Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen

Post-translational modifications (PTMs) strongly influence the structure and function of proteins. Lysine side chain acetylation is one of the most widespread PTMs, and it plays a major role in several physiological and pathological mechanisms. Protein acetylation may be detected by mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a useful and cheaper option. Here, we explored the feasibility of generating mAbs against single or multiple acetylations within the context of a specific sequence. As a model, we used the unstructured N-terminal domain of APE1, which is acetylated on Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture containing all combinations of single or multi-acetylated variants encompassing the 24–39 protein region. Targeted screening of the resulting clones yielded mAbs that bind with high affinity to only the acetylated APE1 peptides and the acetylated protein. No binding was seen with the non-acetylated variant or unrelated acetylated peptides and proteins, suggesting a high specificity for the APE1 acetylated molecules. MAbs could not finely discriminate between the differently acetylated variants; however, they specifically bound the acetylated protein in mammalian cell extracts and in intact cells and tissue slices from both breast cancers and from a patient affected by idiopathic dilated cardiomyopathy. The data suggest that our approach is a rapid and cost-effective method to generate mAbs against specific proteins modified by multiple acetylations or other PTMs.     

Role of the novel metallopeptidase Mop112 and saccharolysin for the complete degradation of proteins residing in different subcompartments of mitochondria

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m- and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space.     

Structural analysis of gelsolin using synchrotron protein footprinting

Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.     

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.     

Protease proteomics: revealing protease in vivo functions using systems biology approaches

Proteases irreversibly modify proteins by cleaving their amide bonds and are implicated in virtually every important biological process such as immunity, development and tissue repair. Accordingly, it is easy to see that deregulated proteolysis is a pathognomic feature of many diseases. Most of the current information available on proteases was acquired using in vitro methods, which reveals molecular structure, enzyme kinetics and active-site specificity. However, considerably less is known about the relevant biological functions and combined roles of proteases in moulding the proteome. Although models using genetically modified animals are powerful, they are slow to develop, they can be difficult to interpret, and while useful, they remain only models of human disease. Therefore, to understand how proteases accomplish their tasks in organisms and how they participate in pathology, we need to elucidate the protease degradome-the repertoire of proteases expressed by a cell, a tissue or an organism at a particular time-their expression level, activation state, their biological substrates, also known as the substrate degradome-the repertoire of substrates for each protease-and the effect of the activity of each protease on the pathways of the system under study. Achieving this goal is challenging because several proteases might cleave the same protein, and proteases also form pathways and interact to form the protease web [Overall, C.M., Kleifeld, O., 2006. Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. Cancer 6 (3), 227-239]. Hence, the net proteolytic potential of the degradome at a particular time on a substrate and pathway must also be understood. Proteomics offers one of the few routes to the understanding of proteolysis in complex in vivo systems and especially in man where genetic manipulations are impossible. The aim of this chapter is to review methods and tools that allow researchers to study protease biological functions using proteomics and mass spectrometry. We describe methods to assess protease expression at the messenger RNA level using DNA microarrays and at the protein level using mass spectrometry-based proteomics. We also review methods to reveal and quantify the activity state of proteases and to identify their biological substrates. The information acquired using these high throughput, high content techniques can then be interpreted with different bioinformatics approaches to reveal the effects of proteolysis on the system under study. Systems biology of the protease web-degradomics in the broadest sense-promises to reveal the functions of proteases in homeostasis and in disease states. This will indicate which proteases participate in defined pathologies and will help targeting specific proteases for disease treatments.     

A functional proteomics screen of proteases in colorectal carcinoma

BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.     

Comparative chemical and immunological characterization of five lipolytic enzymes (carboxylesterases) from rat liver microsomes

The chemical and immunological properties of five closely related microsomal serine hydrolases (carboxylesterases) from rat liver have been compared to evaluate whether they are variants of a single protein or independent proteins. These enzymes represent medium-chain-length acylcarnitine hydrolase, palmitoyl carnitine hydrolase, medium-chain-length monoglyceride hydrolase, and two long-chain monoglyceride hydrolases. All enzymes have similar subunit Mr's (58,000-61,000) and bear one active site per protein subunit, as could be shown by active sites with radioactive bis(4-nitrophenyl)phosphate, and have subsequently been cleft by proteases or by BrCN. The patterns of radioactive peptides obtained after electrophoresis or thin-layer chromatography indicated that the two long chain monoglyceride hydrolases were closely related, while all other hydrolases differed from these and from each other. The two long-chain monoglyceride hydrolases also had identical N- and C-termini that differed from those of the other hydrolases. All hydrolases contain low amounts of hexoses. It is concluded that the hydrolases investigated represent four independent enzymes with differing amino acid sequences. Three of the four hydrolases were microheterogenous. These results were confirmed with an immunological study using rabbit antisera against three of the hydrolases. Heparin-releasable liver lipase was not cross-reactive with the lipolytic enzymes investigated here.     

Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies

Summary In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (HFABP_c), four oligo-peptides of 15–20 amino-acids each and corresponding with different antigenic parts of the human H-FABP_c molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABP_c. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.     

Effects on translation pausing of alterations in protein and RNA components of the ribosome exit tunnel

Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, Crb^CmlA, that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to Crb^CmlA and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome.     

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.     

Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis.

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.     

Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects

Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested.     

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.