Production and characterization of collagenase from a new Amazonian Bacillus cereus strain.

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2-11.0 and 25-50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.

Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

Pro-domain of a cysteine cathepsin contains a highly conserved Ex2Rx2Fx2Nx3Ix3N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

Immunomodulation of Zerumbone via Decreasing the Production of Reactive Oxygen Species from Immune Cells.

BACKGROUND AND OBJECTIVE: Zerumbone has been reported to exert anti-inflammatory, anti-ulcer and anti-hyperglycemic effects but the specific mechanism through which zerumbone exerts its anti-inflammatory action through inhibiting reactive oxygen species was not well studied. Hence, this paper studied the zerumbone capacity to inhibit intracellular and extracellular Reactive Oxygen Species (ROS) produced by whole blood cell, polymorphoneutrophil (PMNs) and macrophage cells due to the zymogen and phorbolmyristerate acetate (PMA) oxidant effect. MATERIALS AND METHODS: Zymogen and PMA based chemiluminescence assay were used to determine the immunomodulatory effect of zerumbone at concentrations (100, 10 and 1 µg mL-1) toward production of Reactive Oxygen Species (ROS) from whole blood, PMNs and macrophage. RESULTS: Zerumbone significantly inhibited intracellular and extracellular ROS production by the zymosan/PMA-activated phagocyte cells with IC50 values of (16.3±0.1, 23.7±0.1 and 4.97±0.1 µg mL-1) against whole blood, PMNs and macrophage respectively. CONCLUSION: The anti-inflammatory activity of zerumbone was so much significant that even strong oxidant (zymogen and PMA) were not able to produce reactive oxygen species when incubated together in phagocytic cells, thus suppress production of ROS. Therefore, it is highly used in herbal medicine as a potent immunomodulatory therapy in various inflammation associated diseases.

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element.

Inhibition of pro-/active MMP-2 by green tea catechins and prediction of their interaction by molecular docking studies.

Matrix metalloproteinases (MMPs) play a crucial role in developing different types of lung diseases, e.g., pulmonary arterial hypertension (PAH). Green tea polyphenolic catechins such as EGCG and ECG have been shown to ameliorate various types of diseases including PAH. Our present study revealed that among the four green tea catechins (EGCG, ECG, EC, and EGC), EGCG and ECG inhibit pro-/active MMP-2 activities in pulmonary artery smooth muscle cell (PASMC) culture supernatant. Based on the above, we investigated the interactions of pro-/active MMP-2 with the green tea catechins by computational methods. In silico analysis revealed a strong interaction of pro-/active MMP-2 with EGCG/ECG, and galloyl group has been observed to be responsible for this interaction. The in silico analysis corroborated our experimental observation that EGCG and ECG are active in preventing both the proMMP-2 and MMP-2 activities. Importantly, these two catechins appeared to be better inhibitors for proMMP-2 in comparison to MMP-2 as revealed by gelatin zymogram and also by molecular docking studies. In many type of cells, activation of proMMP-2 occurs via an increase in the level of MT1-MMP (MMP-14). We, therefore, determined the interactions of MT1-MMP with the green tea catechins by molecular docking analysis. The study revealed a strong interaction of MT1-MMP with EGCG/ECG, and galloyl group has been observed to be responsible for the interaction.

Proteome-wide covalent ligand discovery in native biological systems.

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen.

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

Expression of surface-associated 82kDa-proMMP-9 in primary acute leukemia blast cells inversely correlates with patients' risk.

With its ability to degrade extracellular matrix proteins and activate growth factors and cytokines, matrix metalloproteinase (MMP)-9 is an important regulator of cell function. Previously, we reported that myeloid leukemic cells express a unique 82kDa-proMMP-9 variant on their cell surface that is not affected by its natural inhibitor. In this study, we generated monoclonal antibodies that specifically recognize 82kDa-proMMP-9. Flow cytometry analysis using these antibodies revealed significant surface expression of 82kDa-proMMP-9 in monocytes, but minimal amounts in T and B cells isolated from peripheral blood of nine healthy donors and 22 patients with acute myeloid leukemia (AML). In all AML patients, blasts expressed 82kDa-proMMP-9 at levels of 4%-46%, with significantly higher levels in patients with a better risk defined according to National Comprehensive Cancer Network (NCCN) guidelines (ρ = -0.748, p < 0.001) and favorable phenotype according to the French-American-British classification (p = 0.02) compared with patients with adverse prognoses. Receiver operating characteristic curve analysis confirmed the diagnostic accuracy of 82kDa-proMMP-9 measurement in AML blasts (area under the curve: 0.893 [0.739-1.000], p = 0.019). It led us to define a cutoff value of 11.5% for identifying patients with lower NCCN risk (p = 0.005) and with a tendency toward a higher probability of response to anthracycline-based therapy (p = 0.109) and increased event-free survival (p = 0.24). Thus, 82kDa-proMMP-9 expression on blasts may represent a novel independent marker of prognosis in patients with AML.

Characterization of the effects of C-terminal pro-sequence on self-inactivation of Stereum purpureum endopolygalacturonase I.

Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence. Of note, the optimal pH for pro-endoPG I activity shifted to pH 4.0 from pH 4.5-5.0 of endoPG I. The kinetic parameters indicated that the activity inhibition resulted from a pH-independent decrease of substrate affinity and pH-dependent deterioration of velocity by the pro-sequence. Analysis of site-directed mutations within pro-endoPG I showed that its α-helical structure includes two glutamates (E364 and E366) and alanine (A365), and its orientation by prolines (especially P348) in the pro-sequence played a significant role in its suppression activity. As for mutations in the mature domain, a marked reduction of suppression was observed for enzymes with mutations in H150, R220 and K253, indicating that the pro-sequence interacts with the active cleft by a few ionic bonds.

Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare trypsin can be favored as a dietary supplement in animals and humans.

Reverse zymography alone does not confirm presence of a protease inhibitor.

Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.

A novel effect of imidazole derivative KK-42 on increasing survival of Aeromonas hydrophila challenged prawn Macrobrachium nipponense.

Imidazole derivative KK-42 is well known as the insect growth regulator. Here we find that KK-42 pretreatment could promote the survival of Macrobrachium nipponense infected with Aeromonas hydrophila, which is considered to be possibly related to the prophenoloxidase (proPO), a conserved copper-containing enzyme that plays an important role in defense against pathogens. In this study, a full-length of proPO gene from M. nipponense haemocytes, designated as MnproPO, was firstly cloned and characterized. The full-length cDNA contained 2428 bp with a 2013 bp open reading frame encoding a putative proPO protein of 671 amino acids with a predicted molecular mass of 76.5 kDa and pI of 7.31. It was predicted to possess all the expected features of proPO members, including two putative copper-binding sites with six histidine residues and a thiol ester-like motif. Sequence analysis showed that MnproPO exhibited the highest amino acid sequence similarity (93%) to a proPO of Macrobrachium rosenbergii. The gene was expressed highly in haemocytes and weakly in hepatopancreas. Real-time PCR analysis revealed that the MnproPO expression increased significantly at 3, 12 and 24 h after KK-42 treatment, the PO activity also importantly rose from 6 to 48 h in KK-42-treated prawns and reached the maximum at 24 h with a 2.3-fold higher than that in control group. Injection of A. hydrophila could stimulate the MnproPO transcription and PO activity whether or not the prawns were pretreated by KK-42, the mRNA level increased obviously only at 3 h and 6 h after the bacterium injection (challenged control), but increased constantly during the phase of experiment except at 6 h under the condition of KK-42 pretreatment (challenged treatment group). The change trend of PO activity was basically similar to that of MnproPO expression. Our present results demonstrate that the MnproPO expression as well as PO activity may be induced by KK-42, which is likely one of the molecular mechanisms of KK-42 acts for increasing survival of the prawn infected with A. hydrophila.

Molecular cloning of prophenoloxidase and the effects of dietary ß-glucan and rutin on immune response in hemocytes of the fleshy shrimp, Fenneropenaeus chinensis.

To investigate the effects of dietary ß-glucan (0.5 or 1 g kg⁻¹ diet: 0.5-BG, 1-BG) and rutin (0.5 or 1 g kg⁻¹ diet: 0.5-RT, 1-RT) after 10 days in the absence of pathogen challenge on the immune response of Fenneropenaeus chinensis, we determined total hemocyte count (THC) and the expression of four immune-related genes in hemocytes: those for prophenoloxidase (proPO), peroxinectin (PX), lipopolysaccharide and/or ß-glucan binding protein (LGBP), and c-type lectin (CL). As a prerequisite for subsequent experiments, cDNA encoding proPO of the fleshy shrimp, Fenneropenaeus chinensis (f-proPO) was obtained from hemocytes; it had a full length of 3023 bp, with an open reading frame (ORF) of 2061bp, a 105-bp 5'-untranslated region, and a 906-bp 3'-untranslated region containing the poly A signal. The THCs of shrimp fed ß-glucan of 1 g kg⁻¹ diet, and rutin of 1 g kg⁻¹ diet were significantly higher than that of the control (P < 0.05). The expression of proPO mRNA was slightly downregulated and that of LGBP mRNA was upregulated (except in 1-RT). PX and CL mRNA remained constitutively expressed in all groups. Our results reveal that ß-glucan and rutin dietary supplements have minimal effect on immune response in the absence of pathogen challenge.

Modulation of in vitro activity of zymogenic and mature recombinant human ß-secretase by dietary plants.

The in vitro activity of human recombinant ß-secretase (BACE1) was studied using a fluorogenic substrate based on the cleavage site for the enzyme in the Swedish mutation of amyloid precursor protein. The enzyme was inhibited by a control peptide inhibitor with good repeatability. The enzyme preparation comprised a mixture of pro-enzyme or zymogen and mature enzyme whereby the pro-enzyme sequence forms a 'flap' that can obstruct the binding site. 'Open flap' forms of the zymogen and mature enzyme are active, but the 'closed flap' form of the zymogen is inactive. This mixture of enzyme populations permitted apparent stimulation of enzyme activity under particular conditions, presumably due to facilitating flap-opening of the zymogen. As reported for heparin, enzyme activation was stimulated in the presence of low concentrations of Tween 20 and dimethylsulfoxide before becoming inhibited at higher concentrations. Dietary plant extracts either consistently inhibited (e.g. clove, tea, cinammon) or consistently stimulated (e.g. mushroom, parsley, asparagus) BACE1. Common structural features identified by Fourier transform infrared spectroscopy revealed that BACE1 activity could be explained by differential interactions of either small molecule or polymeric species with mature versus zymogen forms of the enzyme, respectively. Further, enzyme activity could be reversed by mixtures of high and low mass species. These results may have implications for the regulation of ß-secretase activity in vivo by either endogenous or possibly dietary factors and for a potential role of BACE1 in stimulation of the production of amyloid beta peptide in sporadic Alzheimer's disease.

Identification of nitroglycerin-induced cysteine modifications of pro-matrix metalloproteinase-9.

Nitroglycerin (NTG), an important cardiovascular agent, has been shown recently to activate matrix metalloproteinase-9 (MMP-9) in biological systems, possibly leading to destabilization of atherosclerotic plaques. The chemical mechanism for this activation, particularly on the cysteine switch of the pro-form of MMP-9 (proMMP-9), has not been investigated and was examined here using nano-flow liquid chromatography coupled to mass spectrometry. In order to obtain high sequence coverage, two orthogonal enzymes (trypsin and GluC) were employed to digest the protein in parallel. Two complementary activation methods, collision-induced dissociation (CID) and electron-transfer dissociation (ETD), were employed for the identification of various modifications. A high-resolution Orbitrap analyzer was used to enable confident identification. Incubation of NTG with proMMP-9 resulted in the formation of an unstable thionitrate intermediate and oxidation of the cysteine switch to sulfinic and irreversible sulfonic acid derivatives. The unstable thionitrate modification was confirmed by both CID and ETD in the proteolytic peptides produced by both trypsin and GluC. Incubation of proMMP-9 with diethylenetriamine NONOate (a nitric oxide donor) led to sulfonic acid formation, but no observable sulfinic acid modification. Extensive tyrosine nitration by NTG was observed at Tyr-262, in close proximity to an oxidized Cys-256 of proMMP-9. The intramolecular interaction between these two residues toward NTG-induced oxidation was examined using a synthesized peptide representing the sequence in this domain, PWCSTTANYDTDDR, and the modification status was compared against an analog in which Cys was substituted by Ala. We observed a thionitrate product, extensive Cys oxidative modifications and enhanced tyrosine nitration with the Cys peptide but not with the Ala analog. Our results indicated that neighboring Cys and Tyr residues can facilitate each other's oxidation in the presence of NTG.

Intein lacking conserved C-terminal motif G retains controllable N-cleavage activity.

A split-intein consists of two complementary fragments (N-intein and C-intein) that can associate to carry out protein trans-splicing. The Ssp GyrB S11 split-intein is an engineered unconventional split-intein consisting of a 150-amino-acid N-intein and an extremely small six-amino-acid C-intein, which comprises the conserved intein motif G. Here, we show that fusion proteins containing the 150-amino-acid N-intein could be triggered to undergo controllable N-cleavage in vitro when the six-amino-acid C-intein or a derivative thereof was added as a synthetic peptide in trans. More importantly, we discovered, unexpectedly, that the 150-amino-acid N-intein could be induced by strong nucleophiles to undergo N-cleavage in vitro, and in Escherichia coli cells, in the absence of the motif G-containing six-amino-acid C-intein. This finding indicated that the first step of the protein splicing mechanism (acyl shift) could occur in the absence of the entire motif G. Extensive kinetic analyses revealed that both the motif G residues and the Ser+1 residue positively influenced N-cleavage rate constants and yields. The 150-amino-acid N-intein could also tolerate various unrelated sequences appended to its C-terminus without disruption of the N-cleavage function, suggesting that the catalytic pocket of the intein has considerable structural flexibility. Our findings reveal interesting insights into intein structure-function relationships, and demonstrate a new and potentially more useful method of controllable, intein-mediated N-cleavage for protein engineering applications.

Prophenoloxidase from Pieris rapae: gene cloning, activity, and transcription in response to venom/calyx fluid from the endoparasitoid wasp Cotesia glomerata.

Prophenoloxidase (PPO) plays an important role in melanization, necessary for defense against intruding parasitoids. Parasitoids have evolved to inject maternal virulence factors into the host hemocoel to suppress hemolymph melanization for the successful development of their progeny. In this study, the full-length complementary DNA (cDNA) of a Pieris rapae PPO was cloned. Its cDNA contained a 2 076-base pair (bp) open reading frame (ORF) encoding 691 amino acids (aa). Two putative copper-binding sites, a proteolytic activation site, three conserved hemocyanin domains, and a thiol ester motif were found in the deduced amino acid sequence. According to both multiple alignment and phylogenetic analysis, P. rapae PPO gene cloned here is a member of the lepidopteran PPO-2 family. Injection of Cotesia glomerata venom or calyx fluid resulted in reduction of P. rapae hemolymph phenoloxidase activity, demonstrating the ability to inhibit the host's melanization. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of P. rapae PPO-2 in the haemocytes from larvae had not significantly changed following venom injection, suggesting that the regulation of PPO messenger RNA (mRNA) expression by venom was not employed by C. glomerata to cause failure of melanization in parasitized host. While decreased P. rapae PPO-2 gene expression was observed in the haemocytes after calyx fluid injection, no detectable transcriptional change was induced by parasitization, indicating that transcriptional down-regulation of PPO by calyx fluid might play a minor role involved in inhibiting the host's melanization.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

A synthetic peptidoglycan fragment as a competitive inhibitor of the melanization cascade.

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.