Helicobacter pylori infection, serum pepsinogens as markers of atrophic gastritis, and leukocyte telomere length: a population-based study.

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.

Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (Monopterus albus Zuiew).

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.

The panoramic picture of pepsinogen gene family with pan-cancer.

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.

Mannose 6-phosphate-independent targeting of lysosomal enzymes in I-cell disease B lymphoblasts.

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6-phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.

Transcription of the gene for a pepsinogen, PEP1, is regulated by white-opaque switching in Candida albicans.

Cells of Candida albicans WO-1 spontaneously switch between a white and opaque CFU, and this phase transition involves a dramatic change in cellular phenotype. By using a differential hybridization screen, an opaque-specific cDNA, Op1a, which represents the transcript of a gene regulated by switching, has been isolated. The gene for Op1a is transcribed by opaque but not by white cells. The nucleotide sequence of the Op1a cDNA reveals over 99% base homology with an acid protease gene of C. albicans, and the predicted amino acid sequence demonstrates that the product of this gene is a member of the family of pepsinogens, which possess a hydrophobic leader sequence for secretion and two catalytic aspartate domains. Southern blots of both genomic DNA digested with 14 different endonucleases and electrophoretically separated chromosomes were probed with the Op1a cDNA. No polymorphisms were detected in either case between white and opaque cells, suggesting that no genomic reorganization occurs in the proximity of the gene during the white-opaque transition. Although transcription of Op1a correlates with the high levels of extracellular protease activity in opaque cell cultures and the absence of activity in white cell cultures, stimulation of extracellular protease activity by addition of serum albumin is not accompanied by Op1a transcription in cultures of WO-1 white cells or cultures of two additional clinical isolates of C. albicans, suggesting that expression of one or more other protease genes is stimulated in these cases. The results demonstrate that transcription of the Op1a gene is under the rigid control of switching in strain WO-1.

Purification and characterization of pepsinogens from the gastric mucosa of African coelacanth, Latimeria chalumnae, and properties of the major pepsins.

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.

DNA methylation and expression of the rat pepsinogen gene in embryonic, adult, and neoplastic tissues.

The relationship between methylation and expression of rat pepsinogen 1 (Pg1) genes was investigated in various tissues. On Northern blotting with a Pg1 complementary DNA probe, Pg1 mRNA was detected only in the glandular stomach of normal rats. Methylation analysis with Msp1/HpaII and Hha1 revealed tissue specific methylation patterns of Pg1 genes with less methylated in the stomach than in other normal tissues not expressing the genes. During stomach development, there was a progressive increase in the Pg1 mRNA level that almost coincided with change in the mucosal pepsinogen level and progressive demethylation after the onset of transcription. Thus, there was an inverse correlation between methylation and expression of Pg1 genes, suggesting a role of DNA methylation in Pg1 gene regulation during normal differentiation, although not its primary role in gene activation. There was no detectable Pg1 mRNA in either primary or transplanted stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine. The methylation patterns of Pg1 genes were different from those of normal tissues that expressed the gene and of those that did not and no simple correlation was observed between methylation and expression of Pg1 genes. This result is consistent with a previous finding that DNA methylation is deranged in tumor cells.

Purification and molecular cloning of aspartic proteinases from the stomach of adult Japanese fire belly newts, Cynops pyrrhogaster.

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.

Structure, molecular evolution, and hydrolytic specificities of largemouth bass pepsins.

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.

Regulation of pepsinogen gene expression in epithelial cells of vertebrate stomach during development.

Pepsinogens are zymogens of pepsins, aspartic proteases working as digestive enzymes in the vertebrate stomach, of which biological and molecular properties have been extensively studied. In developmental biology, pepsinogens offer excellent molecular markers of differentiation of stomach epithelial cells, since their expression is strictly limited to those cells and there are some isozymes that are expressed in developmental stage-specific manner. It is now well established that the expression of embryonic chicken pepsinogen (ECPg) gene is regulated by epithelial-mesenchymal interactions: it is mesenchyme that determines the expression pattern of ECPg along the digestive tract, by supporting or inhibiting the intrinsically endowed ability of epithelial cells to express it. In the present review article, I will describe recent molecular biological and experimental embryological consequences of our studies on the regulation of ECPg expression by mesenchymal cells, with special attention to the nature of mesenchymal factors and the molecular mechanisms of reactivity of epithelial cells to the mesenchymal influences.

Cloning and sequencing of rhesus monkey pepsinogen A cDNA.

The complete nucleotide sequence of Rhesus monkey (Macaca mulatta) pepsinogen A (PGA) cDNA was determined from two partially overlapping cDNA clones, covering the whole coding sequence and part of the flanking sequences. The nucleotide and deduced amino acid sequences were compared to known PGA sequences from other species. The degree of similarity with human PGA appeared to be 96% at the nucleotide sequence level and 94% at the amino acid sequence level. In the coding region the divergence was highest in the activation peptide. The amino acid sequence similarity between Japanese monkey (Macaca fuscata) PGA and Rhesus monkey PGA was shown to be 99%. Using the cDNA as probe in Southern hybridization of EcoRI-digested human and Rhesus monkey genomic DNAs, PGA patterns with inter-individual differences were observed. The hybridization patterns are compatible with the existence of a PGA multigene family in both species.

Nucleotide sequence and expression in Escherichia coli of cDNA of swine pepsinogen: involvement of the amino-terminal portion of the activation peptide segment in restoration of the functional protein.

A clone, pSPcA2, which carries the full-length swine pepsinogen cDNA was isolated. The coding sequence comprised the signal peptide [15 amino acids (aa)], the activation peptide segment (44 aa) and mature pepsin (327 aa). The deduced amino acid sequence agrees with the published sequence with two exceptions. Asparagine instead of aspartate is present at aa positions 19 and 308. Two types of plasmids, pAS and pUCtacSPc series, were constructed for expressing swine pepsinogen cDNA. These plasmids directed the synthesis of polypeptides which were detected by employing an antibody to swine pepsinogen. However, all the polypeptides formed aggregates and showed no acid protease activity. Only the protein directed by pAS5 regained the acid protease activity after renaturation procedures. The activity was completely inhibited by pepstatin. Furthermore, the renatured pAS5 protein was spontaneously converted to pepsin under acidic conditions. The presence of Arg-8 in the activation peptide segment appears important for the stabilization of the pepsinogen molecule.

Identification of a Glu greater than Lys substitution in the activation segment of human pepsinogen A-3 and -5 isozymogens by peptide mapping using endoproteinase Lys-C.

The isozymogens PGA-3 and PGA-5 of human pepsinogen A were digested with endoproteinase Lys-C. The peptides were separated by reverse-phase HPLC. PGA-5 showed a peak strongly absorbing at 254 nm absent in PGA-3. Analysis of amino acid composition using the Pico-Tag methodology combined with DABITC-sequencing reveals the sequence Tyr-Phe-Pro-Gln-Trp-Lys (peptide 37-43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu greater than Lys mutation at position 43 in the activation segment of PGA-5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.

Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus.

A cDNA was isolated from an adult Haemonchus contortus cDNA expression library the deduced amino acid sequence of which showed significant homology to mammalian pepsinogen sequences. The library was screened with antisera raised against Haemonchus galactose-containing glycoprotein complex, a gut membrane protein complex with aspartyl proteinase activity which has shown considerable potential as a protective antigen. The amino acid sequence obtained corresponded very closely in part to the N-terminal amino acid sequences of two polypetides within the complex. The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages. The cDNA was expressed in E. coli, and antibody produced to the recombinant protein bound to the luminal surface of the gut in the adult parasite. The proteinase may play a central role in digesting the blood meal and is considered a potential sub-unit vaccine candidate.

Expression of protease genes in the gastric mucosa during aging.

The current investigation examines the changes in the expression of pepsinogen C and cathepsin D and E genes in the gastric mucosa during aging and following physiological stimuli of fasting and refeeding. Northern blot analysis of gastric mucosal RNA, isolated from overnight fasted 6-, 12-, and 24-month-old male Fischer 344 rats, revealed that although steady-state mRNA levels of each of these protease remained essentially unchanged between 6 and 12 months of age, in 24-month-old rats the levels were decreased by about 60%, when compared with their younger counterparts. Interestingly, the relative concentration of beta-actin mRNA--but not 18s rRNA--in 12- and 24-month-old rats was also decreased by 23% and 37%, respectively, when compared with 6-month-old animals. In the next set of experiments, groups of young (3 month) and aged (24 month) rats were either fed throughout (controls) or fasted for 48 h and then fed for 6 h and 24 h. Gastric mucosal RNA from each group was assayed for steady-state mRNA levels of pepsinogen C and cathepsin D. Results showed that whereas in young rats fasting decreased pepsinogen C and cathepsin D mRNAs by 80-85%, in aged rats only pepsinogen mRNA was significantly decreased (45%), when compared with the corresponding initial fed controls. In both age groups, refeeding increased pepsinogen C mRNA concentration essentially to the respective initial fed levels. In contrast, cathepsin D mRNA levels in the gastric mucosa of aged rats was affected neither by fasting nor by refeeding. Our current data show that aging not only diminishes the expression of protease genes in the gastric mucosa, but also the expression of one of its structural genes, beta-actin. In addition, responsiveness of these protease genes to the physiological stimuli of fasting and refeeding is also attenuated by aging. We postulate that these age-related changes may in part be due to diminished differentiation of gastric mucosal cells.

Monkey pepsinogens and pepsins. VII. Analysis of the activation process and determination of the NH2-terminal 60-residue sequence of Japanese monkey progastricsin, and molecular evolution of pepsinogens.

Japanese monkey progastricsin was shown to be activated to gastricsin exclusively by a two-step process through an intermediate form. The occurrence of this process was substantiated by the isolation of the intermediate form and released peptides. By NH2-terminal sequence analyses of these protein and peptide species, the amino acid sequence of the 43-residue activation segment (propart) was determined to be as follows: (Formula: see text) The NH2-terminal 26-residue peptide was released first, resulting in generation of the intermediate form. The subsequent release of peptides, residues Nos. 27-40 and 27-43, generated two gastricsins as the final products. This two-step process of activation of Japanese monkey progastricsin is in striking contrast to the one-step activation process occurring exclusively for pepsinogen A of the same monkey species. The course of molecular evolution of pepsinogens including progastricsins was deduced from the amino acid sequences of their activation segments by constructing phylogenic trees. The trees divided pepsinogens into 3 clusters, i.e., pepsinogens A, progastricsins and prochymosin, showing that these three groups diverged from one another very early on in the course of the evolution of pepsinogens.

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.

Developmental changes of DNA methylation pattern of embryonic chick pepsinogen gene.

Embryonic chick pepsinogen (ECPg) is one of the pepsinogen isozymogens and its expression is restricted to epithelial cells of the embryonic chick proventriculus (glandular stomach). To examine whether DNA methylation is involved in the regulation of organ-specific and developmental stage-specific expression of ECPg gene, we analyzed the extent of methylation of ECPg gene in normal embryonic and hatched chick organs using methylation-sensitive restriction enzymes. In the proventriculus some CCGG sites underwent demethylation in the gene region after the onset of transcription of the ECPg gene. By contrast, these sites were kept methylated throughout the development in the other organs which do not express ECPg gene. GCGC sites in the gene region became methylated in organs which do not express the ECPg gene, after the initiation of transcription of the ECPg gene in the proventriculus. In the proventriculus, GCGC sites, which were methylated in other organs, were kept unmethylated throughout the development. The methylation state of CpG sites showed no change in the proventriculus of a chick 2 weeks after hatching when the expression of the ECPg gene had completely ceased. The data presented here demonstrate that the DNA methylation is involved in the regulation of organ-specific expression, but stage-specific expression might be brought about by some other mechanisms.

Biochemical and immunological characterizations of rat pepsinogens and pepsins.

Biochemical and immunological properties of two kinds of pepsinogens isolated from the gastric mucosal extracts of adult Wistar rats were studied. Their activated enzymes were prepared from the zymogens using a DEAE-Sepharose CL-6B column. The isoelectric points of pepsinogens I and II were estimated to be 3.90 and 3.75, respectively, by isoelectric focusing, and those of pepsins I and II to be 3.60 and 3.45, respectively. Amino acid compositions of the two pepsinogens or pepsins were strikingly similar to each other and neither pepsinogen I nor II contained organic phosphate. The biochemical properties of rat preparations compared with porcine pepsinogens A and C and pepsins A [EC 3.4.23.1] and C [EC 3.4.23.3] showed that rat pepsinogens and pepsins resembled porcine pepsinogen C and pepsin C, respectively. Pepsinogens I and II were demonstrated to share a similar immunogenic molecular structure by double diffusion analysis and Laurell immunoelectrophoresis. Rabbit antipepsinogen I serum cross-reacted with the mouse preparation but did not with the rabbit and porcine preparations. The possibility of the genetically controlled occurrence of pepsinogens I and II in the rat is discussed.

Isolation of human, swine, and rat prepepsinogens and calf preprochymosin, and determination of the primary structures of their NH2-terminal signal sequences.

The total RNAs were extracted from human, swine, rat, and calf gastric mucosae, and translated in vitro in the presence of radiolabeled amino acids using a wheat germ cell-free system. Upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the translation products, a protein band with a molecular weight of about 43,000 was obtained in each case as one of the major products. These products could be specifically immunoprecipitated with a corresponding anti-pepsinogen or anti-chymosin antiserum. Radiosequence analysis of these translation products purified by SDS-polyacrylamide gel electrophoresis showed that each of them is a precursor form, i.e., prepepsinogen or preprochymosin, having an amino-terminal extension peptide (signal sequence) comprising 15 (human and swine) or 16 (rat and calf) amino acid residues. The primary structures of these signal sequences were determined to be as follows: (Sequence: see text). These signal sequences share common characteristics with those of other pre-secretory proteins, i.e., the presence of positive charges in the NH2-terminal region, hydrophobic amino acid clusters in the interior part, and amino acids with short side chains at the site of cleavage by the signal peptidase.