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.     

Cathepsin D of mouse leukemia L12010 cells unusual intracellular localization and biochemical properties

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopynic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18.After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45 000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95 000. Both forms are 4–5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss activity after treatment with β-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse luekemia L1210 cells are similar to the situation found for rat thoratic duct lymphocytes.     

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.     

Molecular characterization of cathepsin L from hepatopancreas of the carp Cyprinus carpio

Purified cathepsin L from carp, Cyprinus carpio, consists of a 28 kDa single-chain form that is different from the 24 and 5 kDa mammalian two-chain form. We cloned cathepsin L from carp hepatopancreas. The sequence consisted of a 1490 bp cDNA and a 1014 bp open reading frame, encoding a deduced protein of 337 amino acids that is likely processed to an active enzyme (single-chain form) with 222 amino acids. Its similarity to other types of vertebrate cathepsin L is less than 69%. Mammalian cathepsin L is further processed to a two-chain form, but possibly this is not the case with carp cathepsin L: the P1 site where cleavage occurred in the two-chain form of mammalian cathepsin L contains a serine, while carp cathepsin L processes a valine. Therefore, carp cathepsin L may have a different mechanism of action from mammalian cathepsin L.     

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor.

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm-associated and the seminal plasma inhibitors may confer to them different physico-chemical properties which are postulated to be of functional importance.     

Possible involvement of a sperm-associated body in the process of fertilization in quail

The present paper describes a novel structure, termed the sperm-associated body, which is found both in the lumen at the oviductal infundibulum and in the vitelline membrane of the ovum in the quail Coturnix japonica. The fully developed sperm-associated body, which is about 100 microm long, consisted of two parts; a core of concentric-circular appearance and a cortex of needle-like projections. The outer surface of the body was coated with CaCO3. The body was always accompanied by spermatozoa. About 70 sperm-associated bodies were observed in a single ovum. Electron-microscopically, small numbers of holes were detected in the vitelline membranes of a fertile ovum, and the sperm-associated bodies were always present in these holes. Frequently observed in the vitelline membranes was a disk speculated to be a portion of the inner layer of the membrane partially affected by spermatozoa. However, neither sperm-associated bodies nor spermatozoa were observed there. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding the inner layer of the vitelline membrane and penetrating it.     

A cysteine proteinase secreted from human breast tumours is immunologically related to cathepsin B.

The stable cathepsin B-like cysteine (thiol) proteinase secreted from human breast tumours in culture was shown to be destabilized by mercurial compounds. After such treatment the enzyme cross-reacts in a radioimmunoassay with a monospecific antiserum to human liver cathepsin B. It is suggested that the secreted enzyme may be a precursor form of lysosomal cathepsin B.     

Evidence for a defective thiol protease inhibitor in skeletal muscle of mice with hereditary muscular dystrophy

The thiol protease inhibitor (TPI-d) from hind-limb skeletal muscle of dystrophic 60-day-old male mice (strain 129/ReJ/dy) has been purified to apparent homogeneity and compared with the thiol protease inhibitor (TPI-n) from hind-limb skeletal muscle of normal 60-day-old male littermates. While both TPI-d and TPI-n displayed identical properties on sodium dodecyl sulfate-polyacrylamide gels (14,800 relative mass), analytical isoelectric focusing gels (pI 4.5), and high performance liquid chromatography columns, TPI-d was unable to inhibit papain and cathepsin B after purification by isoelectric focusing. However, a component in the purified TPI-d preparation with an isoelectric point of 4.9 initially masked the functional state of TPI-d, using papain when assayed with the test proteases papain and cathepsins H and L. This inhibitory component was absent from TPI-n preparations. Pure TPI-d was also unable to inhibit in vitro myosin hydrolysis by cathepsin B, whereas TPI-n completely blocked cathepsin B catalyzed myosin hydrolysis. Given the central role of the thiol proteases, especially cathepsin B, in intracellular protein metabolism and the possibility that uncontrolled thiol protease activity in muscle leads to muscle protein breakdown and dystrophy, our data suggest that a modified (defective) thiol protease inhibitor (TPI-d) may be (one of) the end product(s) of the dystrophy gene in mice with the hereditary form of the disease.     

Mechanism of staphylococcal resistance to non-oxidative antimicrobial action of neutrophils: importance of pH and ionic strength in determining the bactericidal action of cathepsin G

The staphylococcalcidal action of highly purified, enzymically inactive human lysosomal cathepsin G was studied. The bactericidal action of cathepsin G was optimal at pH 7.5 and was inhibited by NaCl; concentrations greater than 0.15 M NaCl completely inhibited killing of Staphylococcus aureus. Under optimal conditions (pH, temperature and NaCl concentration) the ED50 (effective dose) of cathepsin G against S. aureus strain 8325-4 was about 3.1 micrograms ml-1. Polymeric teichoic acid may serve as a binding site for cathepsin G by promoting electrostatic interactions since a mutant lacking this surface component exhibited enhanced resistance to the lethal action of cathepsin G, compared to the teichoic-acid-positive parental strain. These results suggest that (i) the ability of cathepsin G to kill intraphagosomal staphylococci may be regulated in part by the ionic strength of the environment and the pH of the maturing phagolysosome, and (ii) that strategies which retard acidification of the developing phagolysosome would promote the staphylococcalcidal action of cathepsin G.     

Cathepsin B-related proteases in the insulin secretory granule

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.     

The human cysteine protease cathepsin V can compensate for murine cathepsin L in mouse epidermis and hair follicles

Mice lacking the ubiquitously expressed lysosomal cysteine protease cathepsin L, show a complex skin phenotype consisting of periodic hair loss and epidermal hyperplasia with hyperproliferation of basal epidermal keratinocytes, acanthosis and hyperkeratosis. The recently identified human cathepsin L-like enzyme cathepsin V, which is also termed cathepsin L2, is specifically expressed in cornea, testis, thymus, and epidermis. To date, in mice no cathepsin V orthologue with this typical expression pattern has been identified. Since cathepsin V has about 75% protein sequence identity to murine cathepsin L, we hypothesized that transgenic, keratinocyte-specific expression of cathepsin V in cathepsin L knockout mice might rescue the skin and hair phenotype. Thus, we generated a transgenic mouse line expressing cathepsin V under the control of the human keratin 14 promoter, which mimics the genuine cathepsin V expression pattern in human skin, by directing it to basal epidermal keratinocytes and the outer root sheath of hair follicles. Subsequently, transgenic mice were crossed with congenic cathepsin L knockout animals. The resulting mice show normalization of epidermal proliferation and normal epidermal thickness as well as rescue of the hair phenotype. These findings provide evidence for keratinocyte-specific pivotal functions of cathepsin L-like proteolytic activities in maintenance of epidermis and hair follicles and suggest, that cathepsin V may perform similar functions in human skin.     

Peptidyl sulfonium salts. A new class of protease inhibitors

The possibility has been examined that peptidylmethyl sulfonium salts might affinity label proteases by an alkyl transfer from sulfur to an active center residue. The synthesis of a number of agents of this type is described as well as initial results of their effect on cysteinyl proteases, papain and cathepsin B. These are readily inactivated by reagents in which the peptidyl portion contains features that promote binding to the proteases such as a penultimate phenylalanine residue. Irreversible inactivation ensues by transfer of the peptidyl portion, not methyl groups. Peptidylmethyl sulfonium salts lose a proton to form an ylide structure which may be the prevalent form at physiological pH values. The ylide may also be the active affinity labeling form of the reagent since the rate of inactivation of cathepsin B increases with pH. In contrast, the action of another affinity labeling reagent for cathepsin B, benzyloxycarbonyl-Phe-AlaCHN2, a diazomethyl ketone, is relatively independent of pH.     

Expression of cysteine proteases in extraembryonic tissues during mouse embryogenesis

The expression of cathepsin B- and L-specific mRNAs as well as active forms of the enzymes was determined in mouse placenta and visceral yolk sac from 7.5 through 17.5 days postconception, a period marked by major anatomic transitions in the mouse conceptus. The level of specific mRNA was determined relative to the 28S ribosomal RNA in a series of multiprobe ribonuclease protection assays using high-specific-activity antisense cathepsin B and L riboprobes. The molecular forms of active cysteine proteases present in the tissues at the time of extraction were detected using a membrane-permeant radiolabeled active site-specific inhibitor, Fmoc-[(125)I(2)]Tyr-Ala-CHN(2). The results of this study show that the expression of active cathepsin L relative to active cathepsin B is significantly higher in visceral yolk sac than in placenta, consistent with a higher proteolytic requirement for the former tissue. Active cathepsin L was highest at Day 9.5 in visceral yolk sac, a stage at which it has been shown that proteolysis in this organ is required for production of amino acids for embryonic protein synthesis. Cathepsin L mRNA was also elevated in the Day 9.5 placenta, but paradoxically this did not result in an increase in cellular active enzyme. An unknown protein, termed p14, highly expressed in placenta, also reacted with the inhibitor. Expression of this protein was highest early during gestation in the ectoplacental cone, suggesting that p14 may be important in the implantation process.     

Calcium requirement and increased association with bovine sperm during capacitation by heparin

The requirement for external Ca+2 during capacitation of ejaculated bovine sperm with heparin and changes in sperm-associated 45Ca+2 during capacitation were investigated in vitro. Sperm capacitation was evaluated by ability to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine. The percentage of sperm which were capacitated during a 4 h incubation with heparin increased exponentially with increased exposure time to 2 mM Ca+2. When sperm were incubated with or without heparin in the presence of 45CaCl2, there was no difference in the amount of 45Ca+2 associated with sperm initially or at 1 h of incubation. Incubation with heparin resulted in a greater amount of sperm-associated 45Ca+2 at 2, 3, and 4 h as compared to sperm incubated without heparin. The amount of 45Ca+2 associated with sperm during capacitation was unaffected by washing with 2 mM EGTA-5 mM LaCl3. Glucose (5 mM) inhibited the effects of heparin on sperm-associated 45Ca+2 and on capacitation. The inhibitory effects of glucose could be overridden by 8-bromo-cAMP. The results suggest that the requirement for external Ca+2 during capacitation with heparin may be related to an increased association of external Ca+2 with sperm.     

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.     

Interactions of labeled epididymal secretory proteins with spermatozoa after injection of 35S-methionine in the mouse

The sequential interactions of epididymal secretory proteins with spermatozoa during epididymal transit were examined. Mice received injections of 35S-methionine, and the radiolabeled luminal fluid and sperm-associated proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis at various times after injection. The majority of the luminal fluid and sperm-associated proteins were found in the caput epididymidis at 8 h; by 7 days, many of these proteins had been transported to the cauda epididymidis. Two classes of epididymal protein-sperm interactions were distinguished on the basis of regional synthesis and secretion. The major class consisted of proteins that were synthesized, secreted, and bound to spermatozoa in the caput epididymidis. In this class, however, the binding of proteins to the spermatozoa was variable. For example, a protein of 25 kDa remained associated with spermatozoa in substantial amounts during epididymal transit, while proteins of 40 and 35 kDa decreased in amount. Other proteins such as a protein of 18 kDa did not remain associated with spermatozoa. Another class of proteins (54, 44, 29 kDa) were synthesized and secreted from all epididymal regions but bound only to caput spermatozoa. Most of the epididymal proteins appeared to be tightly bound to the spermatozoa since spermatozoa already saturated with the unlabeled protein in the distal epididymis remained so even though the spermatozoa were surrounded by labeled proteins in the luminal fluid. These studies demonstrate that a variety of specific interactions occur between epididymal secretory proteins and spermatozoa as they migrate and mature in the epididymis.     

Collagenase activity of cathepsin K depends on complex formation with chondroitin sulfate

Bone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis.     

Analysis of a truncated form of cathepsin H in human prostate tumor cells.

Increased expression of proteases has been correlated with the malignant progression of a variety of tumors. We found a significant increase in cathepsin H expression in high-grade prostatic intraepithelial neoplasia and carcinoma of the prostate. Two forms of cathepsin H, the full-length form (CTSH) and a truncated form with a 12-amino acid deletion in its signal peptide region (CTSHDelta10-21), were identified by cDNA sequence analysis. This deletion occurred not at the genomic level but likely at the RNA processing level. Both forms are expressed in prostate tissues as well as LNCaP, PC-3, and DU-145 prostate cancer cell lines. The deletion within the signal peptide region affected the trafficking of cathepsin H. Fluorescence microscopy, subcellular fractionation, and activity data indicated that the truncated form was perinuclear and secreted and had a reduced lysosomal association as compared with the full-length cathepsin H. Furthermore, the truncated cathepsin H was enzymatically active. Therefore, an increase in overall cathepsin H expression, particularly in the truncated form with a high secretion propensity, may affect cell biological behaviors such as those associated with tumor progression.