Cellular and subcellular localization of progastricsin in calf fundic mucosa: colocalization with pepsinogen and prochymosin

The presence of gastricsin in bovine abomasal juice has been reported previously, but its exact site of origin has not yet been established. Specific polyclonal antibodies were used in the peroxidase-antiperoxidase method or the protein A/gold technique to label cells producing progastricsin. This immunocytolocalization was correlated with that of pepsinogen and prochymosin using specific polyclonal antibodies against those zymogens. The present study clearly established that progastricsin was located exclusively in chief, mucous neck, transitional mucous neck/chief, foveolar epithelial and surface epithelial cells of the calf fundic mucosa. Furthermore, progastricsin was found to be colocalized with pepsinogen and prochymosin in the same secretory granules of these cells. Progastricsin was not observed in parietal, gastric endocrine and undifferentiated neck cells.     

Separation of porcine pepsinogen A and progastricsin. Sequencing of the first 73 amino acid residues in progastricsin.

Porcine pepsinogen A (EC 3.4.23.1) and progastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of progastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of progastricsin. The sequence of this was determined; in addition, the first 30 residues of gastricsin were sequenced. The sequence of the first 73 amino acid residues of progastricsin shows an overall identity with progastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pepsinogen A numbering) is changed to Arg in porcine progastricsin.     

A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin.

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.     

Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

Immunolocalisation of aspartic proteinases in the developing human stomach

The distribution and time of appearance in the developing human stomach of the 4 aspartic proteinases, pepsinogen, progastricsin, slow-moving protease and cathepsin D, all present in gastric carcinoma, has been determined by the peroxidase-antiperoxidase method on formalin fixed paraffin embedded sections of fetal stomach. Slow-moving protease appears to be the dominant enzyme from 12 weeks gestation onward, although progastricsin is also present at this time. Pepsinogen and cathepsin D do not appear until 17-18 weeks.     

Development of gastric acid secretion in pigs from birth to thirty six days of age: the response to pentagastrin

The development of the gastric acid secretory response to pentagastrin was studied using 56 Large White x Landrace pigs, 0-36 days of age, 1.1-13.3 kg body-weight, obtained from 12 litters. Gastric acid secretory capacity was measured using a gastric perfusion technique and intravenous infusion of pentagastrin at dose rates of 2, 4 and 8 micrograms/h per kg. Significant positive linear correlations were found between stomach weight and age, and between stomach weight and body-weight during the 36 day period. The stomach weight to body-weight ratio increased for the first 3 days of age and then decreased during the following 33 days. Basal acid secretion was detected in all unsuckled pigs (n = 9), 2- to 8-h old. Maximal acid outputs in response to pentagastrin in these pigs were 0.16 +/- 0.02 mmol/kg body-weight and 0.034 +/- 0.001 mmol/g stomach weight. For the 56 pigs, significant linear correlations were found between maximal acid output and age, maximal acid output and body-weight, and maximal acid output and stomach weight. There was a significant linear increase in maximal acid output per unit stomach weight during the first 7 days of age, but during the subsequent 29 days the pattern of increase in gastric secretory capacity was slower and curvilinear. In the oldest nine pigs, 24-36 days of age, maximal acid outputs were 0.974 +/- 0.058 mmol/kg body-weight and 0.234 +/- 0.016 mmol/g stomach weight which represents a six to seven-fold increase compared with those determined in pigs at birth. Comparison of gastric acid secretory capacity determined under anaesthesia with that in conscious pigs showed that anaesthesia appeared to suppress basal output but had no effect on pentagastrin stimulated output. Comparison of response to histalog (betazole HCl) and pentagastrin indicated that newborn pigs were more sensitive to histalog but in pigs 9-38 days of age, there were no significant differences in responsiveness to the two secretagogues. These results show that gastric sensitivity to pentagastrin increases rapidly in the first week of life, that the stomach of the newborn pig is more sensitive to histalog than pentagastrin and that studies of the effect of pentagastrin on acid secretion, done under anaesthesia, are comparable to those in the conscious pig.     

Prediction of the tertiary structure of the beta-secretase zymogen

beta-Secretase, also known as BACE, is a transmembrane aspartyl protease, which generates the N terminus of Alzheimer's disease amyloid beta-peptide. The activity of beta-secretase is the rate-limiting step of brain plaques production in vivo, and hence is a potential target for disease modifying drugs for Alzheimer's disease. To better understand the mechanism of action of beta-secretase and help explore novel strategies for drug discovery for Alzheimer's disease, it is important to elucidate the three-dimensional structure of its zymogen. Based on the X-ray structure of the enzyme's protease domain and the X-ray structure of pepsinogen, a model of the three-dimensional structure of the beta-secretase zymogen has been constructed. Comparison of the computed structure of pro-BACE with X-ray structures of pepsinogen and progastricsin (two other pro-aspartyl proteases) reveals a significant difference in the relationship of the pro-segment to the catalytic aspartates. In both pepsinogen and progastricsin a lysine side-chain in the pro-segment forms a salt bridge to the two catalytic aspartates, occupying the position normally occupied by a catalytic water. In the pro-BACE model there is no salt bridge, and the corresponding residue-a proline-does not interact at all with the catalytic residues. These findings can be used to elucidate the recent observations that the pro-domain of beta-secretase does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain. The predicted three-dimensional structure of beta-secretase zymogen and the relevant findings might also provide useful insights for rational design of effective drugs against Alzheimer's disease.     

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.     

Molecular cloning of neonate/infant-specific pepsinogens from rat stomach mucosa and their expressional change during development

To clarify the nature of rat neonate/infant-specific pepsinogens, we carried out their purification and molecular cloning. Prochymosin was found to be the major neonatal pepsinogen. The general proteolytic activity of its active form, chymosin, was, however, lower than those of pepsins A and C which are predominant in adult animals. Molecular cloning of rat prochymosin cDNA was achieved along with cDNA for another neonate-specific pepsinogen, pepsinogen F, although determination of pepsinogen F in neonatal gastric mucosa was unsuccessful, presumably due to its lack of proteolytic activity or different proteolytic specificity. Northern blot analysis confirmed that genes for prochymosin and pepsinogen F are expressed only at neonatal/infant stages and the switching of gene expression to that of pepsinogen C occurred at late infant stages. A phylogenetic tree based on nucleotide sequences showed clearly that pepsinogens fall into four major groups, namely prochymosin and pepsinogen F of the neonate/infant and pepsinogens A and C of adult animals. Although, to date, prochymosin and pepsinogen F were believed to be expressed in only a limited number of mammals, the present results suggest that they might be expressed at the neonatal/infant stage in a variety of mammals.     

Effect of omeprazole on secretion,synthesis and the gene expression of pepsinogen in the guinea pig stomach mucosa

The effects of omeprazole, a proton pump inhibitor, on gene expression, protein synthesis, intracellular storage and secretion of pepsinogen in guinea pig stomach were investigated. After treatment with omeprazole for five days, acid and pepsinogen secretion into the gastric lumen was significantly reduced. Concomitant with this, there was an increase in intracellular pepsinogen as demonstrated by increased pepsin activity in the gastric mucosa, more intense immunohistochemical staining by antibodies specific of pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount for pepsinogen mRNA was reduced as revealed by Northern blotting and in situ hybridization. Ultrastructurally the endoplasmic reticulum of these cells was poorly developed, the findings being consistent with a reduction in protein synthesis. It appears that omeprazole inhibits the secretion of pepsinogen, increasing the intracellular store and leading to the reduction in gene expression probably by a feedback mechanism and consequent reduction in pepsinogen synthesis. Since these changes were most evident in the acid-secreting fundic gland mucosa, as compared with other mucosae secreting only pepsinogen, namely pyloric and duodenal mucosa, it appears probable that these changes are linked with omeprazole-induced reduction in the acid secretion.     

Immunocytochemical localization of rabbit gastric lipase and pepsinogen

Lipase and pepsin activities were determined in rabbit gastric biopsy specimens. Lipase activity was found to be restricted to a small part of the fundic mucosa, near the cardia, whereas pepsin activity spread over about two thirds of the total fundic area, overlapping that of lipase. The cells producing these two enzymes were labeled by immunofluorescence using polyclonal antibodies against rabbit gastric lipase (RGL) or antibodies against rabbit pepsinogen. The immunocytochemical localization showed unequivocally that RGL and pepsinogen, which were both present in the cardial area, were in fact located in different gastric cells. The cells producing pepsinogen were in the lower base of the gastric fundic glands, whereas the cells producing RGL were in the upper base of the same glands. The cells producing pepsinogen and RGL showed no significant morphological differences. In the part of the fundic area, where only pepsin activity was detected, cells producing pepsinogen covered both the lower and the upper base of the gastric glands. No chief cells were observed in the antral mucosa. RGL and pepsinogen could represent useful gastric enzyme markers for cellular differentiation studies.     

The complete amino acid sequence of monkey progastricsin

The complete amino acid sequence of progastricsin from the Japanese monkey (Macaca fuscata) was determined. Progastricsin is composed of 374 residues, including the gastricsin moiety of 331 residues and the activation segment of 43 residues. Upon activation under acidic conditions, progastricsin was converted to gastricsin via the intermediate protein species. NH2-terminal sequence determination of these protein species enabled us to deduce the NH2-terminal 78-residue sequence of progastricsin, including the 43-residue activation segment. The complete sequence of the gastricsin moiety was determined using peptide fragments obtained by several chemical and enzymatic cleavages. The molecular weight of progastricsin was determined to be 40,785. As compared with pepsinogen A of the same monkey species, deletion of 4 residues and insertion of 5 residues were observed. Although monkey progastricsin and pepsinogen A have highly homologous sequences around the two active site aspartyl residues, the homology between these proteins is rather small (49% identity). This indicates that progastricsin diverged from pepsinogen A in the early phase of the evolution of gastric aspartyl proteinases.     

Immunocytochemistry and in situ hybridization studies of pepsinogen C-producing cells in developing rat fundic glands

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.     

Pepsinogen C: a type 2 cell-specific protease

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.     

Gastrin in fetal and neonatal pigs

1. The concentration and molecular profile of gastrin were examined in plasma and tissue extracts of fetal and neonatal pigs from 93 days gestation up to 12 weeks of age and also in the fetal gastric contents. 2. Gastrin was present in the gastrointestinal tract and plasma of fetal pigs at 93 days gestation. The concentration in both plasma and antral extracts increased progressively up to birth and continued to rise postnatally, reaching a peak at about 3 weeks of age in plasma and 6 weeks in the antrum. 3. In blood the major molecular form of gastrin was G34 (up to 80%), while in the antrum the major form was G17 (66-91%). The percentage of G34 in the antrum was highest in later gestation (21%), and reached adult proportion by 8 weeks of age (4%). 4. A considerable amount of gastrin, chiefly G17, was detected in the fetal gastric contents. Synthetic human G17 was stable in fetal gastric contents when incubated at 37 degrees C for 60 min, although, when incubated with gastric contents from a sow, it disappeared within 5 min. 5. It is suggested that the presence of gastrin in fetal gastric contents may be important in stimulation of fetal gut development.     

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human progastricsin. Analysis of intermediates during activation into gastricsin and determination of the amino acid sequence of the propart

Human progastricsin was prepared from extracts of gastric mucosa by chromatography on columns of DEAE-cellulose. The amino acid compositions of progastricsin and gastricsin were determined and calculated on the basis of the molecular weights 38 000 and 32 000 respectively. The activation of progastricsin at pH 2 was investigated and monitored by agarose gel electrophoresis at pH 5.4. Two intermediates were observed. Determination of the amino acid sequence showed that the propart consists of 43 amino acid residues. A pronounced homology with other gastric zymogens was found. With the proenzyme amino acid residue numbering used previously [B. Foltman (1981) Essays in Biochemistry, 17, 52-84] the activation of progastricsin at pH 2 may be summarized as follows. The first cleavage occurs after Phe (p27). At pH 5.4 the peptide remains associated with the protein (intermediate I). Subsequent proteolysis removes the peptides from Leu (p28) to Leu (p45). At pH 5.4 the N-terminal peptide from progastricsin (p2-p27) remains associated with gastricsin (intermediate II) until the propart peptide is hydrolysed to smaller fragments.     

Premature induction of pepsinogen in developing rat gastric mucosa by hormones

Daily injection of ACTH into rats from the age of 9 days caused precocious development of peptic activity in the gastric mucosa. A single injection of hydrocortisone into rats aged 2 to 9 days also evoked premature development of peptic activity in the gastric mucosa. The patterns on polyacrylamide gel electrophoresis of pepsinogen from adults and from infants treated with hormone were quite similar but differed from that of untreated infants. Enzyme from the gastric mucosa of adult rats and enzyme evoked with hormones were more stable than that from infant rats.     

Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.