Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

A cell-penetrating artificial metalloenzyme regulates a gene switch in a designer mammalian cell.

Complementing enzymes in their native environment with either homogeneous or heterogeneous catalysts is challenging due to the sea of functionalities present within a cell. To supplement these efforts, artificial metalloenzymes are drawing attention as they combine attractive features of both homogeneous catalysts and enzymes. Herein we show that such hybrid catalysts consisting of a metal cofactor, a cell-penetrating module, and a protein scaffold are taken up into HEK-293T cells where they catalyze the uncaging of a hormone. This bioorthogonal reaction causes the upregulation of a gene circuit, which in turn leads to the expression of a nanoluc-luciferase. Relying on the biotin-streptavidin technology, variation of the biotinylated ruthenium complex: the biotinylated cell-penetrating poly(disulfide) ratio can be combined with point mutations on streptavidin to optimize the catalytic uncaging of an allyl-carbamate-protected thyroid hormone triiodothyronine. These results demonstrate that artificial metalloenzymes offer highly modular tools to perform bioorthogonal catalysis in live HEK cells.

Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

In Silico Investigation of Traditional Chinese Medicine for Potential Lead Compounds as SPG7 Inhibitors against Coronary Artery Disease.

Coronary artery disease (CAD) is the most common cause of heart attack and the leading cause of mortality in the world. It is associated with mitochondrial dysfunction and increased level of reactive oxygen species production. According to the Ottawa Heart Genomics Study genome-wide association study, a recent research identified that Q688 spastic paraplegia 7 (SPG7) variant is associated with CAD as it bypasses the regulation of tyrosine phosphorylation of AFG3L2 and enhances the processing and maturation of SPG7 protein. This study aims to identify potential compounds isolated from Traditional Chinese Medicines (TCMs) as potential lead compounds for paraplegin (SPG7) inhibitors. For the crystallographic structure of paraplegin, the disordered disposition of key amino acids in the binding site was predicted using the PONDR-Fit protocol before virtual screening. The TCM compounds saussureamine C and 3-(2-carboxyphenyl)-4(3H)-quinazolinone, have potential binding affinities with stable H-bonds and hydrophobic contacts with key residues of paraplegin. A molecular dynamics simulation was performed to validate the stability of the interactions between each candidate and paraplegin under dynamic conditions. Hence, we propose these compounds as potential candidates as lead drug from the compounds isolated from TCM for further study in drug development process with paraplegin protein for coronary artery disease.

The evolutionary conservation of the A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motif metzincins across vertebrate species and their expression in teleost zebrafish.

BACKGROUND: The A Disintegrin-like and Metalloproteinase domain with Thrombospondin-1 motifs (ADAMTS) enzymes comprise 19 mammalian zinc-dependent metalloproteinases (metzincins) with homologues in a wide range of invertebrates. ADAMTS enzymes have a broad range of functions in development and diseases due to their extracellular matrix remodelling activity. Here, we report a detailed characterisation of their evolutionary conservation across vertebrates. RESULTS: Using bioinformatics complemented with de novo sequencing, gene sequences for ADAMTS enzymes were obtained from a variety of organisms. Detailed evolutionary analyses revealed a high level of conservation across vertebrates with evidence of ADAMTS gene expansion during two rounds of whole genome duplication (WGD) in vertebrates, while tandem duplication events and gene loss were also apparent. However, the additional round of teleost-specific WGD did not have a significant effect on ADAMTS gene family members suggesting their conserved roles have remained constant in teleost fish. Quantitative reverse-transcriptase polymerase chain reaction analysis revealed dynamic expression of adamts genes throughout zebrafish embryonic development reflecting the key conserved roles they play in vertebrate embryogenesis. Notably, several adamts mRNAs were maternally expressed with a dramatic increase in mRNA levels coinciding with zygotic expression and organogenesis. Broad adamts mRNA expression was also demonstrated in several adult organs indicating potential roles in adult homeostasis. CONCLUSIONS: Our data highlight the evolution of the ADAMTS gene family through duplication processes across metazoans supplemented by a burst of amplification through vertebrate WGD events. It also strongly posits the zebrafish as a potential model species to further elucidate the function of ADAMTS enzymes during vertebrate development.

Temperature-sensitive liposome-mediated delivery of thrombolytic agents.

BACKGROUND: Clinical efficacy of thrombolytic drugs is limited by lack of specific delivery and requires large therapeutic doses which increase toxicity. Encapsulating these drugs in temperature-sensitive liposomes and applying hyperthermia to deliver thrombolytic agents locally to thrombus might theoretically favourably alter the therapeutic window. The objectives of this study were to formulate liposomes encapsulating thrombolytics and assess thrombolytic activity following hyperthermia. METHODS: Three liposome formulations were investigated: temperature-sensitive liposome (TSL, DPPC:DSPE-PEG2000 (mol% 95:5)), low temperature-sensitive liposome (LTSL, DPPC:MSPC:DSPE-PEG2000 (mol% 85.3:9.7:5)), and traditional temperature-sensitive liposome (TTSL, DPPC:HSPC:Chol:DSPE-PEG2000 (mol% 55:25:15:5)). To characterise temperature-dependent release of high molecular weight cargo from each formulation, fluorescein-conjugated dextrans (70 kDa) were loaded and release was quantified via spectrophotometry. Staphylokinase (SAK), urokinase, and tissue-type plasminogen activator were also loaded individually into each liposome formulation. Leakage at 37 °C and release at 38-44 °C were quantified via chromogenic enzymatic activity assay. Clot lysis was evaluated by measuring mass of blood clots before and after thrombolytic liposome treatment. RESULTS: The LTSL formulation had optimal release characteristics with maximum release at 41.3 °C. Release of dextrans from LTSLs was observed to be 11.5 ± 1.5%, 79.7 ± 1.6%, and 93.6 ± 3.7% after 15 min in plasma at 37°, 39°, and 41.3 °C, respectively. The SAK LTSL had the highest release/leakage ratio and demonstrated greater clot lysis. CONCLUSIONS: The SAK LTSL achieves significant clot lysis in vitro. When combined with local hyperthermia, the SAK LTSL potentially produces sufficient thrombolysis while minimising systemic side effects.

A drug-repositioning screening identifies pentetic acid as a potential therapeutic agent for suppressing the elastase-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues.

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

S1 pocket fingerprints of human and bacterial methionine aminopeptidases determined using fluorogenic libraries of substrates and phosphorus based inhibitors.

Methionyl aminopeptidases (MetAPs) are metallo-dependent proteases responsible for removing of N-terminal methionine residue of peptides and proteins during protein maturation and activation. In this report we use a comprehensive strategy to screen the substrate specificity of three methionyl aminopeptidases: Homo sapiens MetAP-1, Homo sapiens MetAP-2 and Escherichia coli MetAP-1. By utilizing a 65-membered fluorogenic substrate library consisting of natural and unnatural amino acids we established detailed substrate preferences of each enzyme in the S1 pocket. Our results show that this pocket is highly conserved for all investigated MetAPs, very stringent for methionine, and that several unnatural amino acids with methionine-like characteristics were also well hydrolyzed by MetAPs. The substrate-derived results were verified using several phosphonate and phosphinate-based inhibitors.

In-depth quantitative cardiac proteomics combining electron transfer dissociation and the metalloendopeptidase Lys-N with the SILAC mouse.

In quantitative proteomics stable isotope labeling has progressed from cultured cells toward the total incorporation of labeled atoms or amino acids into whole multicellular organisms. For instance, the recently introduced (13)C(6)-lysine labeled SILAC mouse allows accurate comparison of protein expression directly in tissue. In this model, only lysine, but not arginine, residues are isotope labeled, as the latter may cause complications to the quantification by in vivo conversion of arginine to proline. The sole labeling of lysines discourages the use of trypsin, as not all peptides will be quantifiable. Therefore, in the initial work Lys-C was used for digestion. Here, we demonstrate that the lysine-directed protease metalloendopeptidase Lys-N is an excellent alternative. As lysine directed peptides generally yield longer and higher charged peptides, alongside the more traditional collision induced dissociation we also implemented electron transfer dissociation in a quantitative stable isotope labeling with amino acid in cell culture workflow for the first time. The utility of these two complementary approaches is highlighted by investigating the differences in protein expression between the left and right ventricle of a mouse heart. Using Lys-N and electron transfer dissociation yielded coverage to a depth of 3749 proteins, which is similar as earlier investigations into the murine heart proteome. In addition, this strategy yields quantitative information on ∼ 2000 proteins with a median coverage of four peptides per protein in a single strong cation exchange-liquid chromatography-MS experiment, revealing that the left and right ventricle proteomes are very similar qualitatively as well as quantitatively.

Artificial metalloenzymes based on the biotin-avidin technology: enantioselective catalysis and beyond.

Artificial metalloenzymes are created by incorporating an organometallic catalyst within a host protein. The resulting hybrid can thus provide access to the best features of two distinct, and often complementary, systems: homogeneous and enzymatic catalysts. The coenzyme may be positioned with covalent, dative, or supramolecular anchoring strategies. Although initial reports date to the late 1970s, artificial metalloenzymes for enantioselective catalysis have gained significant momentum only in the past decade, with the aim of complementing homogeneous, enzymatic, heterogeneous, and organic catalysts. Inspired by a visionary report by Wilson and Whitesides in 1978, we have exploited the potential of biotin-avidin technology in creating artificial metalloenzymes. Owing to the remarkable affinity of biotin for either avidin or streptavidin, covalent linking of a biotin anchor to a catalyst precursor ensures that, upon stoichiometric addition of (strept)avidin, the metal moiety is quantitatively incorporated within the host protein. In this Account, we review our progress in preparing and optimizing these artificial metalloenzymes, beginning with catalytic hydrogenation as a model and expanding from there. These artificial metalloenzymes can be optimized by both chemical (variation of the biotin-spacer-ligand moiety) and genetic (mutation of avidin or streptavidin) means. Such chemogenetic optimization schemes were applied to various enantioselective transformations. The reactions implemented thus far include the following: (i) The rhodium-diphosphine catalyzed hydrogenation of N-protected dehydroaminoacids (ee up to 95%); (ii) the palladium-diphosphine catalyzed allylic alkylation of 1,3-diphenylallylacetate (ee up to 95%); (iii) the ruthenium pianostool-catalyzed transfer hydrogenation of prochiral ketones (ee up to 97% for aryl-alkyl ketones and ee up to 90% for dialkyl ketones); (iv) the vanadyl-catalyzed oxidation of prochiral sulfides (ee up to 93%). A number of noteworthy features are reminiscent of homogeneous catalysis, including straightforward access to both enantiomers of the product, the broad substrate scope, organic solvent tolerance, and an accessible range of reactions that are typical of homogeneous catalysts. Enzyme-like features include access to genetic optimization, an aqueous medium as the preferred solvent, Michaelis-Menten behavior, and single-substrate derivatization. The X-ray characterization of artificial metalloenzymes provides fascinating insight into possible enantioselection mechanisms involving a well-defined second coordination sphere environment. Thus, such artificial metalloenzymes combine attractive features of both homogeneous and enzymatic kingdoms. In the spirit of surface borrowing, that is, modulating ligand affinity by harnessing existing protein surfaces, this strategy can be extended to selectively binding streptavidin-incorporated biotinylated ruthenium pianostool complexes to telomeric DNA. This application paves the way for chemical biology applications of artificial metalloenzymes.

Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae.

Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2 kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and K(m), V(max) and k(cat) values of 1.86 mM, 0.281 µM/min and 27.9 s(-1), respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process.

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions.

The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of approximately 30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential (15)N/(14)N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.

News from an ancient world: two novel astacin metalloproteases from the horseshoe crab.

In this work, we report the cloning, heterologous expression, and characterization of two novel astacin proteases from the chelicerate Limulus polyphemus (horseshoe crab), designated as LAST (Limulus astacin) and LAST_MAM (Limulus astacin containing a MAM domain), respectively. The expression pattern showed ubiquitous occurrence of LAST_MAM, while LAST was predominantly restricted to the eyes and brain, indicating a function in the nervous system. Both enzymes contain the characteristic metzincin-type zinc-binding region and Met turn. While LAST is made up only of the typical prodomain and astacin-like protease domain, LAST_MAM contains an additional MAM (meprin A5 protein tyrosine phosphatase micro) domain, which so far only has been found in few astacins such as the vertebrate meprin Hydra and squid enzymes, and in a number of other extracellular proteins such as A5 protein and tyrosine phosphatase micro. These gave rise to the designation MAM for this protein module. MAM domains have been shown to be responsible for protein oligomerization in meprin proteases and tyrosine phosphatase micro. Since the horseshoe crab has kept its body plan for almost half a billion years, it is therefore a privileged organism for the study of protease evolution. In this context, we could show by phylogenetic analysis that this protease is not related to the other MAM-domain-containing astacins indicating different evolutionary origins of these proteins. Moreover, we clearly demonstrated the divergent evolvement of the MAM module itself, and not only with regard to proteases. However, there are some unique functional features that are not shared by other members of this protein family. For example, LAST_MAM is the only astacin protease known so far that is active in its zymogen form, indicating that the presence of the N-terminal propeptide does not prevent proteolytic activity.

A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius.

Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

AHL-lactonase (AiiA), a metallo-beta-lactamase produced by Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis, specifically hydrolyzes N-acyl-homoserine lactones (AHLs) secreted by Gram-negative bacteria and thereby attenuates the symptoms caused by plant pathogens. In this study, an aiiA gene was cloned from Bacillus subtilis BS-1 by PCR with a pair of degenerate primers. The deduced 250 amino acid sequence contained two small conserved regions, 103SHLHFDH109 and 166TPGHTPGH173, which are characteristic of the metallo-beta-lactamase family. Homology comparison revealed that the deduced amino acid sequence had a high degree of similarity with those of the known AiiA proteins in the B. cereus group. Additionally, the aiiA gene was expressed in Escherichia coli BL21 (DE3) pLysS and the expressed AiiA protein could attenuate the soft rot symptoms caused by Erwinia carotovora var. carotovora.

Activation of the SspA serine protease zymogen of Staphylococcus aureus proceeds through unique variations of a trypsinogen-like mechanism and is dependent on both autocatalytic and metalloprotease-specific processing.

The serine and cysteine proteases SspA and SspB of Staphylococcus aureus are secreted as inactive zymogens, zSspA and zSspB. Mature SspA is a trypsin-like glutamyl endopeptidase and is required to activate zSspB. Although a metalloprotease Aureolysin (Aur) is in turn thought to contribute to activation of zSspA, a specific role has not been demonstrated. We found that pre-zSspA is processed by signal peptidase at ANA(29) downward arrow, releasing a Leu(30) isoform that is first processed exclusively through autocatalytic intramolecular cleavage within a glutamine-rich propeptide segment, (40)QQTQSSKQQTPKIQ(53). The preferred site is Gln(43) with secondary processing at Gln(47) and Gln(53). This initial processing is necessary for optimal and subsequent Aur-dependent processing at Leu(58) and then Val(69) to release mature SspA. Although processing by Aur is rate-limiting in zSspA activation, the first active molecules of Val(69)SspA promote rapid intermolecular processing of remaining zSspA at Glu(65), producing an N-terminal (66)HANVILP isoform that is inactive until removal of the HAN tripeptide by Aur. Modeling indicated that His(66) of this penultimate isoform blocks the active site by hydrogen bonding to Ser(237) and occlusion of substrate. Binding of glutamate within the active site of zSspA is energetically unfavorable, but glutamine fits into the primary specificity pocket and is predicted to hydrogen bond to Thr(232) proximal to Ser(237), permitting autocatalytic cleavage of the glutamine-rich propeptide segment. These and other observations suggest that zSspA is activated through a trypsinogen-like mechanism where supplementary features of the propeptide must be sequentially processed in the correct order to allow efficient activation.

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.

Molecular cloning and characterization of Ac-MTP-2, an astacin-like metalloprotease released by adult Ancylostoma caninum.

Ac-MTP-2 is an astacin-like metalloprotease secreted by adult Ancylostoma caninum hookworms. Ac-mtp-2 cDNA was cloned by immunoscreening a cDNA library with antisera prepared against adult A. caninum excretory/secretory (ES) products. The full-length Ac-mtp-2 contains 850 bp cDNA encoding a 233 amino acid open reading frame (ORF) with 32% amino acid identity to Ce-NSP-4, a pharyngeal cell-derived secreted metalloprotease of the nematode Caenorhabditis elegans. The predicted ORF contained a conserved Met-turn sequence (SXMHY), but only a partial zinc-binding signature sequence (GXXXEHXRXER instead of HEXXHXXGXXHEXXRXDR) found in other astacins. However, by both gelatin gel electrophoresis and azocasein digestion, the recombinant Ac-MTP-2 exhibited proteolytic activity that was inhibited by the zinc chelator 1,10-phenanthroline and Ac-TMP, a putative tissue inhibitor of metalloprotease that was previously shown to be a highly abundant component of adult A. caninum ES products. By RT-PCR, Western blot Ac-MTP-2 was found only expressed in adult hookworms and secreted in the adult ES products. Immunolocalization with antisera shows that Ac-MTP-2 is located to the esophageal glands (confirming its role as a secretory protein), as well as to the parasite uterus. It is hypothesized that Ac-MTP-2 functions in the extracorporeal digestion of the intestinal mucosal plug lodged in the buccal capsule of the adult parasite.

Molecular characterization and phylogenetic analysis of BjussuMP-I: a RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom.

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation, including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I, a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-I metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases.