMEN

Genes encoding pepsinogens, zymogens of digestive enzyme pepsins, are expressed specifically in the gland epithelial cells of the vertebrate stomach, and their expression is also developmentally regulated, therefore providing a good model for the analysis of transcriptional regulation of genes. In the development of chicken embryonic stomach, the epithelium invaginates into the mesenchyme and forms glands and gland epithelial cells then begin to express embryonic chicken pepsinogen (ECPg) gene. It has been shown that cGATA5 binds directly GATA binding sites located within 1.1-kbp upstream of ECPg gene and activates its transcription. To find more precisely the sequences necessary for ECPg gene transcription, we carried out deletion and mutation analysis with 1.1-kbp upstream region. The results suggest that binding of GATA factor to three GATA binding sites within the upstream region -656 to -419 synergistically regulates ECPg expression in the gland epithelial cells.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Pepsinógenos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Análisis Mutacional de ADN , Células Epiteliales/enzimología , Genes Reporteros , Operón Lac , Mesodermo/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Pepsinógenos/metabolismo , Regiones Promotoras Genéticas , Proventrículo/citología , Proventrículo/embriología , Eliminación de Secuencia

RESUMEN

AIM: To develop conditionally immortalized gastric mucosal cell lines that show distinct types of cell differentiation from transgenic mice harboring temperature-sensitive simian virus 40 (tsSV40) large T antigen. METHODS: Gastric mucosal cells from the transgenic mice were cultured at a permissive temperature (33 degrees C), and proliferative cells were then cloned by colony formation. RESULTS: Eight gastric cell lines showed epithelial-like morphology and grew at 33 degrees C. Three different types of the cell lines have been established: (1) MGE12-1, MGE3-2, and MGE509 cells expressing mRNAs for pit cell markers (gastric mucin and cathepsin E); (2) MGE02, MGE503, and MGE511 cells expressing mRNAs for pit and zymogenic (pepsinogen F) cell markers, and (3) MGE507 and MGE727 cells expressing mRNAs for pit, zymogenic, and parietal (H,K-ATPase alpha-subunit) cell markers. Moreover, the TaqMan assay showed that mRNA levels of mucin, H,K-ATPase alpha-subunit, and pepsinogen F were influenced by nonpermissive temperature (39 degrees C) in MGE503 and MGE727 cells. CONCLUSION: These gastric epithelial cell lines seem to reflect different stages of development of gastric mucosal cells.

Asunto(s)

Antígenos Transformadores de Poliomavirus/metabolismo , Diferenciación Celular/fisiología , Células Epiteliales/citología , Mucosa Gástrica/citología , Animales , Biomarcadores , Western Blotting , Catepsina E/genética , División Celular , Línea Celular Transformada/fisiología , Células Epiteliales/metabolismo , Femenino , Mucinas Gástricas/genética , Mucosa Gástrica/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , Ratones , Ratones Desnudos , Ratones Transgénicos , Pepsinógenos/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura

RESUMEN

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian-plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 degrees C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 degrees C increase in denaturation temperature ( T (m)), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in K (m) and decreased k (cat) compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure-function role for the PSS as it pertains to plant aspartic proteinases.

Asunto(s)

Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Arabidopsis/genética , Ácido Aspártico Endopeptidasas/química , Rastreo Diferencial de Calorimetría , Secuencia de Consenso , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Insulina/química , Insulina/metabolismo , Cinética , Datos de Secuencia Molecular , Pepsina A/metabolismo , Pepsinógenos/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Especificidad por Sustrato , Porcinos , Triticum/enzimología

RESUMEN

Purification of pepsinogen B from dog stomach was achieved. Activation of pepsinogen B to pepsin B is likely to proceed through a one-step pathway although the rate is very slow. Pepsin B hydrolyzes various peptides including beta-endorphin, insulin B chain, dynorphin A, and neurokinin A, with high specificity for the cleavage of the Phe-X bonds. The stability of pepsin B in alkaline pH is noteworthy, presumably due to its less acidic character. The complete primary structure of pepsinogen B was clarified for the first time through the molecular cloning of the respective cDNA. Molecular evolutional analyses show that pepsinogen B is not included in other known pepsinogen groups and constitutes an independent cluster in the consensus tree. Pepsinogen B might be a sister group of pepsinogen C and the divergence of these two zymogens seems to be the latest event of pepsinogen evolution.

Asunto(s)

Evolución Molecular , Pepsina A/química , Pepsina A/genética , Pepsinógenos/química , Pepsinógenos/genética , Animales , Secuencia de Bases , Clonación Molecular , Perros , Activación Enzimática/fisiología , Estabilidad de Enzimas/fisiología , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Pepsina A/aislamiento & purificación , Pepsinógenos/aislamiento & purificación , Filogenia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Estómago/química , Estómago/enzimología , Especificidad por Sustrato/fisiología

RESUMEN

Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudo-pepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S4 through S'3 subsites. Hydrophobic and aromatic amino acids are preferred at the P1 and P'1 positions. Interactions at additional subsites are important in some cases, for example with cleavage of kappa-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones.

Asunto(s)

Quimosina , Precursores Enzimáticos , Pepsinógeno C , Pepsinógenos , Secuencia de Aminoácidos , Animales , Quimosina/química , Quimosina/genética , Quimosina/fisiología , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/fisiología , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pepsinógeno C/química , Pepsinógeno C/genética , Pepsinógeno C/fisiología , Pepsinógenos/química , Pepsinógenos/genética , Pepsinógenos/fisiología , Primates , Inhibidores de Proteasas/metabolismo , Homología de Secuencia de Aminoácido , Terminología como Asunto , Transcripción Genética

RESUMEN

A new combination of chromatographic and electrophoretic methods has been developed for better separation and characterization of human pepsinogens. Pepsinogens isolated from the gastric mucosa of patients with gastric cancer have been separated using fast-protein liquid chromatography (FPLC) on an ionex Uno-Q1 column. Proteolytic active fractions were firstly immunodetected by monoclonal antibodies against PGA and PGC using ELISA and then separated by isoelectric focusation in the acidic pH 2.5-5 gradient with an excellent resolution. The combination FPLC and ELISA followed by IEF enabled to separate ten pepsinogen isoforms. This technique is very suitable for studies of the pepsinogen polymorphism and its role in the gastric diseases.

Asunto(s)

Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Pepsinógenos/genética , Polimorfismo Genético , Ensayo de Inmunoadsorción Enzimática , Humanos , Focalización Isoeléctrica , Pepsinógenos/metabolismo , Neoplasias Gástricas/metabolismo

RESUMEN

Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5'-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3 beta, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes.

Asunto(s)

Regulación de la Expresión Génica , Pepsinógeno A/genética , Pepsinógenos/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Elementos Transponibles de ADN/genética , Humanos , Intrones , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia

RESUMEN

Human pepsinogen (PG) A and C were cloned in Escherichia coli, but the levels of expression were low and unstable. When there were fused to maltose-binding protein (MBP), the fusion proteins (MBP-PGA and MBP-PGC) were expressed as the major products. Although these fused products were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple. MBP-PGA and the PGA segment obtained by factor Xa digestion (designated as r-PGA) possessed proteolytic activities equivalent to native PGA purified from gastric tissue (t-PGA). For PGCs (MBP-PGC, r-PGC and t-PGC) also, the specific activities were almost the same. However, the activities of PGCs were about 3- to 4-hold higher than those of PGAs. In PGA and PGC immunoassay systems, r-PGs (r-PGA and r-PGC) and the EIA kit standard PGs (gastric mucosal PGs) exhibited a good correlation. From these results, r-PGs would seem to be applicable as assay standards without compromising the sensitivity of the immunoassay systems.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Inmunoensayo/métodos , Proteínas de Transporte de Monosacáridos , Pepsinógenos/aislamiento & purificación , Pepsinógenos/metabolismo , Anticuerpos Monoclonales/metabolismo , Proteínas Portadoras , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Unión a Maltosa , Pepsinógenos/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Estándares de Referencia

RESUMEN

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days' gestation. The development of these cells could be classified into four stages: (1) 18.5 days' gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3-4 weeks after birth; (4) 4-8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

Asunto(s)

Fundus Gástrico/embriología , Fundus Gástrico/enzimología , Pepsinógenos/análisis , Pepsinógenos/genética , Animales , Especificidad de Anticuerpos , Células Principales Gástricas/enzimología , Células Principales Gástricas/ultraestructura , Colorantes , Digoxigenina , Femenino , Fundus Gástrico/citología , Mucosa Gástrica/citología , Mucosa Gástrica/enzimología , Mucosa Gástrica/ultraestructura , Hidrazinas , Inmunohistoquímica , Hibridación in Situ , Masculino , Microscopía Inmunoelectrónica , Pepsinógenos/inmunología , Ácido Peryódico , Embarazo , Sondas ARN , ARN Mensajero/análisis , Conejos , Ratas , Ratas Wistar , Proteínas de Plata , Coloración y Etiquetado

RESUMEN

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell-cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh. These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial-mesenchymal interactions in developing digestive organs.

Asunto(s)

Sistema Digestivo/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Mesodermo/fisiología , Proteínas/genética , Transactivadores , Animales , Embrión de Pollo , Técnicas de Cultivo , Inducción Embrionaria , Endodermo/química , Epitelio/fisiología , Proteínas Hedgehog , Pepsinógenos/genética , ARN Mensajero/análisis

RESUMEN

We made fusions between Escherichia coli maltose-binding protein (MBP) and the mammalian aspartic proteinases pepsinogen or procathepsin D. When MBP was at the N-terminus, the fusions were soluble in E. coli. When the order was reversed, the chimeric proteins formed inclusion bodies. The data suggest that the solubility of fusion proteins is controlled by whether the protein domains emerging first from the ribosome normally fold into soluble or insoluble states. The soluble MBP-aspartic proteinase fusions were stable but proteolytically inactive. MBP-pepsinogen, however, was efficiently renatured from 8 M urea in vitro, suggesting that the E. coli cytoplasm does not support folding of the mammalian partner protein to the native state. Thus, inclusion body formation may be the consequence, rather than the cause, of non-native folding in vivo, and in E. coli soluble proteins may fold into states different from those reached in vitro.

Asunto(s)

Transportadoras de Casetes de Unión a ATP , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Portadoras/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Transporte de Monosacáridos , Pliegue de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Ácido Aspártico Endopeptidasas/genética , Proteínas Portadoras/genética , Catepsina D/genética , Catepsina D/metabolismo , Precursores Enzimáticos/genética , Escherichia coli/genética , Cuerpos de Inclusión , Proteínas de Unión a Maltosa , Pepsinógenos/genética , Pepsinógenos/metabolismo , Ingeniería de Proteínas , Solubilidad

Asunto(s)

Catepsina D/genética , Precursores Enzimáticos/genética , Expresión Génica , Pepsinógenos/genética , Renina/genética , Animales , Células CHO , Catepsina D/análisis , Catepsina D/metabolismo , Línea Celular , Cricetinae , Precursores Enzimáticos/metabolismo , Humanos , Pepsinógenos/metabolismo , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Renina/análisis , Renina/metabolismo