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Five types of zymogens of pepsins, gastric digestive proteinases, are known: pepsinogens A, B, and F, progastricsin, and prochymosin. The amino acid and/or nucleotide sequences of more than 50 pepsinogens other than pepsinogen B have been determined to date. Phylogenetic analyses based on these sequences indicate that progastricsin diverged first followed by prochymosin, and that pepsinogens A and F are most closely related. Tertiary structures, clarified by X-ray crystallography, are commonly bilobal with a large active-site cleft between the lobes. Two aspartates in the center of the cleft, Asp32 and Asp215, function as catalytic residues, and thus pepsinogens are classified as aspartic proteinases. Conversion of pepsinogens to pepsins proceeds autocatalytically at acidic pH by two different pathways, a one-step pathway to release the intact activation segment directly, and a stepwise pathway through a pseudo-pepsin(s). The active-site cleft is large enough to accommodate at least seven residues of a substrate, thus forming S4 through S'3 subsites. Hydrophobic and aromatic amino acids are preferred at the P1 and P'1 positions. Interactions at additional subsites are important in some cases, for example with cleavage of kappa-casein by chymosin. Two potent naturally occurring inhibitors are known: pepstatin, a pentapeptide from Streptomyces, and a unique proteinous inhibitor from Ascaris. Pepsinogen genes comprise nine exons and may be multiple, especially for pepsinogen A. The latter and progastricsin predominate in adult animals, while pepsinogen F and prochymosin are the main forms in the fetus/infant. The switching of gene expression from fetal/infant to adult-type pepsinogens during postnatal development is noteworthy, being regulated by several factors, including steroid hormones.

Pepsinogens A and C, and prochymosin were purified from four species of adult New World monkeys, namely, common marmoset (Callithrix jacchus), cotton-top tamarin (Saguinus oedipus), squirrel monkey (Saimiri sciureus), and capuchin monkey (Cebus apella). The occurrence of prochymosin was quite unique since this zymogen is known to be neonate-specific and, in primates, it has been thought that the prochymosin gene is not functional. No multiple form has been detected for any type of pepsinogen except that two pepsinogen-A isozymogens were identified in capuchin monkey. Pepsins A and C, and chymosin hydrolyzed hemoglobin optimally at pH 2-2.5 with maximal activities of about 20, 30, and 15 units/mg protein. Pepsins A were inhibited in the presence of an equimolar amount of pepstatin, and chymosins and pepsins C needed 5- and 100-fold molar excesses of pepstatin for complete inhibition, respectively. Hydrolysis of insulin B chain occurred first at the Leu15-Tyr16 bond in the case of pepsins A and chymosins, and at either the Leu15-Tyr16 or Tyr16-Leu17 bond in the case of pepsins C. The presence of different types of pepsins might be advantageous to New World monkeys for the efficient digestion of a variety of foods. Molecular cloning of cDNAs for three types of pepsinogens from common marmoset was achieved. A phylogenetic tree of pepsinogens based on the nucleotide sequence showed that common marmoset diverged from the ancestral primate about 40 million years ago.

A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro-cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch., (Copyright 1999 Wiley-Liss, Inc.)

Acid cysteine proteinase in the eggs of the silkmoth, Bombyx mori, exists as an inactive proenzyme. This 47-kDa pro-BCP1 zymogen molecule can be processed in vitro into an enzymatically active 39-kDa BCP molecule. In this current study, the maximum rate of processing in vitro was achieved at approximately pH 4.0, at a temperature of 37 degrees C under reducing conditions. The rate of conversion was not affected by increasing concentrations of pro-BCP. We prepared immobilized BCP bound to AH-Sepharose and examined the activation. Immobilized pro-BCP was autolysed, although the rate of processing was slow, indicating that the reaction might be an intramolecular one. Kinetic experiments suggest that the mechanism is likely to involve a stepwise reaction, in which pro-BCP is converted to an active enzyme through intermediate forms releasing small peptides stepwise. The results suggest that autocatalytic cleavage (intramolecular) is a major processing step in the early stage of pro-BCP activation.

We have isolated and sequenced a 1,486-base-pair near full-length cDNA coding for Bombyx egg cysteine proteinase. The cDNA encodes 344 amino acid residues containing a typical signal peptide sequence (16 residues), pro-peptide (104 residues), and the sequence for mature enzyme (224 residues). Sequence alignments show that the egg cysteine proteinase is similar to lobster cysteine proteinase (61% identity), barley cysteine proteinase, Aleurain (52%), rice cysteine proteinase, Oryzain (54%), and rat cathepsin L (59%). The amino-terminal sequencing of the egg cysteine proteinase indicates that the enzyme purified as an inactive form from eggs is a pro-enzyme. Pro-egg cysteine proteinase was detected in other silkmoth tissues such as ovary, fat body, hemocyte, and hemolymph by immunoblotting.

The structure of rabbit procathepsin E was determined by molecular cloning of its cDNA. The proenzyme consisted of 379 amino acids and had structural features common to human and guinea-pig procathepsin E species. The highly conserved tripeptide sequence at the active site of aspartic proteinases, Asp-Thr(Ser)-Gly, is, however, replaced by Asp-Thr-Val in rabbit procathepsin E. To our knowledge, this is the first case of such a variation in aspartic proteinases. The processed form, cathepsin E, hydrolyzed various biologically active peptides maximally at around pH5. Tachykinins, such as substance P and neurokinin A, were hydrolyzed most rapidly, with specific cleavage of sequences essential for their activity. The rates of hydrolysis were several hundred-fold higher than those of cathepsin D. Furthermore, cathepsin E was able to inactivate a functional-domain peptide of fibroblast growth factor, the sequence of which resembles those of tachykinins, and it was active in the generation of functional peptides, such as endothelin and angiotensin I, from their respective precursors. Procathepsin E was detected at high levels in various fetal tissues, such as the liver, stomach and blood cells. At the adult stage, the proenzyme was detectable only in specific tissues, such as the urinary bladder, duodenum and colon. Northern-blot analysis showed similar stage-specific and tissue-specific expression of the mRNA for procathepsin E. Since tachykinins and other suited peptide substrates of cathepsin E have been shown to have mitogenic activity, (pro)cathepsin E may regulate the growth and differentiation of embryonic and fetal tissues by degrading or processing these peptides. The enzyme may also regulate the physiological activities of adult tissues which are mediated by substance P and related tachykinins.

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 processing of human gastric procathepsin E to its mature form, cathepsin E, was studied at pH 3.5. The results revealed the autocatalytic and apparently one-step conversion of procathepsin E to cathepsin E within 10 min of incubation at 14 degrees C under the conditions used. Analyses of the amino acid sequences of both procathepsin E and cathepsin E showed that cleavage occurred at the Met36-Ile37 bond to produce the mature form, cathepsin E. The NH2-terminal amino acid sequence of procathepsin E thus determined was identical with that predicted from the cDNA sequence by Azuma et al. except that the NH2-terminal glutamine residue in the latter was converted into a pyroglutamic acid residue in the former and that the glycine residue at position 2 in the latter sequence was deleted in the former. On the other hand, the NH2-terminal amino acid sequence of cathepsin E was identical with that reported previously by us.

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.

Procathepsin D-II (Mr = 37 500) was purified from Japanese monkey lung at pH 7.0, and was shown to be converted to the active form, cathepsin D-II (Mr = 33 000) via an intermediate (Mr = 35 500) upon treatment at pH 3.0 and 14 degrees C. Procathepsin D-II was shown to be the inactive precursor of cathepsin D-II based on the following results: the former was inactive toward heat-denaturated casein at pH 5.4 whereas the latter was active; the former was not inactivated by diazoacetyl-DL-norleucine methyl ester in the presence of Cu2+ ion at pH 6.0 whereas the latter was inactivated rapidly under the same conditions; and the former had no affinity to pepstatin-Sepharose between pH 5 and 7 whereas the latter was adsorbed to it. With a rabbit antiserum against procathepsin D-II, cathepsin D-II, pepsinogen C and pepsin C of Japanese monkey were each found to give a single precipitin line which fused completely with each other on agarose plate. On the other hand, cathepsin D-I purified from the monkey lung, and pepsinogens A (I, II, III-1, III-2 and III-3) obtained from the monkey gastric mucosa failed to precipitate with the antiserum. With the antiserum against the monkey pepsinogen C, the same results were obtained. Further, procathepsin D-II and pepsinogen C were shown to have the same amino-terminal amino acid sequence, Ala-Val-Val-Lys-Val-Pro-Leu-Lys-Lys-Phe-Lys-. All these results indicate a strong similarity of procathepsin D-II and cathepsin D-II to pepsinogen C and pepsin C, respectively.

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.

A pepsinogen C-like acid protease zymogen was found in Japanese monkey prostate extract and seminal plasma by means of the double immunodiffusion method using rabbit anti-pepsinogen C antiserum, and was purified from the prostate by a combination of ammonium sulfate fractionation, DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and immunoadsorption to an anti-pepsinogen C column. The zymogen was purified 6,400-fold in a yield of 13.1%. The purified zymogen gave a single band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The zymogen was converted to the active form by acid treatment at pH 2.8 for 4 h with concurrent reduction of the molecular weight from 41,000 to 36,000. By the double immunodiffusion method, prostate pepsinogen C-like acid protease zymogen, pepsinogen C, lung procathepsin D-II, and their active forms gave a single, fused precipitin line in agar plate with anti-pepsinogen C antiserum, which did not react with cathepsin D and pepsinogen A. Furthermore, the optimal pH of 2.5-3.0, the effect of pepstatin on the activity, and the amino acid compositions were also in good agreement among these three zymogens, showing that they are very similar protease zymogens.