Characterization of the Endoproteases Appearing during Wheat Grain Development

The pattern of endoproteolytic activities occurring during wheat (Triticum aestivum, cultivar Chinese Spring) grain development was investigated. Total endoprotease activity, assayed in solution with azocasein as a substrate, increased during the early stages of grain development to reach a maximum at 15 d postanthesis that was maintained until the grain was mature. Endoprotease activity was also assayed in gradient polyacrylamide gels co-polymerized with gelatin. The increase in endoproteolytic activity was due to the appearance of up to 18 endoproteolytic bands that were arbitrarily classified into five groups (A, B, C, D, and E). The presence of serine, aspartic, metallo, and, to a lesser extent, thiol proteases in developing wheat grains was demonstrated by the use of class-specific protease inhibitors. The appearance of the different classes of endoproteases during seed development was subject to temporal control; serine proteases were more abundant at early stages and aspartic and metallo proteases were more abundant at later stages. At intermediate stages of development (15-20 d postanthesis), most of the endoproteases were localized in the aleurone, testa, and embryo. The content of acidic thiol proteases was low in the developing starchy endosperm.     

Basidiomycetes harbour a hidden treasure of proteolytic diversity

This survey is the first to investigate the proteolytic potential of a large number of basidiomycetes. Aqueous extracts of 43 basidiomycetes were investigated for their content of proteolytic activities, using gelatin zymography. The activities were characterised qualitatively using class specific inhibitors. All four catalytic classes of proteases were present, with 4% of all activities classified as aspartic, 5% as cysteine, 6% as metallo and 22% as serine proteases, while the remaining activities could not be assigned unambiguously. The majority of the latter were not inhibited by any of the inhibitors used and were termed insensitive. Different proteolytic activities are evenly distributed among members of all orders of basidiomycetes, although some taxa are a richer source of proteases than others. A significant number of the cysteine protease activities shown here have not previously been reported in basidiomycetes. The fungal cysteine and serine protease inhibitors, clitocypin and CNSPI (Clitocybe nebularis serine protease inhibitor), both inhibited a number of activities and even a few activities that were otherwise insensitive to all other inhibitors used, hence indicating their potential for a regulatory role. The number and diversity of proteases in basidiomycetes are seen to be remarkable and encourage further investigation.     

A snapshot of the Ixodes scapularis degradome

Parasitic encoded proteases are essential to regulating interactions between parasites and their hosts and thus they represent attractive anti-parasitic druggable and/or vaccine target. We have utilized annotations of Ixodes scapularis proteases in gene bank and version 9.3 MEROPS database to compile an index of at least 233 putatively active and 150 putatively inactive protease enzymes that are encoded by the I. scapularis genome. The 233 putatively active protease homologs hereafter referred to as the degradome (the full repertoire of proteases encoded by the I. scapularis genome) represent ~1.14% of the 20485 putative I. scapularis protein content. Consistent with observations in other animals, the content of the I. scapularis degradome is ~6.0% (14/233) aspartic, ~19% (44/233) cysteine, ~40% (93/233) metallo, ~28.3% (66/233) serine and ~6.4% (15/233) threonine proteases. When scanned against other tick sequences, ~11% (25/233) of I. scapularis putatively active proteases are conserved in other tick species with ≥ 60% amino acid identity levels. The I. scapularis genome does not apparently encode for putatively inactive aspartic proteases. Of the 150 putative inactive protease homologs none are from the aspartic protease class, ~8% (12/150) are cysteine, ~58.7% (88/150) metallo, 30% (45/150) serine and ~3.3% (5/150) are threonine proteases. The I. scapularis tick genome appears to have evolutionarily lost proteolytic activity of at least 6 protease families, C56 and C64 (cysteine), M20 and M23 (metallo), S24 and S28 (serine) as revealed by a lack of the putatively active proteases in these families. The overall protease content is comparable to other organisms. However, the paucity of the S1 chymotrypsin/trypsin-like serine protease family in the I. scapularis genome where it is ~12.7% (28/233) of the degradome as opposed to ~22-48% content in other blood feeding arthropods, Pediculus humanus humanus, Anopheles gambiae, Aedes Aegypti and Culex pipiens quinquefasciatus is notable. The data is presented as a one-stop index of proteases encoded by the I. scapularis genome.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Identification of potential active-site residues in the Escherichia coli leader peptidase.

Leader peptidase of Escherichia coli cleaves the leader sequence from the amino terminus of membrane and secreted proteins after these proteins insert across the membrane. Despite considerable research, the mechanism of catalysis of leader peptidase remains unknown. This peptidase cannot be classified using protease inhibitors to the serine, cysteine, aspartic acid, or metallo- classes of proteases (Zwizinski, C., Date, T., and Wickner, W. (1981) J. Biol. Chem. 256, 3593-3597). Using site-directed mutagenesis, we have attempted to place leader peptidase in one of these groups. We found that leader peptidase, lacking all of the cysteine residues, can cleave the leader peptide from procoat, the precursor to bacteriophage M13 coat protein. Replacement of each histidine residue with an alanyl residue was without effect on catalysis. Among all the serine and aspartic acid residues, serine 90 and serine 185 as well as aspartic acid 99, 153, 273, and 276 are necessary to cleave procoat in a detergent extract. However, only serine 90 and aspartic acid 153 were required for processing using a highly sensitive in vivo assay. In addition to the residues directly affecting catalysis, aspartic acid 99 plays a role in maintaining the structure of leader peptidase. Replacement of this residue with alanine results in a very unstable leader peptidase protein. This study thus defines two critical residues, serine 90 and aspartic acid 153, that may be directly involved in catalysis and provides evidence that leader peptidase belongs to a novel class of serine proteases.     

Conformational homogeneity in molecular recognition by proteolytic enzymes

Crystal structures for several hundred protease-inhibitor complexes have been analysed and their superimpositions have been used to demonstrate a universal relationship between inhibitor/substrate conformation and molecular recognition by all aspartic, serine, cysteine and metallo proteases. Proteases universally recognize an extended beta strand conformation in all their peptidic (and non-peptidic) inhibitors and substrate analogues without significant exceptions. This conformational homogeneity is illustrated here for a subset of 180 protease-inhibitor structures which are displayed as (a) structural overlays of multiple inhibitors for each of eight aspartic, eight serine, six metallo and five cysteine proteases; (b) single inhibitors each bound to different proteases; and (c) Ramachandran plots of peptide or pseudo-peptide dihedral angle pairs which demonstrate beta strands (Phi -54 degrees to -173 degrees, Psi 24 degrees to 174 degrees ) like those normally found paired in proteins as beta sheets. However, unlike beta sheets, alpha and 3(10) helices, beta and gamma turns, where the folded main chain amide components are intramolecularly hydrogen bonded and thus unavailable for interaction with proteins, an inhibitor/substrate in an isolated beta strand conformation provides maximum exposure of its hydrogen bonding donors/acceptors and side chain components to a putative protease receptor. This analysis highlights the advantages of a strand conformation over other elements of secondary structure for protease recognition and may lead to generic strategies for inhibitor design.     

Characterization of senescence-associated proteases in postharvest broccoli florets.

We characterized the senescence-associated proteases of postharvest broccoli (Brassica oleracea L. var Green King) florets, using class-specific protease inhibitors and gelatin-polyacrylamide gel electrophoresis. Different classes of senescence-associated proteases in broccoli florets were partially characterized for the first time. Protease activity of broccoli florets was depressed by all the inhibitors and showed different inhibition curves during postharvest. The hydrolytic activity of metalloprotease (EC 3.4.24. - ) and serine protease (EC 3.4.21. - ) reached a maximum, 1 day after harvest (DAH), then decreased, while the hydrolytic activity of cysteine protease (EC 3.4.22. - ) and aspartic protease (EC 3.4.23. - ) increased throughout the postharvest senescence based on the calculated inhibition percentage of protease activity. The senescence-associated proteases were separated into seven endoprotease (EP) groups by gelatin-polyacryamide gel electrophoresis and classified into EP1 (metalloprotease), EP2 (metalloprotease and cysteine protease), EP3 (serine protease and aspartic protease), EP4, EP5, EP7 (cysteine protease), and EP6 (serine protease) based on the sensitivity of class-specific protease inhibitors. The proteases EP2, EP3, and EP4 were present throughout the postharvest stages. EP3 was the major EP at all times during senescence; EP4 intensity of activity increased after 2 DAH; EP6 and EP7 clearly increased after 4 DAH. Our results suggest that serine protease activity contributes to early stage (0-1 DAH) and late stage (4-5 DAH) of senescence; metalloprotease activity was involved in the early and intermediate stages (0-3 DAH) of senescence; and cysteine protease and aspartic protease activities participated in the whole process of broccoli senescence.     

Identification of the secreted neutral proteases from Anisakis simplex

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.     

Protease activities in carp retina

Proteases of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae.

The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin.     

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media

When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes. The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2. A metallo protease component (SV3) and an azocaseinase component (SV4) were also present. Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8. Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4. This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit. The method chosen for initial downstream processing influenced both the yield and type of protease purified. When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered. The medium used for the growth of Strep. thermovulgaris also appeared to affect the type of protease produced. A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.     

Protease activity in protein-free NSO myeloma cell cultures

Summary Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45–50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30–35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5–4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.     

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.     

Surface characters and extracellular toxins involved in the pathogenesis of Aeromonas hydrophila

A small number of serotypically distinct strains of A. hydrophila obtained from diseased freshwater fish were examined for their pathogenic properties comprising of cell surface characteristics and extracellular toxins. Test strains exhibited homogeneity in their cell surface characteristics despite being serologically heterogeneous. Studies on extracellular biological activities revealed qualitative and quantitative differences in production of toxins, probably explaining their antigenic diversity. Three distinct proteases, namely heat stable metallo protease, heat labile serine protease and heat labile metallo protease were identified from the strains.