Senarath B. P. Athauda

Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxidized insulin

Proteolytic activity and cleavage specificity of cathepsin E were investigated in a wide range of pHs from 3.0 to 10.5 using the B chain of oxidized insulin as substrate. Contrary to the previous notion that cathepsin E is virtually inactive above pH 6, significant proteolytic activity was observed at pH 7.4 and above. Further, cleavage specificity appeared to change significantly with pH and rather specific cleavage occurred at pH 7.4 and above as compared to pH 5.5 and 3.0. These results suggest that cathepsin E may function in vivo at the physiological pH with a rather restricted specificity.

Distinct cleavage specificity of human cathepsin E at neutral pH with special preference for Arg-Arg bonds.

In order to clarify the potential role of cathepsin E at neutral pH, the cleavage specificity of human cathepsin E was examined at pH 7.4 toward reduced-carboxymethylated(RCm-)ribonuclease A and various bioactive and related peptides. The specificity of the enzyme at pH 7.4 was found to be considerably different from that at acidic pH; preferential cleavages were observed with Arg-X and Glu-X bonds, which are not the major cleavage sites at acidic pH. Moreover, the Arg-Arg bond was found to be the most preferential site of cleavage. This unique specificity observed at pH 7.4 suggests the possibility that cathepsin E might be involved in processing and/or degradation of certain proteins and/or peptides at or near neutral pH in vivo.

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.

Acid Proteinase From Nepenthes distillatoria (Badura)

Plant aspartic proteinases have so far received much less attention in contrast to the well characterized mammalian, fungal and viral aspartic proteinases.1 They are widely distributed in the plant kingdom, and have been detected in seeds, leaves and flowers of different plants as well as in the digestive fluid of some insectivorous species.1 Aspartic proteinases from barley, rice and cardoon flower have been well characterized and their cDNA-derived primary structures have been reported.2–4 However, only a few studies were reported on proteinases of insectivorous plants.5–8 Insectivorous plant Nepenthes distillatoria is available in Sri Lanka in a large quantity and will be a good source of the insectivorous plant proteinases. They are interesting not only from the view point of plant physiology, but also from the view point of structure/function relationship and molecular evolution of aspartic proteinases.

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.

Aspergillus niger var. macrosporus proteinase B. cDNA cloning, expression, and activation of the proenzyme.

Filamentous fungus Aspergillus niger var. macrosporus produces two types of extracellular acid endoproteinases, proteinases A and B, commercially called proctases A and B, respectively (1,2). Proteinase A is a non-pepsin-type acid proteinase (1–7), whereas proteinase B has properties of pepsin-type aspartic proteinase. Proteinase B has a molecular mass of about 35 kDa and is inhibited by specific inhibitors of aspartic proteinases such as pepstatin, diazoacetyl-DL-norleucine methyl ester in the presence of cupric ions, and 1,2-epoxy-3-(p-nitrophenoxy)propane (4,8).

Isolation and characterization of human gastric procathepsin E and cathepsin E.

Human gastric cathepsin E is an aspartic proteinase present in human gastric mucosa and different from pepsinogens A and C and cathepsin D. It has been localized not only to stomach mucosa, but also to erythrocyte membranes and several lymphoid associated tissues and cells including thymus, spleen, macrophages and polymorphonuclear lymphocytes (1,2). However its physiological function and the process of activation remain obscure. Due to its intracellular localization in lymphoid associated tissues and cells it has been suggested to have a role in immune function (3,4). Studies on cathepsin E has not progressed like those on other aspartic proteinases (pepsinogens and cathepsin D), because of difficulties in obtaining a sufficient amount of the purified native enzyme. Therefore, to understand the structure-function relationship of cathepsin E and to clarify its physiological function, it is important to establish a purification procedure of procathepsin E and cathepsin E for further characterization.

Isolation, Characterization, and Structure of Procathepsin E and Cathepsin E from the Gastric Mucosa of Guinea Pig

Cathepsin E [EC 3.4.23.34] is a non-secretory intracellular aspartic proteinase, and its precursor is known as procathepsin E (1,2). (Pro)cathepsin E is a dimeric enzyme different from other aspartic proteinases, consisting of two identical subunits of about 40 kDa (3,4). The enzymatic properties of cathepsin E have been shown to resemble those of pepsins [EC 3.4.23.1]; for example, it has hydrolytic activity at acidic pH, with an optimum at pH 2–3 against protein substrates, and is sensitive to various pepsin inhibitors (5–10).

Effects of Hydrocortisone on the Pepsinogen-Producing Cells in Rat Stomach Mucosa

Pepsinogen is a marker of the terminal differentiation of stomach mucosa. At present, controlling mechanisms of differentiation in stomach mucosa is not fully understood. Previous studies demonstrated that administration of hydrocortisone to developing rats induces a precocious increase in the mucosal pepsinogen level in the stomach (1–3), indicating that glucocorticoids are somehow involved in the differentiation of the stomach mucosa. However, the physiological significance of glucocorticoids in the regulation of pepsinogen gene expression is not well understood. In addition, the effects of glucocorticoids on pepsinogen-producing cells in fully-differentiated stomach mucosa are less clear (4,5). In this study, we examined the effects of hydrocortisone on infant and adult rat stomach mucosa, especially on pepsinogen gene expression and the morphology of pepsinogen-producing cells.