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.

Pepsinogens and pepsins from largemouth bass, Micropterus salmoides: purification and characterization with special reference to high proteolytic activities of bass enzymes.

Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin.

Pepsinogens and pepsins from Japanese seabass (Lateolabrax japonicus).

Three pepsinogens (PG1, PG2, PG3) were highly purified from the stomach of Japanese seabass (Lateolabrax japonicus) by ammonium sulfate fractionation, DEAE-Sephacel anion exchange column chromatography and Sephacryl S-200 gel-filtration. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed that the molecular masses of the three PGs were 35, 37, and 34kDa, and their isoelectric points were 5.3, 5.1, and 4.7, respectively. Zymography analysis showed that the three pepsinogens had different mobilities and enzymatic activities under native conditions. Pepsinogens converted into their active form pepsins under pH 2.0 by one-step pathway or stepwise pathway. All three pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor. The N-terminal amino acid sequences of the three pepsinogens were determined to the 30th, 30th and 28th amino acid residue and those of their corresponding active form pepsins were also determined to the 19th, 18th and 20th amino acid residue, respectively. All amino acid sequences of Japanese seabass PGs revealed high identities to reported fish and mammalian pepsinogens. The effective digestion of fish and shrimp muscular proteins by pepsins indicated their physiological function in the degradation of food proteins.

Study on pepsinogens and pepsins from snakehead (Channa argus).

Three pepsinogens (PG1, PG2, and PG3) were highly purified from the stomach of freshwater fish snakehead (Channa argus) by ammonium sulfate fractionation, anion exchange, and gel filtration. Two-dimensional gel electrophoresis and native-PAGE analysis revealed that their molecular masses were 37, 38, and 36 kDa and their isoelectric points 4.8, 4.4, 4.0, respectively. All of the pepsinogens converted into their active form pepsins under pH 2.0 by one-step pathway or stepwise pathway. The three pepsins showed maximal activity at pH 3.0, 3.5, and 3.0 with optimum temperature at 45, 40, and 40 degrees C, respectively, using hemoglobin as substrate. All of the pepsins were completely inhibited by pepstatin A, a typical aspartic proteinase inhibitor. The N-terminal amino acid sequences of the three pepsinogens were determined to the 34th, 25th, and 28th amino acid residues, respectively. Western blot analysis of the three PGs exhibited different immunological reactions.

Pepsinogens and pepsins from mandarin fish (Siniperca chuatsi).

Four pepsinogens (PG-I, PG-II, PG-III(a), and PG-III(b)) were highly purified from the stomach of the freshwater fish mandarin fish (Siniperca chuatsi) by ammonium sulfate fractionation, anion exchange, and gel filtration. The molecular masses of the four purified PGs were 36, 35, 38, and 35 kDa, respectively. All the pepsinogens converted into their active form pepsins within a few minutes under pH 2.0. The optimum pH and temperature of the four enzymes were 3.0-3.5 and 45-50 degrees C, using hemoglobin as a substrate. The N-terminal amino acid sequences of PG-I and PG-II were determined to the 12th and 17th amino acid residues, respectively. Western blot analysis using antisea bream polyclonal antibodies cross reacted with PG-I, PG-II, and PG-III(b) while no cross reaction with PG-III(a) was detected, suggesting the diversity of pepsinogens in fish.

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.

Purification and partial characterization of canine pepsinogen A and B.

OBJECTIVE: To purify and partially characterize various isoforms of canine pepsinogen (PG) from gastric mucosa. SAMPLE POPULATION: Stomachs obtained from 6 euthanatized dogs. PROCEDURE: Mucosa was scraped from canine stomachs, and a crude mucosal extract was prepared and further purified by use of weak anion-exchange chromatography, hydroxyapatite chromatography, size-exclusion chromatography, and strong anion-exchange chromatography. Pepsinogens were characterized by estimation of molecular weights, estimation of their isoelectric points (IEPs), and N-terminal amino acid sequencing. RESULTS: Two different groups of canine PG were identified after the final strong anion-exchange chromatography: PG A and PG B. Pepsinogens differed in their molecular weights and IER Pepsinogen B appeared to be a dimer with a molecular weight of approximately 34,100 and an IEP of 4.9. Pepsinogen A separated into several isoforms. Molecular weights for the various isoforms of PG A ranged from 34,200 to 42,100, and their IEPs ranged from 4.0 to < 3.0. The N-terminal amino acid sequence for the first 25 amino acid residues for PG A and B had good homology with the amino acid sequences for these proteins in other species. CONCLUSIONS AND CLINICAL RELEVANCE: Canine PG B and several isoforms of canine PG A have been purified. Availability of these PGs will facilitate development of immunoassays to measure PG in canine serum as a potential diagnostic marker for gastric disorders in dogs.

Primary structure, unique enzymatic properties, and molecular evolution of pepsinogen B and pepsin B.

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.

Multiplicities and some enzymatic characteristics of ape pepsinogens and pepsins.

Pepsinogen levels in ape stomachs were comparable to those in macaques and significantly higher than those in the stomachs of other mammals, including carnivores and ruminants. The occurrence of multiple forms of pepsinogens was remarkable. Nine, sixteen, eight, and fourteen pepsinogens were purified or partially purified from the gastric mucosa of a gibbon, orang-utan, gorilla, and chimpanzee, respectively. Most of these were type-A pepsinogens, and only one type-C pepsinogen was identified in each ape. The two types could be readily distinguished by staining for proteolytic activity on polyacrylamide gel electrophoresis (PAGE) in the presence/absence of pepstatin. Type-A pepsinogens were further divided into two subtypes. One subtype, constituting a major group of pepsinogens in apes, exhibited high hemoglobin-digestive activity. The other subtype was specified by a relatively high content of Lys and low hemoglobin-digestive activity. It is likely that pepsinogen-A genes have been duplicated several times as hominoids, including humans, evolved in the primate lineage. The presence of multiple pepsinogens in apes might be advantageous in the efficient digestion of a wide variety of foods.

Purification of two equine pepsinogens by use of high-performance liquid chromatography.

OBJECTIVES: To purify and characterize pepsinogens in equine gastric mucosa. SAMPLE POPULATION: Stomachs collected from 2 healthy horses at necropsy. PROCEDURE: After collection, stomachs were placed immediately in ice before storage at -48 C. After slow thawing, the mucosa was scraped off while the tissue was immersed in 0.1M potassium phosphate (pH 7.4) at 4 C, then was homogenized. The filtered extract was subjected to anion-exchange chromatography. Fractions that were found to contain pepsin or pepsinogen were further chromatographed. Individual fractions were tested for pepsinogen or pepsin content by monitoring proteolytic activity at pH 2 and 3, respectively. Fractions from all columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to confirm molecular weight of pepsinogens and pepsin. RESULTS: Two pepsinogens and at least 1 pepsin were purified from equine gastric mucosa. CONCLUSIONS: On the basis of molecular mass, equine gastric mucosa contains 2 pepsinogens. CLINICAL RELEVANCE: Results of this study will enable future development of an ELISA or radioimmunoassay for use in the diagnosis of equine gastric ulceration.

Purification of recombinant human pepsinogens and their application as immunoassay standards.

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.

Antimicrobial peptides derived from pepsinogens in the stomach of the bullfrog, Rana catesbeiana.

Three antimicrobial peptides, which had strong antimicrobial activity against a broad spectrum of microorganisms, were isolated from the stomach of the bullfrog, Rana catesbeiana. Two of the antimicrobial peptides were found to be derived from the N-terminal sequences of pepsinogen A and C prosequences. The amino acid sequences of the new antimicrobial peptides, named bullfrog pepsinogen A-derived antimicrobial peptide (bPaAP) and bullfrog pepsinogen C-derived antimicrobial peptide (bPcAP), were Gly-Val-Val-Lys-Val-Ser-Arg-Leu-Lys-Gly-Glu-Ser-Leu-Arg-Ala-Arg-Leu (MW 1865.5) and Ile-Ile-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-Ser-Met-Arg-Glu-Val-Met-Arg-A sp-His-Gly-Ile-Lys-Ala-Pro-Val-Val-Asp-Pro-Ala-Thr-Lys-Tyr (MW 3691.6), respectively. The bPaAP and bPcAP adopted 35% and 42% amphipathic alpha-helical structure in 50% trifluoroethanol, respectively, and were non-hemolytic up to a concentration of 200 microg/ml. Synthesized pepsinogen C prosequences of monkey and human, which had similar structural characteristics as bPaAP and bPcAP, also showed antimicrobial activity at concentrations of 10-200 microg/ml. The third peptide was buforin I, previously found in the stomach of the Asian toad, Bufo bufo gargarizans. These findings strongly suggest that peptides derived from the prosequences of pepsinogens, along with buforin I, may contribute to the antimicrobial function of the gastrointestinal mucosa of vertebrates, including human.

Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin.

When the mammalian aspartic proteinases, procathepsin D or pepsinogen, are expressed in Escherichia coli both accumulate in inclusion bodies. While pepsinogen is efficiently refolded in vitro, recovery of procathepsin D is limited by insolubility. We expressed procathepsin D and pepsinogen in E. coli, with E. coli maltose-binding protein (MBP) or thioredoxin (trx) fused to their C-termini (aspartic proteinase-MBP or aspartic proteinase-trx). The fusion proteins were still found in inclusion bodies. However, the recovery of soluble procathepsin D-MBP and procathepsin D-trx after refolding was facilitated by the bacterial fusion partners. Maltose-binding protein was more efficient than thioredoxin in increasing the recovery of soluble protein. The vector, pET23bMBPH6, can be used for general expression of heterologous proteins in E. coli. The vector includes a histidine tag at the C-terminus of MBP to allow one-step purification of the fusion proteins under denaturing conditions. After purification, the protein of interest can be cleaved from MBP with factor Xa protease and separated from the MBP partner. Refolded pepsinogen-MBP and pepsinogen-trx were enzymatically active, but procathepsin D-MBP and procathepsin D-trx were soluble but largely inactive. The results show that the limited recovery of activity upon refolding of procathepsin D is not the consequence of competing aggregation. Thus, the fusions do not necessarily facilitate native refolding, but they do enhance the recovery of soluble protein. Such fusions could provide a system to study, in soluble form, folding states which are otherwise inaccessible because of aggregation and precipitation.

Pepsinogens and pepsins from house musk shrew, Suncus murinus: purification, characterization, determination of the amino-acid sequences of the activation segments, and analysis of proteolytic specificities.

Three pepsinogens, namely, pepsinogens A, C-1, and C-2, were purified from gastric mucosa of adult house musk shrew (Suncus murinus) by conventional chromatographic and gel filtration procedures. The molecular masses were 40, 39, and 41 kDa for pepsinogens A, C-1, and C-2, respectively. Pepsinogen C-2 contains an Asn-linked carbohydrate chain(s) of about 2 kDa. Each pepsinogen was converted to pepsin through an intermediate form under acidic conditions. By NH2-terminal sequence analysis of these protein species, the amino acid sequences of activation segments (proparts) of pepsinogens A and C-1 were determined to be LYKVPLVKKKSLRQNLIENGLLKDFLAKHNVNPASKYFPTE and KVTKVTLKKFKSIRENLREQGLLEDFLKTNHYDPAQKYHFGDF, respectively. The similarity of these two sequences is nearly 50%. Each pepsin cleaved preferentially peptide bonds between hydrophobic and aromatic amino acids, or bonds on either side of these amino acids. Although each activation segment had several sites susceptible to pepsin action, activation proceeded by limited cleavages of the segment, presumably due to the steric inflexibility of the segment in native pepsinogen. The activity of pepsin A was inhibited completely in the presence of a more than equimolar amount of pepstatin, while a hundred-molar excess amount of pepstatin was needed for the complete inhibition of the activity of pepsins C-1 and C-2.

[Liquid chromatographic separation of pepsinogens].

In this work, we reported a method for the purification of pepsinogens from human gastric mucosa with high pressure gel filtration chromatography (HPLC) and medium pressure anion exchange chromatography (MPLC). First, the two fractions of Pg1 and mixture of PGI and PGII were separated from pepsinogens with HPLC on a Bio-Sil SEC-125 column within 50min. Then the mixture of fractions of PGI and PGII was further separated with MPLC on a Bio-Scale Q2 column within 30min.

The primary structure of the major pepsinogen from the gastric mucosa of tuna stomach.

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.

Purification and characterization of turtle pepsinogen and pepsin.

Pepsinogen was purified from the gastric mucosa of soft-shelled turtle (Trionyx sinensis) by a series of chromatographies on DEAE-cellulose, Sephadex G-100, and Q-Sepharose. Upon chromatography on Q-Sepharose, it was separated into nine isoforms. These isoforms showed a relative molecular mass of approximately 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoforms 4 through 9 contained carbohydrate (approx. 2% each). Insofar as they were examined, their NH2-terminal sequences differed only in showing substitution at a few positions. At pH 2.0, they were rapidly activated to the corresponding isoforms of pepsin in a stepwise manner. The nine isoforms showed similar specific activity toward hemoglobin and hydrolyzed N-acetyl-L-phenylalanyl-L-diiodotyrosine, a good substrate for pepsin A, at somewhat different rates. They were inhibited by pepstatin to various extents, more strongly than human pepsin C but less strongly than human pepsin A. All isoforms appeared to have similar cleavage specificity toward oxidized insulin B chain, which resembled those of both human pepsins A and C. A cDNA clone for one of the zymogen isoforms was isolated and sequenced. The amino acid sequence thus deduced was more homologous with those of mammalian pepsinogens A than those of mammalian pepsinogens C or prochymosin.

Purification of pepsinogens from human urine and electrophoretic analysis by caseogram print.

Pepsinogen (PG) A and C were purified from human urine, and analyzed by a highly sensitive detection method, "caseogram print". Purification was achieved by a series of conventional chromatographies and FPLC. A relatively large amount (13.2 mg) of PGA was purified from about 20 liters of urine. Purified PGA was separated by a Mono-Q column into each of its isozymogens. The elution order (PGA-5, 4+3, 2) corresponded to the order of electrophoretic migration. Although the concentration of urinary PGC was very low, a trace amount was purified and visualized by electrophoresis. The urinary and mucosal PGCs migrated at the same position, and urinary PGC was detected as two isozymogens similarly to mucosal PGC, suggesting that urinary and mucosal PGCs may be essentially identical.

Purification and characterization of porcine pepsinogen B and pepsin B.

Porcine pepsinogen B was prepared from extracts of adult porcine fundic mucosa. Immunoelectrophoresis showed no immunochemical cross-reactions between pepsinogen B and other porcine gastric zymogens. Pepsin B was purified after activation of the zymogen. The enzyme showed an optimum of general proteolytic activity at pH 3.0. Activation of pepsinogen B at pH 2 resulted in formation of the covalent intermediate (pseudo-pepsin B) by proteolytic cleavage of bond Met16p-Glu17p (pig pepsinogen A numbering, "p" indicates residues of the prosegment peptide). Pseudopepsin B was stable at pH 2. The intermediate was converted to pepsin B at pH 5.5. The overall activation of pepsinogen B was much slower than found for other investigated gastric zymogens. During the conversion of pepsinogen B to mature pepsin B a segment of 43 amino acid residues was cleaved from the N-terminal of pepsinogen B. The amino acid sequence of the prosegment and the first 24 residues of pepsin B was determined. Relative to porcine pepsinogen A, progastricsin, and prochymosin, the following degrees of identities were observed: 40, 55, and 51%.

Ostrich pepsinogens I and II: purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus.

Pepsins are a series of gastric proteases secreted as inactive precursors (pepsinogens) which are active at acidic pH. The aim of this study was to purify ostrich pepsin(ogen)s and to compare their biochemical and immunological characteristics with those of pepsin(ogen)s of mammalian and avian origin. Ostrich pepsinogens were purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained through acidification, and purified by chromatography on SP-Sephadex C-50. Amino acid compositions, N-terminal sequences, Ouchterlony double-diffusion as well as Western blot analysis were performed. Two pepsinogens were isolated and purified from the proventriculus of the ostrich, pepsinogens I and II. Both pepsinogens and pepsins were purified to homogeneity as shown by PAGE and SDS-PAGE, with SDS-PAGE revealing M(r) values of 40,400 and 41,900 for pepsinogens I and II, respectively. SDS-PAGE revealed M(r) values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. Ostrich pepsinogens I and II were found to have identical N-terminal sequences, with Asp as N-terminal amino acid. Amino acid compositions were obtained for both pepsinogens, with ostrich pepsinogen I being slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Pepsinogen II showed a pI of 4.29. Ostrich pepsinogens I and II were found to be immunologically separate entities, and no cross-reactivity was observed between anti-(ostrich pepsinogen I/II) sera and porcine pepsin/pepsinogen. The study indicates that only two pepsinogens are present in the ostrich. They differ in terms of electrophoretic mobility, molecular mass and immunological reactivity, but have been found to have identical N-terminal sequences. It is concluded that both pepsinogens belong to the pepsinogen A class of aspartyl proteases (EC 3.4.23.1).