Molecular cloning of cDNA for rat cathepsin C. Cathepsin C, a cysteine proteinase with an extremely long propeptide

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The deduced amino acid sequence of cathepsin C comprises 462 amino acid residues: 28 NH2-terminal residues corresponding to the signal peptide, 201 residues corresponding to the propeptide, and 233 COOH-terminal residues corresponding to the mature enzyme region. Four potential glycosylation sites were found, three located in the propeptide region, and one in the mature enzyme region. The amino acid sequence of mature cathepsin C has 39.5% identity to that of cathepsin H, 35.1% to that of cathepsin L, 30.1% to that of cathepsin B, and 33.3% to that of papain. Cathepsin C, therefore, is a member of the papain family, although its propeptide region is much longer than those of other cysteine proteinases and shows no significant amino acid sequence similarity to any other cysteine proteinase.     

Acid proteolytic activities in mouse liver and muscle tissues after treatment with protease inhibitor leupeptin

The activities of acid proteolytic enzymes were assayed in the liver and muscular tissues of mice (Mus musculus) 1, 6 and 24 hr after the administration of a protease inhibitor leupeptin (i.p., 15.5 mg/kg body wt). Leupeptin administration induced a strong inhibition of cathepsin B and a moderate inhibition of cathepsin C and acid autolytic rate in mouse liver 1 hr after injection. Thereafter the inhibition reduced and disappeared during 24 hr. The activity of cathepsin D was increased in liver 6 and 24 hr after injection. The activity of beta-glucuronidase was not affected by the leupeptin treatment. The administration of leupeptin did not affect the rate of acid autolysis and the activities of cathepsin C and D in cardiac and skeletal muscles. A slight increase in cathepsin B activity was observed 1 hr after leupeptin treatment in calf muscles. The cause of both tissue and enzyme specific changes after leupeptin treatment is discussed.     

Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.

Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

The crystal and molecular structures of a cathepsin K:chondroitin sulfate complex

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 Å. The overall structure reveals an unusual “beads-on-a-string”-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K_d, of about 10 nM was determined for the interaction between cathepsin K and C4-S.     

A comparative study of two kinds of cathepsin D-type proteinases from rat gastric mucosa

The localization of cathepsin D-like acid proteinase in the rat stomach and other tissues was studied, and its biochemical properties were compared with those of rat gastric cathepsin D (EC 3.4.23.5). Cathepsin D-like acid proteinase existed overwhelmingly in the mucosal layer and was hardly detected in the gastric juice. Its subcellular distribution profile was very similar to that of acid phosphatase, but not to that of pepsinogen. This proteinase-like enzyme activity was also found in rat splenic extract. These results strongly suggest that the proteinase is a lysosomal enzyme. In addition, cathepsin D-like acid proteinase demonstrated an in vitro transition of molecular species during storage at -30 degrees C. Although this molecular change was distinctive in ion-exchange column chromatography and susceptibility to some enzyme inhibitors, it was not accompanied by a significant decrease in molecular weight. To compare cathepsin D-like acid proteinase with ordinary cathepsin D, gastric cathepsin D was newly purified to apparent homogeneity in polyacrylamide gel electrophoresis. Its biochemical properties demonstrate that this is a true cathepsin D in rat gastric mucosa. Moreover, this cathepsin D activity was not abolished by treatment with antiserum specific to cathepsin D-like acid proteinase or pepsinogen. From these results, we can conclude that the proteinase is a lysosomal acid proteinase different from newly purified gastric cathepsin D.     

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.     

Bovine cathepsins S and L: isolation and amino acid sequences.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.     

Selective cleavage of peptide bonds by cathepsins L and B from rat liver

The selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gln, or Asn, in position P1. enhanced the susceptibility of the peptide bond to cathepsin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.     

Various properties of cathepsin G and elastase from swine peripheral blood neutrophils

Using gel filtration through Sephadex G-100 and bioaffinity chromatography on contrical-Sepharose, cathepsin G and elastase were isolated from pig peripheral blood neutrophil granules and purified to homogeneity. Both enzymes hydrolyzed the total histone from calf thymus as well as synthetic substrates--tert-butoxy-L-alanine p-nitrophenyl ester (elastase) and benzoyltyrosine ethyl ester (cathepsin G). The use of natural and synthetic protease inhibitors showed that both enzymes were related to the group of serine proteases. The molecular mass of the cathepsin G subunit as determined by SDS polyacrylamide gel electrophoresis is 28-29 kD, that of elastase--30-31 kD. The pH optima for the hydrolysis of proteinaceous and synthetic substrates for cathepsin G and elastase are 8.0-8.5 and 7.0-7.5, respectively. The isoelectric points for elastase and cathepsin G are 9.7-10.0 and greater than 10, respectively; the temperature optima--30-40 degrees C and 50-60 degrees C, respectively. The amino acid composition of the two enzymes from pig granulocytes revealed a high content of arginine and was similar to that of human granulocytes.     

The primary structure and tissue distribution of cathepsin C.

A cDNA for rat cathepsin C (dipeptidylaminopeptidase I) was isolated. The encoded protein is composed of the signal peptide of 28 residues, the propeptide of 201 residues and the mature enzyme region of 233 residues. The amino acid sequence of the mature enzyme region has 39.5 to 30.5% identity to other papain family proteinases. Cathepsin C is, therefore, belongs to papain family, although its propeptide region is much longer than those of other cysteine proteinases and show no significant sequence similarity to any other cysteine proteinase. The mRNA and protein for cathepsin C are broadly distributed in rat tissues, but the relative proportions of cathepsin C and other cysteine proteinases are found to vary from tissue to tissue.     

Naturally occurring inhibitors of intracellular proteinases

The papain inhibitor isolated from chicken egg white inhibits the enzymatic activity of cathepsin B1 and cathepsin C. The inhibitor bears two nonoverlapping reactive sites: one binds cathepsin B1, papain, ficin, and bromelain, the other one cathepsin C. The inhibitor decreases the degree of an immunologic hypersensitive reaction, the so-called Arthus reaction. A statistically significant inhibition of this immunologically developed inflammation occurs only if the inhibitor is applied intradermally and simultaneously with the provoking dose of the antigen to rabbits sensitized to the same antigen. The pepsin inhibitor from the body walls of the roundworm Ascaris lumbricoides inhibits the proteolytic activity of cathepsin E. This inhibitor covalently bound to Sepharose 4B was used for affinity chromatography of cathepsin E. A cathepsin D inhibitor was isolated from potato tubers and its inhibitory and chemical characteristics were studied. The inhibitor does not inhibit either cathepsin E or pepsin yet inhibits trypsin in the alkaline pH-range. The molecular weight of the inhibitor is 21 790 and its molecule consists of 199 amino acid residues. The sequence of 17 amino acid residues was determined by Edman degradation of the inhibitor molecule.     

Hydrolase activities increase in the rat aorta with growth and aging but not in liver and kidney

We examined specific activities (based on DNA) of six glycosidases and cathepsin C in aorta, kidney, and liver from male rats of 2, 6, 10, and 14 months of age. The premise was that assessing cellular catabolism of arterial and nonvascular tissues over age might more fully clarify the impact of age (and growth) alone upon vascular wall metabolism. All aortic glycosidases increased significantly (P less than 0.05) over the holding period as follows: neutral alpha-glucosidase, up 93%; beta-galactosidase, up 102%; N-acetyl-beta-glucosaminidase, up 119%; alpha-mannosidase, up 77%; beta-glucuronidase, up 65%; acid alpha-glucosidase, up 95%. Cathepsin C specific activity was unchanged as was aortic DNA content; total protein content increased 136%. In the kidney, all glycosidase specific activities declined over age with decreases ranging 39-55%; cathepsin C was unchanged. In the liver, neutral alpha-glucosidase increased 12%, acid alpha-glucosidase was unchanged, and the four remaining glycosidases decreased an average of 5-35% by 14 months of age. Liver cathepsin C decreased 44% over this period. Thus, enhancement of hydrolase baseline activities prevails during growth and aging in rat aortic tissue whereas hydrolases of kidney and liver tissues generally decline.     

Comparative studies of two types of acid proteases from rat gastric mucosa and spleen

Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; these two types of acid proteases are quite different proteins existing in the individual tissues.     

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.     

Ovarian cysteine proteinases in the teleost Fundulus heteroclitus: molecular cloning and gene expression during vitellogenesis and oocyte maturation

The cysteine proteinases cathepsins B and L are members of the multigene family of lysosomal proteases that have been implicated in the processing of yolk proteins (YPs) in teleost oocytes. However, the full identification of the type of cathepsins expressed in fish ovarian follicles and embryos, as well as their regulatory mechanisms and specific function(s), are not yet elucidated. In this study, cDNAs encoding cathepsins B, L, F, K, S, Z, C, and H have been isolated from the teleost Fundulus heteroclitus, and the analysis of their deduced amino acid sequences revealed highly similar structural features to vertebrate orthologs, and confirmed in this species the existence of cathepsin L-like, cathepsin B-like, and cathepsin F-like subfamilies of cysteine proteinases. While all identified cathepsins were expressed in ovarian follicles, the corresponding mRNAs showed different temporal expression patterns. Thus, similar mRNA levels of cathepsins L, F, S, B, C, and Z were found throughout the oocyte growth or vitellogenesis period, whereas those for cathepsin H and K appeared to decrease as vitellogenesis advanced. During oocyte maturation, a transient accumulation of cathepsins L, S, H, and F mRNAs, approximately a 3-, 1.5-, 1.6-, and 6-fold increase, respectively, was detected in ovarian follicles within the 20-25 hr after hormone stimulation, coincident with the maximum proteolysis of the oocyte major YPs. The specific temporal pattern of expression of these genes may indicate a potential role of cathepsin L-like and cathepsin F proteases in the YP processing events occurring during fish oocyte maturation and/or early embryogenesis.     

Cathepsin Q, a novel lysosomal cysteine protease highly expressed in placenta

The complete nucleotide sequence of a novel cathepsin cDNA derived from rat placenta was determined and is termed cathepsin Q. The predicted protein of 343 amino acid is a member of the family C1A protease related to cathepsin L. Rat cathepsin Q and its mouse counterpart were found highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryonic tissues. It is predicted that cathepsin Q will differ in catalytic specificity to another placental-specific protease, cathepsin P, indicating that these enzymes will have unique proteolytic functions in extra-embryonic tissues.     

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.     

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.     

Cloning,expression, purification,and activity of dog (Canis familiaris) and monkey (Saimiri boliviensis) cathepsin S

Cathepsin S is the key protease responsible for the removal of the invariant chain from MHC class II molecules and, as such, has attracted much attention as a target for developing immunosuppressive drugs. To help in testing candidate compounds, the monkey (Saimiri boliviensis) and dog (Canis familiaris) cathepsin S cDNAs have been cloned. The monkey cDNA sequence encodes a 330 amino acid protein with 95% homology to human cathepsin S. The dog cDNA sequence encodes a 331 amino acid protein with 91% homology to human cathepsin S. The amino acid differences do not have a major effect on the hydrolysis of the substrate Z-VVR-AMC, but may affect the substrate specificity. As for human and bovine cathepsin S, activity against Z-VVR-AMC extends into the neutral pH range. These parameters are important for understanding the role of cathepsin S in different species and for testing inhibitors in animal models of autoimmunity.     

Immunochemical similarity between a gastric mucosa non-pepsin acid proteinase and neutrophil cathepsin E of the rat

Antiserum against a rat gastric mucosa non-pepsin acid proteinase precipitates rat neutrophil cathepsin E, with a precipitation curve essentially similar to that of the gastric enzyme. Taken together that the antiserum precipitates a cathepsin E-like acid proteinase from rat spleen (Muto, N., Yamamoto, M. and Tani, S. (1987) J. Biochem. (Tokyo) in press), the data indicate that the non-cathepsin D acid proteinases in rat neutrophils, gastric mucosa and spleen are immunochemically closely related. In contrast with the earlier data, cathepsin E from rabbit neutrophils exhibited a maximal activity at around pH 3.0-3.2 and preferred hemoglobin to albumin as substrate, which supports that the non-cathepsin D acid proteinases in the rat tissues are relevantly classified as cathepsin E.