Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites

Tetanus toxin is synthesized by Clostridium tetani as a 151-kDa peptide chain. The primary gene product is processed post-translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52-kDa light chain and a 98-kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single-chain toxin has been subjected to limited proteolysis with various enzymes. The new N-termini have been determined by Edman degradation and the C-termini by isolation of short C-terminal peptide fragments and subsequent analysis of the sequence and composition. All two-chain toxins result from proteolytic nicking within the 17-residue segment of residues 445-461. Thus, the protease(s) of the culture broth cleave on the C-terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N-termini. Trypsin and clostripain first attack the C-terminal of Arg454 and later Arg448, whereas endoproteinase Arg-C cleaves the former bond only. Chymotrypsin and endoproteinase Glu-C each split a single peptide bond, i.e. that located after Tyr452 and Glu449, respectively. Papain gives rise to a large number of cleavages within the 17-residue segment, the new C-terminus being Thr445 or Asn446 and the new N-terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N-terminal Pro1 and C-terminal Asp1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various two-chain toxins was 5-11 times that of the single-chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat-brain homogenate. The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39-45), provide the detailed protein chemical characterization of the tetanus isotoxins.     

The tetanus toxin light chain inhibits exocytosis

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two-chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half-maximal effect at about 5 nmol/l. Heavy chain (up to 11 nmol/l) and unprocessed single-chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single-chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.     

Chains and fragments of tetanus toxin, and their contribution to toxicity

1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes associated with the nicking and activation of botulinum neurotoxin type E

Secondary and tertiary structural parameters of type E botulinum neurotoxin in the unactivated single-chain and activated two-chain (i.e., after proteolytic cleavage) forms were analyzed using circular dichroism, derivative absorption and fluorescence spectroscopy. The estimated secondary structures (22 and 20% alpha-helix, 44 and 44% beta-pleated sheets, and 34 and 36% random coils for the single- and two-chain neurotoxins, respectively) indicated that virtually no change occurred upon nicking of the single-chain neurotoxin. About 57% of the 70 Tyr residues were exposed in the single-chain form, which increased to 62% in the two-chain form. Fluorescence quenching experiments with neutral, anionic and cationic quenchers indicated that about 40% of the maximum accessible fluorescent Trp residues were exposed on the surface of the single-chain neurotoxin as compared to only 20% in the case of the two-chain neurotoxin. Acrylamide was the most effective quencher with a fraction accessibility of 0.56 and 0.48 of maximum accessible Trp fluorescence residues in the single and two-chain forms of the neurotoxin, respectively. Native polyacrylamide gel electrophoresis of the two forms of the neurotoxin revealed greater mobility for the two chain form. This indicates that the surface charges in the single-chain neurotoxin were altered upon nicking. These observations suggest that nicking of the single-chain type E neurotoxin results in refolding and redistribution of the surface charges of the neurotoxin.     

Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.     

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.     

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.     

Urokinase-related proteins in human urine. Isolation and characterization of single-chain urokinase (pro-urokinase) and urokinase-inhibitor complex

Urokinase-related proteins were purified from 60-liter batches of human urine collected into the protease inhibitor aprotinin to prevent proteolytic degradation. Three homogeneous species were obtained by chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, Sephadex G-100, benzamidine-Sepharose, and immunoadsorption on a murine anti-human urokinase monoclonal antibody. One urokinase-related protein with Mr 95,000 representing a complex of two-chain urokinase with an inhibitor accounts for about 70% of the total urokinase-related antigen in urine. Nucleophilic agents dissociate the complex into active two-chain urokinase and a protein with Mr 45,000-50,000 which is immunologically distinct from urokinase. Approximately 25% of the urinary urokinase-related antigen represents a single-chain molecule with Mr 54,000. This highly purified single-chain molecule was obtained with a yield of 5 micrograms/liter of urine. Only trace amounts (less than 5%) of the urokinase-related antigen were recovered as free two-chain urokinase. The urinary single-chain urokinase-related protein has no specific affinity for fibrin. It has a very low activity on Pyroglu-Gly-Arg-p-nitroanilide, a urokinase-specific synthetic substrate, but directly activates plasminogen following Michaelis-Menten kinetics with Km = 0.7 microM and kcat = 0.0011 S-1. The single-chain molecule is rapidly converted to active two-chain urokinase by plasmin. Active two-chain urinary urokinase has a very high amidolytic activity and activates plasminogen with Km = 60 microM and kcat = 1.4 S-1. It is concluded that the urokinase-related proteins in human urine consist of about 25% of single-chain urokinase (10-20 micrograms/liter) and of about 75% two-chain urokinase (40-50 micrograms/liter), the bulk of which is complexed to an inhibitor. Because even in freshly voided urine most of the urokinase-related antigen is already converted to two-chain urokinase, urine does not seem to be a suitable source for the large-scale purification of single-chain urokinase. In view of the very significant intrinsic plasminogen-activating properties of single-chain urokinase, it should not be considered to be a proenzyme form of urokinase. The dramatic differences of its kinetic constants from those of urokinase render the designation single-chain urokinase equally inadequate. Consequently, the designation "single-chain urokinase-type plasminogen activator" was recently adopted by the International Committee on Thrombosis and Haemostasis (Annual Meeting, San Diego, CA, July 13-14, 1985).     

Recombinant forms of tetanus toxin engineered for examining and exploiting neuronal trafficking pathways

Tetanus toxin is a fascinating, multifunctional protein that binds to peripheral neurons, undergoes retrograde transport and trans-synaptic transfer to central inhibitory neurons where it blocks transmitter release, thereby, causing spastic paralysis. As a pre-requisite for exploiting its unique trafficking properties, a novel recombinant single chain was expressed at a high level in Escherichia coli as a soluble, easily purifiable protein. It could be activated with enterokinase to produce a dichain that matched native toxin in terms of proteolytic and neuroinhibitory activities, as well as induction of spastic paralysis in mice. Importantly, nicking was not essential for protease activity. Substitution of Glu(234) by Ala created a protease-deficient atoxic form, which blocked the neuroparalytic action of tetanus toxin in vitro, with equal potency to its heavy chain; but, the mutant proved >30-fold more potent in preventing tetanus in mice. This observation unveils differences between the intoxication processes resulting from retrograde transport of toxin in vivo and its local uptake into peripheral or central nerves in vitro, dispelling a popularly held belief that the heavy chain is the sole determinant for efficient trafficking. Thus, this innocuous mutant may be a useful vehicle, superior to the heavy chain, for drug delivery to central neurons.     

Detection of bacterial toxins with monosaccharide arrays

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.     

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.     

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.     

EFFECTS OF BOTULINUM AND TETANUS TOXINS ON CHOLINE ACETYLTRANSFERASE ACTIVITY IN SKELETAL MUSCLE IN THE MOUSE

Abstract— The effects of botulinum and tetanus toxins on the activity of choline acetyltransferase present in the motor nerve terminals of fast and slow skeletal muscle in the mouse were investigated. There was no change in the activities of choline acetyltransferase in either muscle after the injection of botulinum toxin but tetanus toxin caused a rise in the activity of the enzyme in fast muscle. Botulinum toxin is known to inhibit the release of acetylcholine and whilst neuromuscular transmission is blocked the motor nerves sprout and form new end-plates. Tetanus toxin has been shown to cause hyperactivity of motor neurons. The nerve growth caused by the botulinum toxin did not result in increased choline acetyltransferase levels in the muscles, whereas the synaptic hyperactivity caused by tetanus was associated with increased enzyme levels.     

Rapid, simplified method for production and purification of tetanus toxin

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Exploiting cell death pathways by an E. coli cytotoxin: autophagy as a double-edged sword for the host

Cytotoxic necrotizing factor 1 is a bacterial protein toxin from Escherichia coli that is able to activate the Rho GTPases and to hinder apoptosis and mitotic catastrophe. Upon exposure to toxin, cells undergo a complex framework of changes, including cytoskeleton remodeling and multinucleation. These cells also show a high survival rate for long periods of time and improve both their macropinocytotic scavenging activities and microautophagy. Only at the very end, probably when "feeding" materials are exhausted, do these cells die by autophagy. Taking into account the complex role of bacterial protein toxins in the infectious processes, we indicate the CNF1 activity as a Janus-faced paradigm of those bacteria that hijack cell fate to their own benefit. This could somehow be linked to the hypothesized connection between certain bacterial toxins and cancer onset.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

The single-chain form of tissue-type plasminogen activator has catalytic activity: studies with a mutant enzyme that lacks the cleavage site

Tissue-type plasminogen activator (t-PA), the serine protease responsible for catalyzing the production of plasmin from plasminogen at the site of blood clots, is synthesized as a single-chain polypeptide precursor. Proteolytic cleavage at the C-terminal side of Arg275 generates a two-chain form of the enzyme whose subunits are held together by a single disulfide bond. We have measured the activities of both forms of the wild-type enzyme, as well as that of a mutant enzyme (Arg275----Gly), created by oligonucleotide-directed mutagenesis, that cannot be cleaved into a two-chain form. Both types of single-chain t-PAs are enzymatically active and exhibit identical Vmax and Km values when assayed with synthetic peptide substrates, indicating that the single amino acid change had no effect on the amidolytic activity of the enzyme. However, cleavage of wild-type t-PA into the two-chain form results in increased activity both on a peptide substrate and on the natural substrates Lys- and Glu-plasminogen in the absence or presence of stimulation by soluble fibrin. The enhanced activity is due to a 3-5-fold increase in the Vmax of the cleaved enzyme, rather than to any change in the Km values for the various substrates. During incubation with plasminogen, the single-chain form of wild-type t-PA is converted to the two-chain form by plasmin generated during the reaction. This conversion, from the less active form of the enzyme, results in a reaction that displays biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.     

Internalization of exogenous cystatin F supresses cysteine proteases and induces the accumulation of single-chain cathepsin L by multiple mechanisms

Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.     

Single-chain tissue-type plasminogen activator is a substrate of mouse glandular kallikrein 24

Leydig cells of the adult mouse testis express at a detectable level three distinct glandular (tissue) kallikrein genes: mKlk21, mKlk24, and mKlk27. Recently, the proteins encoded by these genes were characterized using active recombinant proteases, but their roles in the mouse testis remained to be determined. The present study showed that among the proteases, mK24 markedly enhanced the activity of human recombinant single-chain tissue-type plasminogen activator when the two were incubated together. This activation was found to be due to proteolytic conversion of the single-chain enzyme to a two-chain form. The expression of tissue-type plasminogen activator in interstitial Leydig cells was demonstrated by RT-PCR and immunohistochemical analyses. The primary culture medium of adult male testicular Leydig cells contained immunoreactive substances recognized by anti-mK24 antibodies. In addition, the same medium was capable of converting the single-chain plasminogen activator to the two-chain protein. These results suggest that mK24 may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis, due not only to its own activity, but also to that of plasmin produced by the single-chain tissue-type plasminogen activator-converting activity of mK24.