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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.

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., (Copyright 2000 Academic Press.)

The complete primary structure of the major component of tuna pepsinogens was determined by conventional protein chemistry methods. It was composed of a prosegment of 37 residues and a pepsin moiety of 323 residues, having a relative molecular mass of 39,364. The essential aspartyl residues in the active site and the three disulfide bonds common to other pepsinogens were conserved; however, several unique substitutions and/or deletions characteristic of tuna pepsinogen were found at various positions, especially in the prosegment and subsite regions, as compared with the sequences of other pepsinogens, which may affect the rate of activation of the zymogen, and/or the catalytic function and substrate specificity of the enzyme. Tuna pepsinogen is the least acidic among pepsinogens. The sequence identity between tuna pepsinogen and other pepsinogens ranged from 45 to 52%. A phylogenetic tree based on the primary structures suggested that tuna pepsinogen diverged from the pepsinogen A and prochymosin groups in an early period of pepsinogen evolution.

Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are two factors considered to be involved in the morphogenesis of several organs. To understand the role of HGF and KGF in the stomach development, we analyzed changes in the levels of expression of the genes for the two growth factors and their receptors in the fetal rat stomach by competitive RT-PCR. Resembling our previous results for HGF, the expression of the genes for KGF and its receptor was observed in the mesenchyme and epithelium of 16.5 day fetal stomach, respectively, indicating the possibility that KGF mediates the epithelial-mesenchymal interaction in the early stage of stomach development. The developmental profile of the expression of the genes for the two growth factors and their receptors were different, indicating a difference in their roles; the genes for HGF and c-met, the receptor for HGF, are expressed mainly during the morphogenetic period, while the genes for KGF and its receptor mainly after the morphogenetic period. Thus, it is probable that HGF controls the proliferation of epithelial cells during the morphogenetic process. The expression of the genes for KGF and its receptor is not correlated with epithelial proliferation during morphogenesis, but it does appear to be linked with epithelial differentiation. These results, together with the absence of significant mitogenic effect of KGF on the epithelial cells of the fetal rat glandular stomach in vitro, suggest a role for KGF as a differentiation factor. In addition, the expression profile of the genes for KGF and its receptor towards the end of fetal period appears to be closely correlated with that of mesenchymal cell proliferation, suggesting another role for the growth factor that is not regulated by the epithelial-mesenchymal interaction.

Five pepsinogens were purified from the gastric mucosa of eight species of Asian macaques. The chromatographic behavior of each pepsinogen was essentially the same but differed from human and other mammalian pepsinogens. The major pepsinogen in each species was pepsinogen A-1, accounting for 29-48% of the total. Amino acid compositions and some enzymatic properties of derived pepsins were similar for the various monkey species. This high degree of similarity confirms that these species are closely related to one another.

Adrenalectomy caused a significant decrease in the mucosal level of pepsinogen in the adult rat stomach. This decrease was mainly due to a reduction in the level of the main component of rat pepsinogen isozymogens, namely, Pg1 and in the level of expression of Pg1 mRNA, with a maximal effect being evident 48 hours after operation. Immunohistochemical analysis revealed that the level of expression of Pg1 in the pepsinogen-producing cells was decreased throughout the stomach mucosa and this decrease was especially marked in the immature chief cells located in the glandular neck of the fundic mucosa. Adrenalectomy also increased the proliferation of mucosal cells and suppressed the expression of cathepsin E and class III mucin, markers of differentiated stomach mucosa. All these changes were reversed by hydrocortisone replacement and the extent of the recovery of the level of Pg1 mRNA was dependent on the dose of hydrocortisone, indicating the transcriptional control of the Pg1 gene by hydrocortisone. The observed results suggest that the continuous presence of glucocorticoids is necessary for active transcription of Pg1 gene in fully differentiated stomach mucosa and that glucocorticoids are important regulators of both the function and the morphology of stomach mucosal cells.

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 for pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount of 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.

Procathepsin E and progastricsin were purified from the gastric mucosa of the guinea pig. They were converted to the active form autocatalytically under acidic conditions. Each active form hydrolyzed protein substrates maximally at around pH 2.5. Pepstatin inhibited cathepsin E very strongly at an equimolar concentration, whereas the inhibition was much weaker for gastricsin. Molecular cloning of the respective cDNAs permitted us to deduce the complete amino acid sequences of their pre-proforms; preprocathepsin E and preprogastricsin consisted of 391 and 394 residues, respectively. Procathepsin E has unique structural and enzymatic features among the aspartic proteinases. Lys at position 37, which is common to various aspartic proteinases and is thought to be important for stabilizing the activation segment, was absent at the corresponding position, as in human procathepsin E. The rate of activation of procathepsin E to cathepsin E is maximal at around pH 4.0. It is very different from the pepsinogens and may be correlated with the absence of Lys37. Native procathepsin E is a dimer, consisting of two monomers covalently bound by a disulfide bridge between 2 Cys37. Interconversion between the dimer and the monomer was reversible and regulated by low concentrations of a reducing reagent. Although the properties of the dimeric and monomeric cathepsins E are quite similar, a marked difference was found between them in terms of their stability in weakly alkaline solution: monomeric cathepsin E was unstable at weakly alkaline pH whereas the dimeric form was stable. The generation of the monomer was thought to be the process leading to inactivation, hence degradation of cathepsin E in vivo.

The developmental changes in the expression of monkey pepsinogens and structural differences between the polypeptides were investigated. Monkey pepsinogens included five different components, namely, pepsinogens A-(1-4) and progastricsin. Their respective relative levels and specific activities changed significantly during development. The sequential expression of genes for type-A pepsinogens was particularly noteworthy. Pepsinogen A-3 was the major zymogen at the newborn stage, accounting for nearly half of the total pepsinogens at this stage. Pepsinogen A-2 became predominant at the 4-month stage, and pepsinogen A-1 predominated at the juvenile and adult stages. Enzymatic properties of pepsinogens A-1, A-2 and A-3 were similar but not identical to those of pepsinogen A-4 and progastricsin, in particular with respect to the activation processes. Each pepsin digested various protein substrates but some differences in specificity were evident. cDNA clones for five pepsinogens were isolated, and the nucleotide sequences were determined. Each cDNA contained leader, pro, and pepsin regions that encoded 15, 47, and 326 amino acid residues, respectively, with the exception of the cDNA for progastricsin in which the pro and pepsin regions encoded 43 and 329 amino acid residues, respectively. Type-A pepsinogens exhibited a high degree of similarity, with over 96% of bases in the nucleotide sequences of the protein-coding regions being identical. Northern analysis revealed that the level of expression of genes for type-A pepsinogens and for progastricsin was significant at the fetal stage and increased with development.

In order to clarify the structure and development of rabbit pepsinogens, purification and molecular cloning of these proteins were performed at various developmental stages. Several pepsinogens were isolated, and they were classified as pepsinogens F and M, and into pepsinogen groups I, II, and III. The relative levels and specific activities of the various pepsinogens changed significantly during development. Pepsinogens F and M were present only at the early postnatal stage, and their level was higher than those of other pepsinogens at this stage. Pepsinogens in groups I, II, and III were the predominant zymogens at the late postnatal stage. cDNA clones encoding all of these pepsinogens were obtained, with the exception of pepsinogens I and M, and the nucleotide sequences were determined. Each cDNA contained a leader region (signal peptide), a pro-region (activation segment), and a pepsin region, of 15, 44, and 328 residues, respectively, with the exception of the cDNA for pepsinogen F in which the pro- and pepsin regions were composed of 43 and 330 residues, respectively. Pepsinogens in groups II and III exhibited a high degree of similarity with one another, whereas many substitutions were found in pepsinogen F. A unique substitution in the activation segment of pepsinogen F, namely, Gly----Asp at position 21, was found, which made the structural features of this segment more specific. A phylogenic tree was constructed from the differences in nucleotide sequences and showed clearly that each pepsinogen in groups II and III could be classified as pepsinogen A, a major pepsinogen in mammals. Pepsinogen F diverged significantly from these groups and may be a new type of pepsinogen. Northern analysis revealed that the expression of the gene for pepsinogen F was restricted to the early postnatal stage, and the expression of genes for pepsinogens in groups II and III was detected predominantly at later stages, a result that shows the switching of gene expression from fetal pepsinogen to adult pepsinogens during development.

The amino acid sequences in the NH2-terminal region and some other parts of human gastric cathepsin E were investigated. The NH2-terminal sequencing revealed that the cathepsin E preparation which had been activated at pH 4.0 contained one major and one minor isozymes in an approximate molar ratio of 3:1. The NH2-terminal sequence of the former was very similar to but partly different from that predicted from cDNA sequencing by Azuma et al., whereas the latter had an NH2-terminal sequence identical with the predicted sequence. These results provide structural evidence for the presence of at least two isozymic forms in human gastric cathepsin E. In addition, the site of carbohydrate attachment was elucidated by isolation and analysis of a glycopeptide fraction from an enzymatic digest of cathepsin E. A single carbohydrate chain was deduced to be attached to the asparagine residue at position 34 in the major isozyme and to the corresponding asparagine residue in the minor isozyme.

Pepsinogens were purified from the gastric mucosa of the crab-eating monkey, Macaca fascicularis. Eight pepsinogens were shown to be present disc-electrophoretically and they were termed pepsinogens I-a, I-b, III-1-a, III-1-b, III-2-a, III-2-b, III-3, and C, based on the nomenclature used for Japanese monkey pepsinogens. The molecular weights were 43,000 for pepsinogens I-a and I-b, 40,000 for pepsinogens III-1-a, III-1-b, III-2-a, III-2-b, and III-3, and 38,000 for pepsinogen C, as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Pepsinogens I-a and I-b contained carbohydrate amounting to about 4-5% by weight. Each was activated to pepsin by acidification at pH 2.0. Pepsinogen III-1 (a mixture of III-1-a and III-1-b) yielded a single pepsin, i.e. pepsin III-1, and pepsinogen III-2 (a mixture of III-2-a and III-2-b) also gave a single pepsin, i.e. pepsin III-2. The molecular weights were estimated to be 38,000 for pepsins I-a and I-b, 35,000 for pepsins III-1, III-2, and III-3, and 34,000 for pepsin C. Optimal pHs toward acid-denatured hemoglobin were 1.9, 2.3, 2.0, 2.0, and 2.3 for pepsins I-a, III-1, III-2, III-3, and C, respectively. Pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), and p-bromophenacyl bromide inhibited each pepsin. Amino acid compositions of the pepsinogens and pepsins were determined. Pepsinogen C and pepsin C were distinct from the other pepsinogens and pepsins in their high ratios of glutamic acid to aspartic acid, and leucine to isoleucine. Amino acid sequences of the amino (N)-terminal 14 residues of pepsinogens were determined by the manual Edman procedure. One to three substitutions of amino acids were observed in the 14-residue segments among the pepsinogens except for pepsinogen C. There were 7 amino acid substitutions between pepsinogens C and III-3. These results suggest that the amino acid substitutions in the N-terminal region contribute considerably to the heterogeneity of pepsinogens.

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N-acetyl-L-phenylalanyl-L-diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+, 1,2-epoxy-3-(p-nitrophenoxy)propane and p-bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins. The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2-terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28-56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.

Five pepsinogens were purified from gastric mucosa of Japanese monkey by DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Each was shown to be homogeneous by polyacrylamide disc gel electrophoresis. They were designated as pepsinogens I, II, III-1, III-2, and III-3, respectively, based on the elution profile on DEAE-cellulose chromatography. The molecular weights of pepsinogens I and II were 48,000 and 43,000, respectively, and those of the other three were 40,000. Each pepsinogen was converted to pepsin [EC 3.4.23.1] by acidification, and some characteristics, e.g. the pH dependence of activity, sensitivity to various inhibitors, stability to alkali, and hydrolytic activity toward N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine (APDT), were determined. The characteristics of pepsins I and II were the same, and those of pepsins III-1, III-2, and III-3 were similar. Pepsin III-3 showed high stability to alkali (pH 8.0), while the others were less stable. Each pepsin hydrolyzed APDT and was inhibited by acid protease-specific inhibitors, e.g. pepstain, diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), and p-bromophenacyl bromide. The compositions of pepsins I and II were the same, indicating that they are the same protein, and those of pepsins III-1, III-2, and III-3 resembled that of human pepsin. The diversity of pepsinogens and pepsins is discussed in comparison with pepsinogens and pepsins from other animals.

A new pepsinogen component, pepsinogen C, was purified from the gastric mucosa of Japanese monkey. The chromatographic behavior of this component on DE-32 cellulose was coincident with that of pepsinogen III-2 previously reported (1), and final purification was performed by large-scale polyacrylamide disc gel electrophoresis. The molecular weight was 35,000 as determined by gel filtration. The ratios of glutamic acid to aspartic acid and of leucine to isoleucine were higher than those of other Japanese monkey pepsinogens. The activated form, pepsin C, had a molecular weight of 27,000 and contained a large number of glutamic acid residues. The optimal pH for hemoglobin digestion was 3.0. Pepsin C could scarcely hydrolyze the synthetic substrate, N-acetyl-L-phenylalanyl-3, 5-diiodo-L-tyrosine (APDT). 1, 2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), p-bromophenacyl bromide, and diazoacetyl-DL-norleucine methyl ester (DAN) inhibited pepsin C [EC 3.4.23.3] in the same way as pepsin III-3 of Japanese monkey. The susceptibility to pepstatin of pepsin C was lower than that of pepsin III-3, and 500 times more pepstatin was required for the same inhibitory effect. The classification and nomenclature of Japanese monkey pepsinogens and pepsins are discussed.

A cathepsin D-like acid proteinase from human gastric mucosa was purified for the first time to homogeneity as examined by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 85,000 by gel filtration on Sephadex G-150 and, after treatment with 2-mercaptoethanol, about 38,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. These results indicate that the native enzyme is composed of two apparently identical monomeric units. The protein band could also be stained with the Schiff reagent after periodate oxidation, indicating that the proteinase is a glycoprotein. Analyses showed that the enzyme contains approximately 4 glucosamine and 16 mannose residues per molecule (M.W. 85,000). The amino acid composition generally resembled those of cathepsins D except for the lower contents of basic amino acid residues, especially lysine. It also showed some similarity to pepsinogens. The optimal pH was 2.3--3.5 with hemoglobin as a substrate. The enzyme was strongly inhibited, like pepsin, by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as well as by pepstatin and diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. The proteinase was stable in alkaline solution up to pH 9.0, and was rather stable to sequential acidification and neutralization. The acidification resulted in an increase of electrophoretic mobility towards the anode.