Takae Towatari

Novel epoxysuccinyl peptides Selective inhibitors of cathepsin B, in vitro

A series of new epoxysuccinyl peptides were designed and synthesized to develop a specific inhibitor of cathepsin B. Of these compounds, N‐(L‐3‐trans‐ethoxycarbonyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐030) and N‐(L‐3‐trans‐propylcarbamoyloxirane‐2‐carbonyl)‐L‐isoleucyl‐L‐proline (compound CA‐074) were the most potent and specific inhibitors of cathepsin B in vitro. The carboxyl group of proline and the ethyl ester group or n‐propylamide group in the oxirane ring were necessary, the ethyl ester group or the n‐propylamide group being particularly effective for distinguishing cathepsin B from other cysteine proteinases such as cathepsins L and H, and calpains.

Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

New derivatives of E‐64 (compound CA‐030 and CA‐074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K 1 values for cathepsin B were about 2–5 nM, like that of E‐64‐c, whereas their initial K 1 values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA‐030 or CA‐074 into rats, compound CA‐074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA‐030 does not show selectivity for cathepsin B, although both compounds CA‐030 and CA‐074 show complete selectivity for cathepsin B in vitro.

Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin–proteasome pathway

To elucidate the mechanisms of microgravity‐induced muscle atrophy, we focused on fast‐type myosin heavy chain (MHC) degradation and expression of proteases in atrophied gastrocnemius muscles of neonatal rats exposed to 16‐d spaceflight (STS‐90). The spaceflight stimulated ubiquitination of proteins, including a MHC molecule, and accumulation of MHC degradation fragments in the muscles. Semiquantitative reverse transcriptase‐polymerase chain reaction revealed that the spaceflight significantly increased mRNA levels of cathepsin L, proteasome components (RC2 and RC9), polyubiquitin, and ubiquitin‐conjugating enzyme in the muscles, compared with those of ground control rats. The levels of μ‐calpain, m‐calpain, cathepsin B, and cathepsin H mRNAs were not changed by the spaceflight. We also found that tail‐suspension of rats for 10 d or longer caused the ubiquitination and degradation of MHC in gastrocnemius muscle, as was observed in the spaceflight rats. In the muscle of suspended rats, these changes were closely associated with activation of proteasome and up‐regulation of expression of mRNA for the proteasome components and polyubiquitin. Administration of a cysteine protease inhibitor, E‐64, to the suspended rats did not prevent the MHC degradation. Our results suggest that spaceflight induces the degradation of muscle contractile proteins, including MHC, possibly through a ubiquitin‐dependent proteolytic pathway.

Molecular cloning and sequencing of cDNA for rat cathepsin L

A near full‐length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (M r 37 685) containing a typical signal sequence (N‐terminal 17 residues), pro‐peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants: Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well‐conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxy‐nucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G‐50 and HPLC DEAE‐5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N‐terminal amino acid sequences and elution positions on ion‐exchange chromatography of the products. The K i values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N‐terminal region of cystatin for inhibitory activities on cysteine proteinases.

Cellular proteases involved in the pathogenicity of enveloped animal viruses, human immunodeficiency virus, influenza virus A and Sendai virus.

In enveloped viruses, post-translational proteolytic activation is a critical step for the fusion activity and thus for the infectivity of the virus. In addition to the membrane receptors for the viruses, proteolytic activation is indispensable for effective virus spread in the infected host and it is a prime determinant for pathogenicity. Here we described the host cellular processing proteases, tryptase Clara and tryptase TL2, which proteolytically activate the infectivity of influenza A and Sendai viruses in the respiratory tract and HIV-1 in human CD4+ T cells, respectively. A novel trypsin-like protease, designated tryptase Clara, was purified from rat lung. The enzyme is localized in Clara cells of the bronchial epithelium and is secreted into the airway lumen. The enzyme specifically recognizes the consensus cleavage motif Gln(Glu)-X-Arg of influenza A and Sendai viruses and proteolytically activates the envelope fusion glycoproteins of the progeny viruses extracellularly in the airway lumen. Human mucus protease inhibitor and pulmonary surfactant in airway fluid inhibited the proteolytic activation of these viruses and also suppressed multiple cycles of viral replication in vitro. These results suggest that an imbalance between the amount of tryptase Clara and that of endogenous inhibitors in airway fluid is a prime determinant for pneumopathogenicity of the viruses. Therefore supplementing an endogenous inhibitor at therapeutic doses may protect against virus infection. In HIV-1 infection, binding of the gp120 envelope glycoprotein to the CD4 receptor is not sufficient in itself to allow virus entry, and an additional component(s) in the membrane is required for cell infection as a cofactor. We isolated a serine protease named tryptase TL2, in the membrane of CD4+ lymphocytes, which specifically binds to the V3 loop of HIV-1 gp120 as a cofactor. After binding, tryptase TL2 proteolytically processed gp120 into two protein species of 70 and 50 kDa and the cleavage was suppressed by a neutralizing antibody against the V3 loop. The amino acids that constitute the cleavage sites in the V3 loop of almost all HIV isolates are variable, but they are restricted to those which are susceptible to chymotryptic and/or tryptic enzyme. The multi-substrate specificity of tryptase TL2, which has tryptic and chymotryptic specificities, may correspond tot he variability of the V3 loop. The selective cleavage of the V3 loop by tryptase TL2 may lead to a conformational change of gp120, resulting in the dissociation of gp120 from gp41, exposing the fusogenic domain of the transmembrane protein gp41 following virus-host cell fusion.

Primary structure study of rat liver cathepsin B -- a striking resemblance to papain.

Summary Crystalline rat liver cathepsin B, a lysosomal thiol protease, is composed of two polypeptide chains with molecular weights of ca . 25,000 and 5,000, linked by disulfide bridge(s). The chains were separated by gel filtration after reduction and carboxymethylation. Sequence analyses of the two chains revealed that the enzyme has an extremely high sequence homology to papain, a plant thiol protease, especially in the active site related areas.

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their M r values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N‐terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176–177 (Asp‐Ser) were not present at the C‐terminus and alanine was replaced by proline at residue 125. Asn‐108 in the heavy chain is modified with carbohydrate.

The human mucus protease inhibitor and its mutants are novel defensive compounds against infection with influenza A and Sendai viruses.

Tryptase Clara, a trypsin-like protease localized exclusively in and secreted by Clara cells of the bronchial epithelium, is a prime host factor that processes viral envelope glycoproteins and determines the infectivity of influenza A and Sendai viruses (H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma, The Journal of Biological Chemistry, 1992, Vol. 267, pp. 13573-13579). We report here that human mucus protease inhibitor (MPI), a major inhibitor of granulocyte elastase in the lining fluid of the human respiratory tract, significantly inhibited induction of the infectivity of influenza A and Sendai viruses by tryptase Clara in vitro and multicycles of mouse-adapted influenza A virus replication in rat lungs in vivo. Recombinant MPI and the C- but not the N-terminal domain of MPI inhibited both the activity of tryptase Clara and the induction of virus infection by tryptase Clara. The 50% inhibitory concentrations of MPI and the C-terminal domain peptide (Pro50-Ala107) of MPI for tryptase Clara were 7.4 and 61.6 nM, respectively, with Sendai virus envelope glycoproteins as the substrate. Studies on deletion mutants of the C-terminal domain of MPI revealed that the minimal size of MPI required for the inhibition of tryptase Clara is the peptide Lys60-Ala107. Studies involving site-directed mutagenesis of the C-terminal domain of MPI indicated that the Leu72-Met73 site of MPI is the inhibitory site for tryptase Clara. Substitution of residue Leu72 with a basic amino acid significantly increased in the inhibitory activity of the C-terminal domain of MPI, but further substitution of residue Met73 with various amino acids in these mutants reduced the inhibitory activity. Since there is evidence suggesting that the concentration of MPI in respiratory fluid is insufficient for prevention of virus infection, the administration of MPI, the recombinant C-terminal domain of MPI, and their mutants, with residue Leu72 substituted with residues Arg72 and Lys72, may be useful for treatment of such pneumotropic virus infections.

Separation of a new protease from cathepsin B1 of rat liver lysosomes

The intracellular proteases known to be capable of hydrolyzing high molecular weight proteins are lysosomal cathepsin D (EC 3.4.4.23) and B1 (EC 3.4.4.-) [1-5] and seryl-proteases (Group Specific Proteases; GSP), which we recently found in mitochondrial membranes [6-8] . Of these, cathepsin B 1 and GSP are probably most important for intracellular protein degradation under physiological conditions, because they can hydrolyze certain native proteins under weakly acidic and weakly alkaline conditions, respectively, whereas cathepsin D is only active under strongly acidic conditions and only attacks denatured proteins. Cathepsin B1 has been defined as a protease possessing the following characteristics [2-5,9] : (1) activity with some high mol. wt. proteins and a-N-benzoyl-DLarginine-p-nitroaniline (BAPA), (2) a mol. wt. of about 25 000, (3) a pH optimum of 5.0-6.0 and (4) inhibition by leupeptin. However, we observed that the activities of crude cathepsin B1 on BAPA and on an enzyme protein used as substrate were affected differently by heat treatment. Therefore, wepurified cathepsin B1 to find out whether these activities were due to the same protein. As described in this paper, using circulating long column chromatography of Sephadex G-75 as the final purification step, we separated into two proteases from the most pure B1 fraction which has been published [19], the first peak is active toward BAPA and aldolase, and the second peak is active toward glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), ornithine-ketoacid