Endopeptidase Substrates Research Articles

The Enzyme Gene Expression of Protein Utilization and Metabolism by Lactobacillus helveticus CICC 22171.

The purpose of this study was to explore the hydrolytic ability of Lactobacillus helveticus CICC 22171 with regard to protein and the expression of enzyme genes during protein utilization. The results revealed that the strain hydrolyzed casein from the C-terminal, reached the maximum level in 6 h, and the number of amino acids in the hydrolyzed peptide was 7–33. The molecular weight was 652.4–3432.74 kDa. Hydrophobic peptides produced by hydrolysis were the source of β-casein bitterness. Leucine and glutamine were the preferred cleavage points after 1 h; tyrosine and tryptophan subsequently increased. The first step of hydrolysis was controlled by PrtP and PrtM genes and coordinated with the action of PrtH1 and PrtH2. The transport system consisted of DtpT, OppB, OppD and OppF. The hydrolytic third step endopeptidase system consisted of the aminopeptidases (PepN, PepC, PepM and PepA), the endopeptidases (PepE, PepF and PepO); the dipeptidases (PepV and PepD), the tripeptidase PepT; the proline peptidases (PepX, PepP, PepQ, PepR and PepI). The expression of CEP genes was significantly different, and the expression level of genes related to the transport system significantly increased from 0 to 1 h. The specificity of the substrate and action site of endopeptidase was abundant.

The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity

The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.

Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity

Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

Isolation, purification and biochemical characterization of dipeptidyl peptidase-III from germinated Vigna radiata seeds

Genome analysis of plants indicated majority of putative protease genes that need characterization at enzymatic and molecular level. Proteases execute important role in seed development but knowledge of dipeptidylpeptidases in seeds is limited. A dipeptidylpeptidase-III that cleaves Arg-Arg from Arg-Arg-4mβNA was purified from germinated Mung bean seeds. Screening of cereals and legumes for DPP-III revealed highest activity in Phaseolus vulgaris and Vigna radiata (variety P.9531) seeds germinated for 48h. Homogenate (10%) was acid precipitated upto pH 4.2 and then subjected to 0–55% ammonium sulphate precipitation followed by successive chromatographies on cation exchanger, gel filtration and anion exchanger. Enzyme purity was confirmed by native polyacrylamide gel electrophoresis and in situ gel assays. DPP-III is a heterodimer of ∼60kDa with two subunits of 32.8kDa and 27kDa on SDS-PAGE. DPP-III worked optimally at pH 8.0 and at 37°C with pH stability between 7.5 to 9.5. Arg-Arg-4mβNA was preferred substrate and none of endopeptidase and monopeptidase substrates were hydrolysed. Inhibitors studies revealed it as thiol protease with involvement of metals at active site. The enzyme might be involved in plant's development by generating/deactivating bioactive peptides. Further studies on functional characterization and molecular structure are in progress in our laboratory.

Prolyl endopeptidase activity in bronchoalveolar lavage fluid: a novel diagnostic biomarker in a guinea pig model of invasive pulmonary aspergillosis

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.

Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype

BackgroundClostridium botulinum strains that produce botulinum neurotoxin type E (BoNT/E) are most commonly isolated from botulism cases, marine environments, and animals in regions of high latitude in the Northern hemisphere. A strain of C. botulinum type E (CDC66177) was isolated from soil in Chubut, Argentina. Previous studies showed that the amino acid sequences of BoNT/E produced by various strains differ by < 6% and that the type E neurotoxin gene cluster inserts into the rarA operon.ResultsGenetic and mass spectral analysis demonstrated that the BoNT/E produced by CDC66177 is a novel toxin subtype (E9). Toxin gene sequencing indicated that BoNT/E9 differed by nearly 11% at the amino acid level compared to BoNT/E1. Mass spectrometric analysis of BoNT/E9 revealed that its endopeptidase substrate cleavage site was identical to other BoNT/E subtypes. Further analysis of this strain demonstrated that its 16S rRNA sequence clustered with other Group II C. botulinum (producing BoNT types B, E, and F) strains. Genomic DNA isolated from strain CDC66177 hybridized with fewer probes using a Group II C. botulinum subtyping microarray compared to other type E strains examined. Whole genome shotgun sequencing of strain CDC66177 revealed that while the toxin gene cluster inserted into the rarA operon similar to other type E strains, its overall genome content shared greater similarity with a Group II C. botulinum type B strain (17B).ConclusionsThese results expand our understanding of the global distribution of C. botulinum type E strains and suggest that the type E toxin gene cluster may be able to insert into C. botulinum strains with a more diverse genetic background than previously recognized.

Molecular ruler of tripeptidylpeptidase II: Mechanistic principle of exopeptidase selectivity

The structure of tripeptidylpeptidase II (TPPII) has shown that it belongs to the group of exopeptidases which use a double-Glu motif to convey aminopeptidase activity. TPPII has been implicated in vital biological processes. At least one of these, antigen processing, requires the involvement of its endopeptidase activity. In order to understand the extent and molecular basis of this unusual functional promiscuity we have performed a systematic kinetic analysis of wild type Drosophila melanogaster TPPII and five point mutants of the double-Glu-motif (E312/E343) involving natural substrates. Unlike the known double-Glu motives of other exopeptidases, the double-Glu motif of TPPII is distinctly asymmetrical: E312 is the crucial determinant of the aminotripeptidolytic ruler mechanism. It both blocks the active-site cleft at substrate position P4 and forms a salt bridge with the N-terminus of the substrate. In contrast, E343 forms a much weaker salt bridge than E312 and it does not have a blocking role. An endopeptidase substrate can bind at relatively high affinity if the length of the substrate permits binding to several S′ sites. However, the lacking alignment of the substrate by the double-Glu motif causes the endopeptidolytic Kcat/KM of TPPII to be very low.

Extraction of Peptidase Substrates by the Isolated Perfused Rat Lung

Peptidases in the lung are well placed to have an important role in regulating levels of circulating endogenous and therapeutic peptides. They also present a first-pass metabolic barrier for peptides delivered to the lung for systemic absorption. The activities of five peptidases were surveyed in the pulmonary circulation of the asanguinous isolated perfused rat lung (IPRL) using synthetic substrates and selective inhibitors. Extraction ratios (ER) were calculated for the substrates of: aminopeptidase N (AMN), ER 0·37 ± 0·04; dipeptidyl peptidase IV (DPP), ER 0·69 ± 0·05; and angiotensin-converting enzyme (ACE), ER 0·40 ± 0·02. These activities were inhibited (> 95%) by the selective inhibitors bestatin, diprotin A, and captopril, respectively. Substrates for neutral endopeptidase 24.11 (NEP) and carboxypeptidase M (CPM) were minimally degraded with ER of 0·02 and 0·00, respectively. The low activity of NEP, a major membrane bound endopeptidase, indicates that the lungs may not contribute greatly to degradation of either systemic NEP substrates, or exopeptidase-resistant peptides absorbed from the peripheral lung. In contrast peptides susceptible to the exopeptidases AMN, DPP, and ACE, will be substantially degraded during passage through the lung. The absence of CPM activity in the pulmonary circulation of the asanguinous IPRL implies that basic carboxypeptidase activity in blood perfused lungs reported in the literature is more likely to be the result of plasma carboxypeptidase N activity.

Ala657 and Conserved Active Site Residues Promote Fibroblast Activation Protein Endopeptidase Activity via Distinct Mechanisms of Transition State Stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN AMINOPEPTIDASE FROM EIMERIA TENELLA

Our previous investigation demonstrated the expression in Eimeria tenella sporulated oocysts of an aminopeptidase (AP) with strong homology to AP N. To further understand the role of proteases during development, we investigated the molecular and biochemical properties of E. tenella AP. Greater than 95% AP activity was present in a soluble extract during sporulation of oocysts with highest activity in fully sporulated oocysts. The AP activity was inhibited by the AP inhibitors bestatin and 1,6-phenanthroline, but not by serine protease inhibitors. The AP had specificity for synthetic endopeptidase substrates that contain arginine, alanine, or glycine at the N terminus. Partial purification of the enzyme yielded a major protein band with an Mr of about 106 kDa and an isoelectric point (Ip) of 5.1. Reverse transcription-polymerase chain reaction indicated that the gene for AP is expressed during sporulation, but expression is absent or greatly reduced in the sporozoites and merozoites. On the basis of the deduced gene structure, the predicted Mr is 110 kDa with a pI of 5.59. Database search indicates that the E. tenella AP shares significant homology with the AP from Apicomplexan taxa: Toxoplasma gondii, Cryptosporidium parvum, and Cryptosporidium hominis. Together, these results confirm the presence of a cytosolic AP related to AP N, which is expressed and active during sporulation of E. tenella oocysts.

CHARACTERIZATION OF EXCRETORY/SECRETORY ENDOPEPTIDASE AND METALLO-AMINOPEPTIDASES FROM TAENIA CRASSICEPS METACESTODES

Cysticercosis is caused by Taenia spp. metacestodes, which must survive in the host tissues to complete their life cycle. Their survival depends on their control of host immune responses. Because many parasites use proteases to modulate host responses, we examined culture media from Taenia crassiceps metacestodes for protease activity using peptide substrates. We identified prominent aminopeptidase activity at neutral pH, which was inhibited by chelating agents and partially inhibited by the aminopeptidase inhibitor, bestatin. Endopeptidase substrates were optimally cleaved at slightly acidic pH and endopeptidase activity was inhibited by cysteine protease inhibitors. Gel filtration FPLC and subsequent visualization by silver staining revealed a metallo-aminopeptidase of molecular weight 21 kDa and cysteine proteases of Mr 70 and 64 kDA. Recombinant IL-2 was digested when incubated with parasite culture supernatants, but not with control media. IL-2 degradation was completely inhibited by 1,10 phenanthroline and partially inhibited by bestatin, suggesting that a metallo-aminopeptidase was responsible. Incubation of human IgG with culture supernatants resulted in complete degradation of IgG, which was blocked by cysteine protease inhibitors. These observations demonstrate that Taenia spp. metacestodes secrete a number of proteolytic enzymes, which may target molecules from the host immune system and assist in evasion of the host immune response.

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice: A comparative study

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's α-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547–55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10–100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10–100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1–1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B 2 receptor antagonist HOE 140 (0.1 μmol/kg) and captopril (100 μg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B 2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.

PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress.

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5alphaDeltapepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme inEscherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya: IMPLICATIONS IN CYTOSOLIC PROTEIN DEGRADATION

Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to detect 20 S proteasomal activity from Archaea to mammals. An o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing activity was detected in Escherichia coli although it lacks 20 S proteasomes. We identified PepN, previously characterized as the sole alanine aminopeptidase in E. coli, to be responsible for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase. First, extracts from an ethyl methanesulfonate-derived PepN mutant, 9218, did not cleave Suc-LLVY-AMC and L-Ala-para-nitroanilide (pNA). Second, biochemically purified PepN cleaves a wide variety of both aminopeptidase and endopeptidase substrates, and L-Ala-pNA is cleaved more efficiently than other substrates. Studies with bestatin, an aminopeptidase-specific inhibitor, suggest differences in the mechanisms of cleavage of aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes whole proteins, casein and albumin. Finally, an E. coli strain with a targeted deletion in PepN also lacks the ability to cleave Suc-LLVY-AMC and L-Ala-pNA, and expression of wild type PepN in this mutant rescues both activities. In addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria harboring 20 S proteasomes, to be an aminopeptidase homologous to E. coli PepN, by mass spectrometry analysis. "Sequence-based homologues" of PepN include well characterized aminopeptidases, e.g. Tricorn interacting factors F2 and F3 in Archaea and puromycin-sensitive aminopeptidase in mammals. However, our results suggest that eubacterial PepN and its homologues displaying aminoendopeptidase activities may be "functionally similar" to enzymes important in downstream processing of proteins in the cytosol: Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.

Toward Computer-Based Cleavage Site Prediction of Cysteine Endopeptidases

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer-based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool 'PEPS' (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rule-based endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin L-deficient and wild-type mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.

Analysis of recombinant Phex: an endopeptidase in search of a substrate.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.

Comparison of Different Peptidase Substrates for Evaluation of Microbial Quality of Aerobically Stored Meats

Different aminopeptidase and endopeptidase substrates were assessed for the detection of enzymatic activity of microorganisms collected from the surface of aerobically cold-stored pork and beef. The most sensitive substrates were fluorogenic Ala-7-amino-4-methylcoumarin (Ala-AMC) or Leu-AMC and colorogenic Ala-p-nitroanilide (Ala-pNA). Activity on natural oligopeptides, e.g., bradykinin or αs1 casein fragment 1 to 23, was very low. The correlation coefficient (r) between log surface counts of 66 meat samples and log fluorescence or absorbance after incubation of surface microbial cells for 2 h with Ala-AMC, Leu-AMC, and Ala-pNA was 0.89, 0.83, and 0.82, respectively. A distinct yellow color was obtained with Ala-pNA when the surface count was ∼106 CFU/cm2. Although correlation and sensitivity was better, no clear advantage is obtained with the use of the fluorogenic Ala-AMC or Leu-AMC instead of Ala-pNA, a substrate proposed by Alvarado et al. (J. Food Sci. 57:1330, 1992) for rapidly assessing the microbial quality of refrigerated meat. The correlation coefficient (r) between time of cold storage and surface count was 0.69.

Characterization of Endopeptidase Activity of Tripeptidyl Peptidase-I/CLN2 Protein Which Is Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH2 is readily hydrolyzed by Cln2p/TPP-I (Kcat/Km = 7.8 s−1 mM−1). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH2Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.

Autocatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.

Secretion of an Aminopeptidase during Transition of Third- to Fourth-Stage Larvae of Ascaris suum

Protease activity was identified in culture fluids collected during in vitro development of L3 to L4 larval stages of Ascaris suum. Fluorogenic peptide substrates with unblocked N-termini were specifically hydrolyzed indicating aminopeptidase activity; a terminal arginyl residue was preferred. Culture fluids did not hydrolyze fluorogenic peptide substrates with blocked N-termini (endopeptidase substrates). The aminopeptidase activity was inhibited by 1,10-phenanthroline (metalloprotease inhibitor) and by amastatin and bestatin (aminopeptidase inhibitors); AEBSF (serine protease inhibitor), Z-phe-ala-FMK and E-64 (cysteine protease inhibitors), and pepstatin A (aspartyl protease inhibitor) had little effect on activity. The apparent molecular weight of the aminopeptidase was estimated by sucrose density gradient centrifugation at 293 kDa. The aminopeptidase displayed an acidic isoelectric point of 4.7. The peak secretion of the aminopeptidase was temporally associated with molting and suggests a function for the protease in this complex process.