Purification and characterization of goat pepsinogens and pepsins

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH₂-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.     

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.     

Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences

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.     

Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

Purification, characterization, and amino acid sequences of pepsinogens and pepsins from the esophageal mucosa of bullfrog (Rana catesbeiana)

Two pepsinogens (pepsinogens 1 and 2) were purified from the esophageal mucosa of the bullfrog (Rana catesbeiana), and their molecular weights were determined to be 40,100 and 39,200, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 70-residue sequences of both pepsinogens are the same, including the 36-residue activation segment. Furthermore, a cDNA clone encoding frog pepsinogen was obtained and sequenced, which permitted deduction of the complete amino acid sequence (368 residues) of one of the pepsinogen isozymogens. The calculated molecular weight of the protein (40,034) coincided well with the values obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results are incompatible with the previous report (Shugerman R. P., Hirschowitz, B. I., Bhown, A. S., Schrohenloher, R. E., and Spenney, J. G. (1982) J. Biol. Chem. 257, 795-798) that the major pepsinogen isolated from the bullfrog esophageal gland is a unique "mini" pepsinogen with a molecular weight of approximately 32,000-34,000. The two pepsinogens were immunologically indistinguishable from each other and related to human pepsinogen C. The deduced amino acid sequence was also more homologous with those of pepsinogens C than those of pepsinogens A and prochymosin. These results indicate that the frog pepsinogens belong to the pepsinogen C group. They were both glycoproteins, and therefore, this is the first finding of carbohydrate-containing pepsinogens C. Both pepsinogens were activated to pepsins in the same manner by an apparent one-step mechanism. The resulting pepsins were enzymatically indistinguishable from each other, and their properties resembled those of tuna pepsins.     

Pepsinogen C and pepsin C from gastric mucosa of Japanese monkey. Purification and characterization.

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.     

Olfactory discrimination of urine odors from five species by tufted capuchin (Cebus apella)

Urine collected from New World monkeys (tufted capuchin, squirrel monkey, cotton-top tamarin) and Old World monkeys (rhesus macaque, Japanese macaque), was used as the odor stimuli. Two adult tufted capuchins were trained on a successive odor-discrimination task with two odors, 30 trials each, in one session per day. Responses to one of the two odors (S+) were reinforced by sweet water. The monkeys failed to discriminate between the urine from the two species of macaques but could discriminate among the urine from the three species of New World monkeys. Furthermore, similarity of urine odors was analyzed by multi-dimensional scaling (MDS) and a cluster analysis. These analysis suggested that the tufted capuchin can distinguish differences among New World monkeys but not between the macaques. The natural distribution of the tufted capuchin overlaps with that of other New World monkeys, but it does not overlap with those of Old World monkeys. Consequently, it can be concluded that this difference in olfactory recognition in the tufted capuchin reflects their sympatric and allopatric relationships with other species.     

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.     

Difference of activation processes and structure of activation peptides in human pepsinogens A and progastricsin

The activation processes of two human pepsinogens A (pepsinogens 3 and 5) and progastricsin were compared with special attention to pepsinogens 3 and 5. Each zymogen was converted to pepsin in a stepwise manner through intermediate forms. In pepsinogens A, the major cleavage site was the Leu23-Lys24 bond and this cleavage was suggested to occur intramolecularly. When each of the pepsins A was added to the corresponding pepsinogen A exogenously, the latter was rapidly converted to pepsin, releasing the 47-residue intact activation segment. In this case, the Leu47-Val48 bond connecting the activation segment with the pepsin moiety was cleaved by an intermolecular reaction. On the other hand, when the pepsinogen A-pepstatin complex was attacked by each corresponding pepsin A added exogenously, significant cleavage by an intermolecular reaction occurred at the Asp25-Phe26 bond, generating the Phe26-intermediate form. These shifts of the cleavage sites in pepsinogens A depending on the activation conditions are likely to correlate with the conformation of the activation segment. These results can be explained consistently in terms of a proposed molecular model of activation.     

Occurrence of cathepsin D isozymes with different specificities in monkey skeletal muscle

Cathepsin D was highly purified from the skeletal muscle of Japanese monkey (Macaca fuscata fuscata) by a procedure including affinity chromatography on concanavalin A-Sepharose and pepstatin-Sepharose, and then resolved into ten isozymes (A through J) by isoelectric focusing. When examined for specificity toward oxidized insulin B chain, isozyme A was highly specific and cleaved exclusively the Leu15-Tyr16 bond, whereas isozyme F was less specific, cleaving the Leu15-Tyr16 and Glu13-Ala14 bonds, with slower cleavages at several other bonds. These results demonstrate for the first time the occurrence of isozymes with different specificities among cathepsin D isozymes obtained from a single source.     

The chromosomal location of rDNA in selected lower primates.

Hybridization in stiu was used to identify the chromosomes that carry rDNA in representative lower primates, including the baboons, Papio cynocephalus and Papio hamadryas; the colobus monkey, Colobus polykomos; the tree shrew, Tupaia glis; the lemur, Lemur fulvis; the saki, Pithecia pithecia; the marmoset, Saguinus nigricollis, and the spider monkey, Ateles geoffroyi. The marker chromosome, common to the Cercopithecines studied to date, carries the rDNA in the baboons. Another marker chromosome carries rDNA in a South American species, the spider monkey. A multichromosomal distribution of rDNA was demonstrated in the tree shrew, lemur, saki, and marmoset. None of the rDNA-containing chromosomes in the prosimians and New World monkeys show homology to the chromosomes that carry rDNA in the Hominids, Pongids, or Old World monkeys.     

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Molecular Genetics of Spectral Tuning in New World Monkey Color Vision

Although most New World monkeys have only one X-linked photopigment locus, many species have three polymorphic alleles at the locus. The three alleles in the squirrel monkey and capuchin have spectral peaks near 562, 550, and 535 nm, respectively, and the three alleles in the marmoset and tamarin have spectral peaks near 562, 556, and 543 nm, respectively. To determine the amino acids responsible for the spectral sensitivity differences among these pigment variants, we sequenced all exons of the three alleles in each of these four species. From the deduced amino acid sequences and the spectral peak information and from previous studies of the spectral tuning of X-linked pigments in humans and New World monkeys, we estimated that the Ala → Ser, Ile → Phe, Gly → Ser, Phe → Tyr, and Ala → Tyr substitutions at residue positions 180, 229, 233, 277, and 285, respectively, cause spectral shifts of about 5, −2, −1, 8, and 15 nm. On the other hand, the substitutions His → Tyr, Met → Val or Leu, and Ala → Tyr at positions 116, 275, and 276, respectively, have no discernible spectral tuning effect, though residues 275 and 276 are inside the transmembrane domains. Many substitutions between Val and Ile or between Val and Ala have occurred in the transmembrane domains among the New World monkey pigment variants but apparently have no effect on spectral tuning. Our study suggests that, in addition to amino acid changes involving a hydroxyl group, large changes in residue size can also cause a spectral shift in a visual pigment. Received: 17 July 1997 / Accepted: 7 December 1997     

Comparison between prochymosin and pepsinogen from lamb and calf

1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 2. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. Lamb prochymosin and pepsinogen are both glycosylated.     

On the activation of the canine pepsinogens.

Acidification induces a conversion of canine pepsinogens by a sequential mechanism to the active pepsins. Activation in the presence of pepstatin, which strongly inhibits the pepsins but does not prevent the first step of activation, allows the isolation of the peptide released in this first step. This peptide inhibits the milk clotting activity of canine and also porcine pepsin. Canine pepsins obtained in the absence of pepstatin were characterized by amino acid composition, molecular weight, and activity against hemoglobin and milk and compared with those of other mammalian pepsins.     

Amphibian pepsinogens: purification and characterization of xenopus pepsinogens, and molecular cloning of Xenopus and bullfrog pepsinogens

Two pepsinogens (Pg C and Pg A) were isolated from the stomach of adult Xenopus laevis by Q-Sepharose, Sephadex G-75, and Mono-Q column chromatographies. Autolytic conversion and activation of the purified Pgs into the pepsins were examined by acid treatment. We determined the amino acid sequences from the NH2-termini of Pg C, pepsin C, Pg A, and pepsin A. Based on the sequences, the cDNAs for Pg C and Pg A were cloned from adult stomach RNA, and the complete amino acid sequences of the Pg C and Pg A were predicted. In addition, a Pg A cDNA was cloned from the stomach of adult bullfrog Rana catesbeiana, and the primary structure of the Pg A was predicted. Molecular phylogenetic analysis showed that such anuran Pg C and Pg A belong to the Pg C group and the Pg A group in vertebrates, respectively. The molecular properties of Pg C and Pg A, such as size, sequences of the activation peptide and active site, profile of autolytic activation, and pH dependency of proteolytic activity of the activated forms, pepsin C and pepsin A, resemble those of Pgs found in other vertebrates. However, the hemoglobin-hydrolyzing activity of Xenopus pepsin C is completely inhibited in the presence of equimolar pepstatin, an inhibitor of aspartic proteinases. Thus, the Xenopus pepsin C differs significantly from other vertebrate pepsins C in its high susceptibility to pepstatin, and closely resembles A-type pepsins.     

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.     

Development and annotation of shotgun sequence libraries from New World monkeys

The draft genome sequences of several primates are available, providing insights into evolutionary and anthropological research. However, genomic resources from New World monkeys are conspicuously lacking. To date, the genomes of only two platyrrhine species, the common marmoset and the Bolivian squirrel monkey, have been fully sequenced. This is especially limiting for comparative genomics research, considering that New World monkeys are the most speciose primate group, and platyrrhine genetic diversity is comparable to that of the catarrhines (i.e. apes and Old World monkeys). Here, we present the generation and annotation of numerous sequence reads from the genomes of Spider monkey (Ateles belzebuth), Owl monkey (Aotus lemurinus) and Uakari (Cacajao calvus), representing the three platyrrhine families, Atelidae, Cebidae and Pitheciidae, respectively. These sequencing reads were developed from gDNA shotgun libraries containing over 3000 individual sequences with an average length of 726 bps. Of these sequences, 1220 contain <20% repeats, and thus are potentially highly useful phylogenetic markers for other platyrrhine species. Among them, a large number of sequencing reads were found to match unique regions within the human (2462 sequences) and the marmoset (2829 sequences) genomes. In particular, the majority of these sequencing reads are from putatively neutrally evolving intergenic regions. Thus, they are likely to be highly informative for inferring neutral evolutionary patterns and genomic evolution for other New World monkeys.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.