Autocatalytic processing of procathepsin E to cathepsin E and their structural differences

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.     

Structural evidence for two isozymic forms and the carbohydrate attachment site of human gastric cathepsin E

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

Purification and further characterization of enteropeptidase from porcine duodenum

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.     

Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.     

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.     

Identification and reverse genetic analysis of mitochondrial processing peptidase and the core protein of the cytochrome bc1 complex of Caenorhabditis elegans, a model parasitic nematode

Mitochondria could be a good target for anti-parasitic drugs. The alpha and beta subunits of mitochondrial processing peptidase (MPP) and the core subunits of the cytochrome bc1 complex, UCR-1 and UCR-2, are homologous to one another and are important for mitochondrial functions. However, our knowledge of these proteins in nematodes is very limited. Caenorhabditis elegans, a free-living nematode, has six genes coding for proteins homologous to these subunits. On primary structure comparison, and immunochemical and enzymological analyses, the gene products were assigned as follows: Y71G12B.24, alpha-MPP; ZC410.2, beta-MPP; F56D2.1, UCR-1; VW06B3R.1, T10B10.2; and T24C4.1, UCR-2. The primary structures of beta-MPP and UCR-1 from Brugia malayi, a parasitic nematode causing human filariasis, were deduced from their cDNA structures. Phylogenetic analysis showed that the UCR-1s from both C. elegans and B. malayi were less related to mammalian UCR-1s than to MPPs from various organisms. MPP and the bc1 complex are essential for the life cycle of C. elegans, because their reverse genetic inhibition is lethal. This suggests the possibility that these proteins are also essential for the viability of B. malayi and other parasitic nematodes, and are potential targets for anti-parasitic agents.     

Characterization of a membrane-bound arginine-specific serine protease from rat intestinal mucosa

Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol. Chem. 269, 32985-32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences determined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes.     

Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum

The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.     

The primary structure of Aspergillus niger acid proteinase A

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.     

A comparative study on the NH2-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins

Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.