Identification of latent procathepsin H in microsomal lumen: characterization of proteolytic processing and enzyme activation

Procathepsin H in kidney and liver microsomal lumen was identified to have a molecular mass of 41 kDa by immunoblot analysis. The proenzyme was then concentrated by applying the microsomal contents to a concanavalin A-Sepharose column. When the concanavalin A-adsorbed fraction was incubated at pH 4.0 at 20 degrees C, the activity measured with synthetic substrate increased 3.5 times over that of the control after 24 h incubation. Immunoblot analysis showed that acidic treatment caused the disappearance of procathepsin H. Thus the proenzyme might be processed to the mature enzyme under acidic conditions. The marked increase of enzymatic activity and the conversion of proenzyme were completely blocked with pepstatin which is a potent inhibitor of aspartic proteases. These results suggested that a protease for processing procathepsin H might be cathepsin D, a major lysosomal aspartic protease. Therefore, procathepsin H seems to be synthesized first in the enzymatically inactive form in endoplasmic reticulum and successively converted into the active form in lysosomes during biosynthesis.     

Mutations of the C-terminal end of cathepsin K affect proenzyme secretion and intracellular maturation

Transfection of the human cathepsin K cDNA into CHO cells results in the expression of mature catalytically active 27-kDa protein and in cells secreting the 39-kDa proenzyme form. Monensin, which neutralizes the pH of acidic organelles, was found to inhibit intracellular processing of the proenzyme and to stimulate its secretion into the culture medium. Brefeldin A caused alterations in immunofluorescence staining consistent with interference of lysosomal targeting and inhibited both intracellular processing and secretion of cathepsin K. Inhibition of glycosylation by tunicamycin also abolished cathepsin K maturation. Furthermore, the processing of the proenzyme to the mature form was abolished by a single mutation of the terminal Met(329) to Ala. The triple mutation of Ser(325), Pro(327), and Met(329) (all to Ala) inhibited both maturation and secretion, using either transient or stable expression systems. The results indicate that intracellular maturation and secretion of cathepsin K can be affected differentially by various treatments and by mutations of the C-terminal end of the protein. These results are consistent with the involvement of both the secreted proenzyme and the intracellularly processed enzyme in cathepsin K-mediated processes.     

Cynomolgus Monkey (Macaca fascicularis) Cathepsin K: Cloning, Expression, Purification, and Activation

Methodology for the production of recombinant active cynomolgus monkey (Macaca fascicularis) cathepsin K (EC 3.4.22.38) was elucidated. The cDNA encoding the cathepsin K was cloned from femaleM. cynomolgusmonkey mRNA. The deduced amino acid sequence ofM. cynomolguspreprocathepsin K from the cDNA sequence showed 94.2% identity to human preprocathepsin K. Sequence differences occurred only in the prepro- domains; the mature domains were identical. The recombinantM. cynomolguscathepsin K was expressed as a secreted proenzyme using baculovirus-infected SF21 insect cells having the predicted N-terminus (LYPEEILDTH … ), indicating proper cleavage of the secretion sequence. Purified monkey procathepsin K was activated under autocatalytic conditions at pH 4.0. The mature enzyme was composed of mixture of enzymes having N-termini of Gly¹¹³and Arg¹¹⁴. The molecular weight was determined to be 23,668.3 Da by MALDI-TOF-MS which is consistent with the absence of carbohydrate on the mature enzyme. These results indicate that monkey procathepsin K is able to autoactivate and produces a mature enzyme which is identical to that of human cathepsin K. Since the sequence of monkey and human mature cathepsin K are identical and thein vitroactivation mechanisms appear to be indistinguishable, monkeys are predicted to be a good animal model for evaluating cathepsin K inhibitorsin vivoas therapeutic agents for diseases characterized by excessive bone loss, such as osteoporosis.     

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.     

Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases

In this study a pathway for the synthesis of triacylglycerol (TAG) within the lumen of the endoplasmic reticulum has been identified, using microsomes that had been preconditioned by depleting their endogenous substrates and then fusing them with biotinylated phosphatidylserine liposomes containing CoASH and Mg(2+). Incubating these fused microsomes with tri[(3)H] oleoylglycerol and [(14)C]oleoyl-CoA yielded microsome-associated triacylglycerol, which resisted extensive washing and had a [(3)H]:[(14)C] ratio close to 2:1. The data suggest that the precursor tri[(3)H]oleoylglycerol was hydrolyzed by microsomal lipase to membrane-bound di[(3)H]oleoylglycerol and subsequently re-esterified with luminal [(14)C]oleoyl-CoA. The accumulation of TAG within the microsomes, even when overt diacylglycerol acyltransferase (DGAT I) was inactive, is consistent with the existence of a latent diacylglycerol acyltransferase (DGAT II) within the microsomal lumen. Moreover, because luminal synthesis of TAG was carnitine-dependent and markedly reduced by glybenclamide, a potent carnitine acyltransferase inhibitor, microsomal carnitine acyltransferase appears to be essential for trafficking the [(14)C]oleoyl-CoA into the microsomal lumen for subsequent incorporation into newly synthesized TAG. This study thus provides the first direct demonstration of an enzymatic process leading to the synthesis of luminal triacylglycerol, which is a major component of very low density lipoproteins.     

Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats

The rat vasopressin precursor, synthesized in the reticulocyte lysate system under the direction of in vitro transcribed mRNA, is processed and correctly delivered to the lumen of added microsomal vesicles. Translation of mRNA for the mutant (Brattleboro) vasopressin precursor which lacks a translational stop codon as a consequence of a frame-shift mutation, gives rise to a mutated protein (B-mutant precursor) with a C-terminal poly(lysine) sequence encoded by the poly(A) tail. Upon addition of microsomal membranes, the mutated precursor has access to the lumen of the vesicles as indicated by removal of the signal peptide; however, the C-terminal part with the poly(lysine) tail remains outside the vesicles as shown by its sensitivity to proteinase K. When a modified RNA, including a stop codon located similarly to that found in the cDNA encoding the normal precursor, is translated in the presence of microsomal membranes, the resulting product (S-mutant precursor) is refractory to proteolysis by exogenously added proteinase K. Analysis of the microsomal membranes indicates, however, that the C-terminus of the S-mutant precursor is still anchored within membranes. For studying the intracellular transport of the mutated precursor Xenopus laevis oocytes were injected with various RNA constructs. To monitor the transport steps from the endoplasmic reticulum to the Golgi compartment an RNA encoding a glycosylation site within the S-mutant precursor sequence was constructed. The resulting GS-mutant precursor is synthesized in the oocyte but not secreted into the incubation medium, completely in contrast to the normal vasopressin precursor which can be detected in the incubation bath 4 h after injection of the respective RNA. The sensitivity of the GS-mutant precursor carbohydrate side chain to endoglycosidase H treatment suggests that the mutated precursor does not reach the Golgi apparatus.     

Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid.

The cysteine protease cathepsin K is believed to play a key role in bone resorption as it has collagenolytic activity and is expressed predominantly and in high levels in bone resorbing osteoclast cells. The addition of nitric oxide (NO) and NO donors to osteoclasts in vitro results in a reduction of bone resorption, although the mechanism of this effect is not fully understood. The S-nitroso derivatives of glutathione (GSNO) and N-acetylpenicillamine (SNAP) and the non-thiol NO donors NOR-1 and NOR-3 all inhibited the activity of purified cathepsin K in a time- and concentration-dependent manner (IC(50) values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 microM, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NO donors with similar potencies. GSNO at 100 microM also completely inhibited the autocatalytic maturation at pH 4.0 of procathepsin K to cathepsin K. The inhibition of cathepsin K by GSNO was rapidly reversed by DTT, but inhibition by NOR-1 was not reversed by DTT, and analysis of the inhibited cathepsin K for S-nitrosylation using the Greiss reaction gave negative results in both cases. Analysis of the protein by electrospray liquid chromatography/mass spectrometry showed that the inhibition of cathepsin K by GSNO resulted in a mass increase of 306 +/- 2 Da, consistent with the formation of a glutathione adduct. Prior inhibition of cathepsin K by the active site thiol-modifying inhibitor E-64 blocked the modification by GSNO, indicating that the glutathione adduct is likely formed at the active site cysteine. Treatment of cathepsin K with NOR-1 resulted in a mass increase of between 30 and 50 Da, corresponding to the oxidation of a cysteine to sulfinic and sulfonic acids. Cotreatment of cathepsin K with NOR-1 plus the sulfenic acid reagent dimedone resulted in a mass increase of approximately 141 Da, which is consistent with the formation of a dimedone adduct. This result demonstrates that the NOR-1-dependent formation of cathepsin K sulfinic and sulfonic acids occurs via a sulfenic acid. These results show that inhibition of cathepsin K activity and its autocatalytic maturation represent two potential mechanisms by which NO can exert its inhibitory effect on bone resorption. This work also shows that oxidative thiol modifications besides S-nitrosylation should be considered when the effects of NO and NO donors on critical thiol-containing proteins are investigated.     

Identification of a precursor form of cathepsin D in microsomal lumen: characterization of enzymatic activation and proteolytic

A precursor form of cathepsin D with 45 kDa was demonstrated in the rat liver microsomal lumen by immunoblotting analysis. The microsomal fraction containing procathepsin D which passed through a pepstatin-Sepharose resin showed no appreciable activity of cathepsin D. The in vitro incubation of this fraction at pH 3.0 resulted in a gradual increase of proteolytic activity toward hemoglobin as substrate and also, the proteolytic conversion of procathepsin D to the mature form was concomitantly observed. The proteolytic processing step was sensitive to pepstatin. These results suggest that procathepsin D is inactive in the endoplasmic reticulum and may be converted to the active forms by autoproteolytic processing mechanism at acidic pH during biosynthesis.     

Topographic studies of microsomal and pure prostaglandin H synthase

Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Expression of the proteinase specialized in bone resorption, cathepsin K, in granulomatous inflammation

BACKGROUND: The cysteine proteinase cathepsin K has aroused intense interest as the main effector in the digestion of extracellular matrix during bone resorption by osteoclasts. The enzyme is not a housekeeping lysosomal hydrolase, but is instead expressed with striking specificity in osteoclasts. In this work, we present evidence for the association of cathepsin K with the granulomatous reaction. Granulomas are inflammatory tissue reactions against persistent pathogens or foreign bodies. We came across cathepsin K while working on Echinococcus granulosus, a persistent tissue-dwelling, cyst-forming parasite that elicits a granulomatous response. MATERIALS AND METHODS: The walls of hydatid cysts from infected cattle were solubilized. Strong proteolytic activity was detected in the extracts. The proteinase responsible was purified by anion exchange and gel filtration. The purified protein was subjected to N-terminal sequencing, and its identity further confirmed by Western blotting, with a cathepsin K-specific antibody. The same antibody was used to localize the proteinase in paraffin-embedded sections of the parasite and the local host response. RESULTS: A proteinase was purified to near homogeneity from hydatid cyst extracts. The enzyme was unequivocally identified as host cathepsin K. Both the proenzyme and the mature enzyme forms were found. Cathepsin K was then immunolocalized both to the parasite cyst wall and to the epithelioid and giant multinucleated cells of the host granulomatous response. CONCLUSIONS: In the granulomatous response to the hydatid cyst, cathepsin K is expressed by epithelioid and giant multinucleated cells. We propose that, by analogy with bone resorption, cathepsin K is secreted by the host in an attempt to digest the persistent foreign body. Both processes, bone resorption and granulomatous reactions, therefore tackle persistent extracellular material (the bone matrix or the foreign body), and utilize specialized cells of the monocytic lineage (osteoclasts or epithelioid/giant cells) secreting cathepsin K as an effector.     

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole.

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.     

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).     

Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 degrees C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Involvement of the carboxyl-terminal propeptide of beta-glucuronidase in its compartmentalization within the endoplasmic reticulum as determined by a synthetic peptide approach

The proenzyme form of beta-glucuronidase is compartmentalized in large quantities within the endoplasmic reticulum by binding to the esterase, egasyn. Also, the propeptide of the proenzyme form of beta-glucuronidase is likely located at the carboxyl terminus. We have, therefore, tested if this carboxyl-terminal peptide is important in binding to egasyn. A polyclonal antibody to a 30-mer synthetic peptide, corresponding to the carboxyl-terminal 30 amino acids of pro-beta-glucuronidase, provided evidence that egasyn binds to the carboxyl terminus of beta-glucuronidase. This antibody interacted with proenzyme beta-glucuronidase-egasyn complexes in which one, two, or three egasyn molecules were bound to the beta-glucuronidase tetramer, but not with those complexes (M4) which contained four egasyn molecules. We interpret these results as indicating that all available carboxyl termini of the beta-glucuronidase proenzyme tetramer are shielded by egasyn in the M4 complexes. The same antibody did not recognize the mature lysosomal form of beta-glucuronidase, indicating that only the proenzyme form of microsomal beta-glucuronidase contains the original carboxyl terminus. Also, the synthetic 30-mer was found to be a specific and potent inhibitor (50% inhibition at 1.3 microM) of the esterase activity of purified egasyn but exhibited little inhibitory activity toward other purified esterases including a rat trifluoroacetylated esterase or egasyn esterase from another species. Together, these data describe a potent interaction of the exposed carboxyl terminus of precursor glucuronidase with the esterase catalytic site of egasyn, which in turn results in the specific localization of glucuronidase within the lumen of the endoplasmic reticulum.     

Identification of subcellular compartments involved in biosynthetic processing of cathepsin D.

We have assigned the biosynthetic processing steps of cathepsin D to intracellular compartments which are involved in its transport to lysosomes in HepG2 cells. Cathepsin D was synthesized as a 51-kDa proenzyme. After formation of 51-55-kDa intermediates due to processing of N-linked oligosaccharides, procathepsin D was proteolytically processed to an intermediate 44-kDa and the mature 31-kDa enzyme. The intersection of the biosynthetic pathway of cathepsin D with the endocytic pathway was labeled with horseradish peroxidase and monitored biochemically by 3,3'-diaminobenzidine cytochemistry. Horseradish peroxidase was used either as a fluid-phase marker to label the entire endocytic pathway or conjugated to transferrin (Tf) to label endosomes only. Directly after biosynthesis cathepsin D was accessible neither to horseradish peroxidase nor Tf-horseradish peroxidase. Newly synthesized 51-55-kDa species of cathepsin D present in the trans-Golgi reticulum were accessible to both horseradish peroxidase and Tf-horseradish peroxidase. The accessibility of trans-Golgi reticulum to both endocytosed horseradish peroxidase and Tf-horseradish peroxidase was monitored by colocalization with a secretory protein, alpha 1anti-trypsin. The proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurred in compartments which were fully accessible to fluid-phase horseradish peroxidase. Tf-horseradish peroxidase had access to only 20% of 44-kDa cathepsin D while it had no access to 31-kDa cathepsin D. In contrast, the 31-kDa species was completely accessible to fluid-phase horseradish peroxidase. We conclude that proteolytic processing of 51-55-kDa to 44-kDa cathepsin D occurs in endosomes, whereas the processing of 44-31-kDa cathepsin D takes place in lysosomes.     

Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.     

Recombinant human cathepsin H lacking the mini chain is an endopeptidase

Human procathepsin H was expressed in the form of inclusion bodies in Escherichia coli. Following refolding and autocatalytic activation, a recombinant cathepsin H form lacking the mini chain was produced. Removal of the mini chain completely abolished aminopeptidase activity of the enzyme and largely increased its endopeptidase activity (approximately 40-fold). Similarly to cathepsin S, Bz-FVR-AMC (k(cat)/K(m) value of 1070 mM(-1) s(-1)) was found to be the preferred substrate of recombinant cathepsin H. However, substrate inhibition was observed at a higher substrate (Z-FR-AMC, Bz-FVR-AMC) concentration. Endopeptidase activity of recombinant cathepsin H was seen also with the protein substrate insulin beta-chain with the major cleavage site between Glu13-Ala14. Recombinant human cathepsin H was inhibited by chicken cystatin, stefin A, and stefin B with the K(i) values in the range of 0.05-0.1 nM, which is slightly tighter than the inhibition of purified cathepsin H by the same inhibitors. These results thus indicate that the cathepsin H mini chain is essential for the aminopeptidase activity of the enzyme but has only a minor effect on the inhibition by cystatins.     

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.