Rat Liver Cathepsin Research Articles

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC50) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC50, 12 nM) and L (IC50, 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.

Characterization of CAA0225, a Novel Inhibitor Specific for Cathepsin L, as a Probe for Autophagic Proteolysis

We screened a series of new epoxysuccinyl peptides for the development of a lysosomal cathepsin L-specific inhibitor. Among the compounds tested, (2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide} (compound CAA0225) was the most potent inhibitor of cathepsin L. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). To assess the contribution of cathepsin L to lysosomal proteolysis, we evaluated autophagy, which is the process of lysosomal self-degradation of cell constituents. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions CAA0225 significantly inhibited degradation of long-lived proteins; however, the magnitude of inhibition was comparable to that in the presence of CA-074-OMe, which is a cathepsin B-specific inhibitor. Thus the contributions of cathepsin L and cathepsin B to autophagic protein degradation of cytoplasmic proteins are nearly equal. During autophagy, microtubule-associated protein IA/IB light chain 3-II (LC3-II) and gamma-aminobutyric acid (A) receptor-associated protein (GABARAP)-II, which are specific markers of autophagosomal membranes that engulf cytoplasmic components, also undergo degradation upon fusion of autophagosomes with lysosomes. CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore we conclude that cathepsin L does not play a general role in the degradation of proteins in the lumen of autophagosomes, but rather is involved specifically in the degradation of autophagosomal membrane markers.

The structures of asparagine-linked oligosaccharides of rat liver cathepsin L reflect the substrate specificity of lysosomal alpha-mannosidase.

We have studied the structures of asparagine-linked oligosaccharides of cathepsin L purified from rat liver in detail. The oligosaccharides released from rat liver cathepsin L on glycopeptidase-F treatment were tagged with 2-aminopyridine at their reducing ends. The pyridylamino (PA) derivatives were separated into seven fractions according to molecular size by normal-phase HPLC. The structure of each oligosaccharide thus isolated was analyzed by reversed-phase HPLC and characterized by ion-spray mass spectrometry and high-resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our results indicate that the asparagine-linked oligosaccharides of rat liver cathepsin L are of the oligomannose type, having two to six mannose residues. Among them, the five major ones are Manalpha1-6Manbeta1-4-GlcNAcbeta1-4GlcNAc, Manalpha1 -6Manalpha1-6Manbeta1-4GIcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)-Manalpha1-6Manbeta1- 4GlcNAcbeta1-4GlcNAc, Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4Glc-NAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-++ +2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4Glc-NAc. Their structures are shown to be products of Man6GlcNAc2 hydrolysis with lysosomal alpha-mannosidase.

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases.

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10(7) M-1 sec-1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10(5) M-1 sec-1 range due to lower k(cat) values, with the Km's being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN2 was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k + 0 = 1,100,000 M-1 sec-1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable frame-work for designing sensitive, selective substrates and inhibitors of cysteine proteinases.

Purification and characterization of cathepsin H fromhepatopancreas of carp Cyprinus carpio

1. 1. Cathepsin H was purified about 5400-fold from hepatopancreas of carp Cyprinus carpio) by the method involving ammonium sulfate fractionation, and chromatography on S-sepharose, DEAE-Sephacel, Ultrogel AcA54, Concanavalin A-Sepharose 4B and GPC on Protein-Pak 125. 2. 2. The purified cathepsin H gave a single protein band on analytical-PAGE, but migrated as two bands of 27,000 and 23,000 mol. wt on SDS-PAGE. 3. 3. Cathepsin H had a pH and temperature optimum of 6.5 and 45°C using Arg-MCA as a substrate, respectively, and was activatedby sulfhydryl compounds and inhibited by cysteine protease inhibitors and metal compounds having high reactivities at cysteine residue. 4. 4. The carp hepatopancreas cathepsin H immunoreacted with the monospecific antibody against rat liver cathepsin H, and did not react with the antibodies against carp hepatopancreas cathepsin B and L by the method of immunoelectrophoretic blotting.

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29.

Purification and characterization of cathepsin J from rat liver

Cathepsin J has been partially purified [Liao, J. C. R. & Lenney, J. F. (1984) Biochem. Biophys. Res. Commun. 124, 909-916], but its detailed properties are still unknown. In this study, we have purified cathepsin J completely and characterized it. It was purified to homogeneity from the mitochondrial-lysosomal fraction of rat liver by acid treatment, followed by ammonium sulfate precipitation (20-65%), and chromatographies on S-Sepharose, ConA-Sepharose, Affi-gel 501, HPLC DEAE-5PW and HPLC TSK G3000SW. Cathepsin J was found to be a lysosomal high-molecular-mass cysteine protease of about 160 kDa consisted of two different subunits. One subunit (alpha subunit) was a glycoprotein with a molecular mass of 19-24 kDa which was reduced to 19 kDa by treatment with endoglycosidase F. It has the amino acid sequence LPESWDWRNVR at its N-terminus, which was very similar to those at the N-termini of rat cathepsins B, H and L. The other subunit (beta subunit) was a glycoprotein with a molecular mass of 17 kDa, which was reduced to 14 kDa by treatment with endoglycosidase F. It had DTPANETYPDLLG at its N-terminus, which had no similarity with the N-terminal sequences of other cathepsins. Cathepsin J showed strong affinity for synthetic substrates such as N-benzyloxycarbonyl-phenylalanyl-arginine 4-methyl-coumaryl-7-amide and glycyl-arginine beta-naphthylamide. It was activated by thiol reagents and chloride ion and was inhibited by cysteine protease inhibitors. However, its initial inhibition constant Ki(initial) by N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3- methylbutylamide (E-64-c) was 1800 nM, which was 100-500 times those of cathepsins B and L. Many properties of cathepsin J were similar to those of cathepsin C (dipeptidylaminopeptidase I) reported as a lysosomal cysteine protease with dipeptidyl-aminopeptidase activity [McDonald, J. K., Reilly, T. J. & Ellis, S. (1964) Biochem. Biophys. Res. Commun. 16, 135-140]. Furthermore, antiserum against rat liver cathepsin C reacted with rat liver cathepsin J. These findings suggested that cathepsin J is identical with cathepsin C.

Inhibition of cathepsins B, H and L by rat salivary cystatins.

The inhibition constants (Ki) for rat liver cathepsins B, H and L of three rat submandibular salivary cystatins (designated as RSC-1, RSC-2 and RSC-3), induced by chronic isoproterenol treatment, were determined. These three cystatins inhibited the enzyme activity of cathepsin L most strongly, whereas they had much larger Ki values for cathepsins B and H.

Multiple Proteolytic Action of Rat Liver Cathepsin B: Specificities and pH-Dependences of the Endo- and Exopeptidase Activities1

Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.

Discrimination between rat thiostatin (T‐kininogen) and one of its cystatin‐like inhibitory fragments by a monoclonal antibody, and localization of the epitope

A monoclonal antibody (mAb D3) raised against rat thiostatin (T-kininogen) strongly interacted with a fragment, identified as cystatin-like domain 3, which inhibits cysteine proteinases but did not recognize intact, native thiostatin. The antigen-antibody reaction requires cleavage of the single peptide chain of thiostatin in its inter-domain 2-3 region. This mAb can also differentiate between the two molecular varieties of thiostatin, reacting only with immobilized domain 3 from T1 thiostatin, which differs from the T2 variety by only 10 out of 125 residues. mAb D3 did not react with an N-terminally truncated domain 3 of T1 thiostatin prepared by submaxillary gland kallikrein k10 proteolysis. This suggests that the epitope, or an essential part of it, is located on a stretch of 12 residues at the N-terminal of the T1 thiostatin domain 3. This sequence in T1 thiostatin differs from that in T2 thiostatin by four amino acids, two of which are arginyl residues in T1. Chemical modification of these residues located at positions 246 and 250 decreased the reactivity of T1 domain 3 towards the antibody, suggesting that at least one of them is a critical residue of the epitope. Arginine 246 is part of a small disulfide loop between cysteines 245 and 248 which is also necessary for antibody recognition. This antibody does not change the inhibitory properties of purified domain 3 towards papain or rat liver cathepsin L, indicating that the N-terminal part of domain 3 is not involved in inhibition. mAb D3 was used to demonstrate the presence of inhibitory thiostatin fragments in ascites fluid but not in plasma from normal or turpentine-injected rats.

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.

ATP-activated, high-molecular-mass proteinase-I from rat skeletal muscle is a cysteineproteinase-α 1-macroglobulin complex

From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated ‘proteinase I’, which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [ 14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of ‘proteinase I’ is similar to α-macroglubulin. Immunoelectrophoretic analyses of ‘proteinase I’ with antisera to ratα 1-macroglobulin,α 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex ofα 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar ‘proteinase’ has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against ‘proteinase I’. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of α-macroglobulin and cysteine proteinases.

An immunocytochemical study on co-localization of cathepsin B and atrial natriuretic peptides in secretory granules of atrial myoendocrine cells of rat heart.

To examine localization of cathepsin B, a representative lysosomal cysteine protease, in atrial myoendocrine cells of the rat heart, immunohistochemistry at the light and electron microscopic level was applied to the atrial tissue, using a monospecific antibody for rat liver cathepsin B. In serial semi-thin sections, immunoreactivity for cathepsin B and atrial natriuretic peptides (ANP) was detected in the para-nuclear region of atrial myoendocrine cells. Several large granules and many fine granules in the region of the cells were positively stained by the cathepsin B antibody. Gold particles indicating cathepsin B antigenicity labeled secretory granules in the cells, which were also labeled by those indicating ANP, using thin sections of the Lowicryl K4M-embedded material. Moreover, some granules labeled densely by immunogold particles for cathepsin B seemed to be lysosomes. By double immunostaining using thin sections of the Epon-embedded material, gold particles indicating cathepsin B and ANP antigenicities were co-localized in secretory granules of the cells. By enzyme assay, activity of cathepsin B was three times higher in atrial tissue than ventricular tissue. The results suggest that co-localization of cathepsin B and ANP in secretory granules is compatible with the possibility that cathepsin B participates in the maturation process of ANP.

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.

Cathepsin E from rat neutrophils: Its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0–3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.

Action of rat liver cathepsin B on bradykinin and on the oxidized insulin A-chain

Rat liver cathepsin B was tested for its peptide-bond specificity against bradykinin and the oxidized insulin A-chain. Bradykinin was shown to be resistant to the action of cathepsin B. One possible reason for this resistance is the proline content of the peptide and the discrimination against proline residues at three or four subsites of cathepsin B. Oxidized insulin A-chain was degraded by a peptidyl dipeptidase activity. Three dipeptides were cleaved from the C-terminal part of the insulin A-chain after having been incubated for 2 h (molar ration E:S = 1:2800) and six dipeptides were released after a longer digestion (10 h, E:S = 1:575).

Purification and some properties of bound cysteine proteinase inhibitor from cornified cells of newborn rat epidermis

Cysteine proteinase inhibitor exists in two forms in terminally differentiated keratinocytes. One is readily soluble in 20 mM sodium phosphate buffer but the other is bound to the plasma membrane and is poorly soluble. The cysteine proteinase inhibitor (CPI) from the membrane was extracted from cornified epidermal layers of 2-day-old rats and its properties were compared with those of soluble CPI. This CPI (bound CPI) was solubilized in alkaline 8 M urea containing 2-mercaptoethanol from the residual tissue exhaustively treated with buffered 4 M urea. CPI was separated from keratin by ammonium sulfate precipitation and purified by means of papain affinity chromatography, ion exchange column chromatography and gel filtration. Bound CPI had an M r value of about 16 000, a p I value of 3.8 and was unstable at above 80°C, while soluble CPI was of M r 13 000 and stable at above 80°C. Both CPIs were stable at 4°C in the range of 3.0–9.0. Bound CPI contained half cystine and the ratio of acidic-to-basic amino acids was 3.18. Bound CPI inhibited rat liver cathepsins B, H, and L but did not inhibit the activity of noncysteine proteinases. Papain activity was inhibited by bound CPI at three sites, noncompetitively, and the K i value was calculated to be 0.11 nM.

Specificity of rat liver cathepsin D.

The specificity of highly purified rat liver cathepsin D was investigated by analyzing the digests of denatured proteins. At the P1 site, cathepsin D prefers hydrophobic residues except Ile and Val, that are branched at the beta-carbon. Strong and weak hydrophobicities are required at P1' and P2 sites, respectively. A lower protency for beta-turn formation is essential for the sequence around the P1 site.

Immunohistochemical detection of Cathepsin D in human neuroontogenesis

An antiserum against rat liver cathepsin D, which was previously found to cross-react with human brain cathepsin(s) D, was used to immunolocalize the enzyme in developing CNS man. Cathepsin D protein was found in neurons as well as in glial cells. The aerliest occurrence of cathepsin D was observed in neuroblasts at the 12th gestational week. During pre-, peri-, and postnatal neuroontogenesis, there is a strong increase in the amount of immunoreactive material.

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.

The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with approximately equal to 880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome.