Midgut cysteine protease-inhibiting activity in Trichoplusia ni protects the peritrophic membrane from degradation by plant cysteine proteases

The action of plant cysteine proteases on the midgut peritrophic membrane (PM) of a polyphagous herbivorous lepidopteran, Trichoplusia ni, was studied. Proteins in PMs isolated from T. ni larvae were confirmed to be highly resistant to the serine proteinases trypsin and chymotrypsin, but were susceptible to degradation by plant cysteine proteases, which is consistent with the known molecular and biochemical characteristics of the T. ni PM proteins. However, the PM proteins were not degraded by plant cysteine proteases in larvae or in the presence of larval midgut fluid in vitro. With further biochemical analysis, cysteine protease-inhibiting activity was identified in the midgut fluid of T. ni larvae. The cysteine protease-inhibiting activity was heat resistant and active in the tested pH range from 6.0 to 10.0, but could be suppressed by thiol reducing reagents or reduced by treatment with catalase. In addition to T. ni, cysteine protease-inhibiting activity was also identified from two other polyphagous Lepidoptera species, Helicoverpa zea and Heliothis virescens. In conclusion, results from this study uncovered that herbivorous insects may counteract the attack of plant cysteine proteases on the PM by inhibiting the potentially insecticidal cysteine proteases from plants in the digestive tract. However, the biochemical identity of the cysteine protease-inhibiting activity in midgut fluid has yet to be identified.     

Isolation,Purification, and Properties of Cysteine Proteinase from <Emphasis Type="Italic">Bombyx mori</Emphasis> L. Eggs

Silkworm moth (bombyx) egg cysteine proteinase with maximal activity at pH 3.0 was purified by chromatography and isoelectrofocusing. On SDS-electrophoresis in polyacrylamide gel the purified enzyme showed a single band of molecular mass 50 kD. Isoelectrofocusing revealed that the bombyx egg cysteine proteinase exists in two forms with pI values of 5.95 and 6.43, respectively. The enzyme activity was sensitive to inhibition by iodoacetamide and p-chloromercuribenzoate but resistant to EDTA, pepstatin, and phenylmethylsulfonyl fluoride. The cysteine proteinase hydrolyzes storage proteins of bombyx eggs but it was inactive with respect to N-benzoyl-D,L-arginine-p-nitroanilide (BAPNA). It is a cathepsin L-like enzyme.     

Purification of cadmium-binding proteins from related species of terrestrial helicidae (gastropoda,mollusca): A comparative study

Summary Three species of terrestrial Helicidae (Helix pomatia, Cepaea hortensis andArianta arbustorum) were fed cadmium-rich diet in the laboratory. The snails accumulated high amounts of the metal in their hepatopancreas. Most cadmium and some zinc were found, after centrifugation, in the soluble fractions from which a cadmium-binding protein was isolated for each species by ion exchange and gel chromatography. The proteins contained different amounts of cadmium, but little or no zinc, and showed high absorption at 254 nm indicating the presence of cadmium-mercaptide bonds. After gel filtration, a molecular weight of 12000 was found for cadmium-binding proteins fromHelix pomatia andArianta arbustorum, whereas a molecular weight of 10 000 was found for a cadmium-binding protein fromCepaea hortensis. SDS-polyacrylamide gel electrophoresis showed one single band for each protein fromHelix pomatia andArianta arbustorum and suggested a molecular weight of 11000 for both species. Amino acid analysis revealed, for each protein, high amounts of cysteine (12–20%), glycine (15–19%), and serine (12–14%), and moderately elevated contents of lysine (9–13%) and alanine (4–8%), but no methionine and only traces, if any, of aromatic amino acids. The ratios of cadmium to cysteine were 1:5, 1:10 and 1:3 in the proteins fromHelix pomatia,Cepaea hortensis andArianta arbustorum, respectively. Some features of the isolated proteins resembled mammalian metallothioneins. Most characteristics, however, differed from true metallothioneins and were similar to cadmium-binding proteins found in some marine molluscs.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

A SELDI‐TOF MS procedure for the detection,quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.     

Characterization of Glycoprotein H and L of Human Herpesvirus 7

The genes encoding the glycoproteins H (gH) and L (gL) of human herpesvirus 7 (HHV-7) have been identified. The gH open reading frame (ORF) was 2,070 base pairs in length and encoded a predicted 690 amino-acid protein. The gH contained characteristics of a transmembrane glycoprotein including 10 consensus N-linked glycosylation sites, 12 cysteine residues, a potential amino-terminal signal sequence and a predicted transmembrane segment located near the carboxyl terminus. The gL ORF was 738 base pairs in length and encoded a predicted 246 amino-acid protein. Four possible N-glycosylation sites and 6 cysteine residues existed within gL. The predicted amino-acid sequences of the HHV-7 gH and human herpesvirus 6 variant A (HHV-6A) gH gene products exhibited 23.6% identity to each other, and those of the gL gene products had 26.0% identity. Upon in vitro translation of the gL gene, the addition of microsomal membranes resulted in two modified products with molecular weights of 32 kDa and 35 kDa from the unmodified initial translation product of 26 kDa. An amino-terminal portion of gH and the full length of gL were expressed as glutathione S-transferase fusion proteins, and these proteins were used to raise immune sera in mice. Lysates of cells infected with HHV-7 were subjected to immunoprecipitation analysis. Approximate molecular weights of 33, 37, 80 and 90 kDa polypeptides were immunoprecipitated with antibodies against the gH protein. Antibodies against the gL protein polypeptides with the same molecular weights were also precipitated, and were observed with the antibodies against the gH protein. These results suggest that HHV-7 gH and gL may form a heterodimeric complex with each other in HHV-7 infected cells, as has been reported for other herpesviruses.     

Molecular characterization of a gene encoding a cysteine-rich protein preferentially expressed in anthers of Lycopersicon esculentum

The tomato (Lycopersicon esculentum Mill.) cDNA clone TomA5B was isolated by differential screening of a cDNA library prepared from anthers at late meiosis to tetrad formation. The 5B gene is present in a single copy in the tomato genome. Expression is developmentally regulated and tissue specific. RNA accumulation was detected from premeiosis through tetrad release in the tapetal cell layer of the anther with low levels of RNA detected in petals and early stages of pistil development. The protein deduced from the DNA sequence analysis is predicted to have a molecular mass of 11.1 kDa and a secretory signal sequence, suggesting it is a secreted protein. The deduced 5B protein has a pattern of cysteine residues that is similar to other proteins that have stamen-specific expression and to a superfamily of seed proteins. The 5B protein is unique in that there is no amino acid sequence similarity to other proteins beyond the similar cysteine motif.     

Vacuole Cysteine Proteases and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Degradation during Monocarpic Senescence in Cowpea Leaves

Characterisation of proteases degrading ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO, EC: 4.1.1.39) was studied in the cowpea leaf during monocarpic senescence 3 and 9 d after flowering (DAF), representing early and mid pod fill. The stage at 3 DAF coincided with decrease in the metabolic parameters characterising senescence, i.e., contents of total soluble proteins, RuBPCO, and leaf nitrogen. At 9 DAF, there was a decline in total soluble proteins and an appearance of a 48 kDa cysteine protease. Characterisation of the proteases was done using specific inhibitors. Subcellular localisation at 3 DAF was studied by following the degradation of RuBPCO large subunit (LSU) in the vacuole lysates using immunoblot analyses. Cysteine proteases played a predominant role in the degradation of RuBPCO LSU at the crude extract level. At 9 DAF, expression of cysteine protease isoforms was monitored using polyclonal antibodies against papain and two polypeptides of molecular masses 48 and 35 kDa were observed in the vacuole lysates. We confirmed thus the predominance of cysteine proteases in the vacuoles during different stages of pod development in cowpea leaf.     

Structural and functional conservation profiles of novel cathepsin L-like proteins identified in the Drosophila melanogaster genome

Cathepsin L is a cysteine protease which degrades connective tissue proteins including collagen, elastin, and fibronectin. In this study, five well-characterized cathepsin L proteins from different arthropods were used as query sequences for the Drosophila genome database. The search yielded 10 cathepsin L-like sequences, of which eight putatively represent novel cathepsin L-like proteins. To understand the phylogenetic relationship among these cathepsin L-like proteins, a phylogenetic tree was constructed based on their sequences. In addition, models of the tertiary structures of cathepsin L were constructed using homology modeling methods and subjected to molecular dynamics simulations to obtain reasonable structure to understand its dynamical behavior. Our findings demonstrate that all of the potential Drosophila cathepsin L-like proteins contain at least one cathepsin propeptide inhibitor domain. Multiple sequence alignment and homology models clearly highlight the conservation of active site residues, disulfide bonds, and amino acid residues critical for inhibitor binding. Furthermore, comparative modeling indicates that the sequence/structure/function profiles and active site architectures are conserved.     

The disulfide oxidoreductase SdbA is active in Streptococcus gordonii using a single C‐terminal cysteine of the CXXC motif

Recently, we identified a novel disulfide oxidoreductase, SdbA, in the oral bacterium Streptococcus gordonii. Disulfide oxidoreductases form disulfide bonds in nascent proteins using a CXXC catalytic motif. Typically, the N‐terminal cysteine interacts with substrates, whereas the C‐terminal cysteine is buried and only reacts with the first cysteine of the motif. In this study, we investigated the SdbA C⁸⁶P⁸⁷D⁸⁸C⁸⁹ catalytic motif. In vitro, SdbA single cysteine variants at the N or C‐terminal position (SdbA_C86P and SdbA_C89A) were active but displayed different susceptibility to oxidation, and N‐terminal cysteine was prone to sulfenylation. In S. gordonii, mutants with a single N‐terminal cysteine were inactive and formed unstable disulfide adducts with other proteins. Activity was partially restored by inactivation of pyruvate oxidase, a hydrogen peroxide generator. Presence of the C‐terminal cysteine alone (in the SdbA_C86P variant) could complement the ΔsdbA mutant and restore disulfide bond formation in recombinant and natural protein substrates. These results provide evidence that certain disulfide oxidoreductases can catalyze disulfide bond formation using a single cysteine of the CXXC motif, including the buried C‐terminal cysteine.     

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox‐sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard‐cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in‐gel electrophoresis and isotope‐coded affinity tagging. In total, 65 and 118 potential redox‐responsive proteins were identified in ABA‐ and MeJA‐treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra‐molecular disulfide bonds. Most of the proteins fall into the functional groups of ‘energy’, ‘stress and defense’ and ‘metabolism’. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA‐ and MeJA‐treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non‐fermenting 1‐related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox‐regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.     

Genetic Design of Stable Metal-Binding Biomolecules,Oligomeric Metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT—the thiol group of cysteine is easily oxidized and proteolysis occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies, which were dissolved with dithiothreitol, and the purified apo-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities that were, respectively, two and four times higher than those of the hMT-II monomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.     

The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH‐redoxin family

NrdH‐redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH‐redoxins have a CVQC active site motif and belong to the thioredoxin‐fold protein family. As for other thioredoxin‐fold proteins, the pK_a of the nucleophilic cysteine of NrdH‐redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK_a value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH‐redoxins were determined, but structural insights explaining the relatively low pK_a remained elusive. We subjected C. glutamicum NrdH‐redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK_a of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N‐terminal of the active site further lowers the cysteine pK_a. However, site‐directed mutagenesis data show that the major contribution to the lowering of the cysteine pK_a comes from the positive charge of the lysine and not from the additional Lys‐Cys hydrogen bond. In 12% of the NrdH‐redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK_a. All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N‐terminally of the active site dynamically regulate the pK_a of the nucleophilic cysteine in NrdH‐redoxins.     

Protonation states of active‐site lysines of penicillin‐binding protein 6 from Escherichia coli and the mechanistic implications

The protonation states of the two active‐site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S‐aminoethylated cysteine (γ‐thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd ‐carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free‐base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pK_a values were compared with the pK_a values calculated computationally, using molecular‐dynamics simulations and a thermodynamic cycle. Study of the γ‐thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half‐reactions. Proteins 2014; 82:1348–1358. © 2013 Wiley Periodicals, Inc.     

Extracellular matrix‐associated proteome changes during non‐host resistance in citrus–Xanthomonas interactions

Non‐host resistance (NHR) is a most durable broad‐spectrum resistance employed by the plants to restrict majority of pathogens. Plant extracellular matrix (ECM) is a critical defense barrier. Understanding ECM responses during interaction with non‐host pathogen will provide insights into molecular events of NHR. In this study, the ECM‐associated proteome was compared during interaction of citrus with pathogen Xanthomonas axonopodis pv. citri (Xac) and non‐host pathogen Xanthomonas oryzae pv. oryzae (Xoo) at 8, 16, 24 and 48 h post inoculation. Comprehensive analysis of ECM‐associated proteins was performed by extracting wall‐bound and soluble ECM components using both destructive and non‐destructive procedures. A total of 53 proteins was differentially expressed in citrus–Xanthomonas host and non‐host interaction, out of which 44 were identified by mass spectrometry. The differentially expressed proteins were related to (1) defense‐response (5 pathogenesis‐related proteins, 3 miraculin‐like proteins (MIR, MIR1 and MIR2) and 2 proteases); (2) enzymes of reactive oxygen species (ROS) metabolism [Cu/Zn superoxide dismutase (SOD), Fe‐SOD, ascorbate peroxidase and 2‐cysteine‐peroxiredoxin]; (3) signaling (lectin, curculin‐like lectin and concanavalin A‐like lectin kinase); and (4) cell‐wall modification (α‐xylosidase, glucan 1, 3 β‐glucosidase, xyloglucan endotransglucosylase/hydrolase). The decrease in ascorbate peroxidase and cysteine‐peroxiredoxin could be involved in maintenance of ROS levels. Increase in defense, cell‐wall remodeling and signaling proteins in citrus–Xoo interaction suggests an active involvement of ECM in execution of NHR. Partially compromised NHR in citrus against Xoo, upon Brefeldin A pre‐treatment supported the role of non‐classical secretory proteins in this phenomenon.     

Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.     

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

Summary TheRhizobium meliloti fdxN gene, which is part of thenifA-nifB fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix^– phenotype of anR. meliloti fdxN: :[Tc] mutant could be rescued by theR. leguminosarum fdxN gene, whereas no complementation was observed withnif-associated genes encoding ferredoxins fromBradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum andRhodobacter capsulatus. In addition to these heterologous genes, severalR. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of theR. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X₂-C-X₂-C-X₃-C,nif-associated ferredoxins, includingR. meliloti FdxN, are characterized by two different cysteine motifs. Six additional amino acids separate the second (Cys₄₂) and the third cysteine (Cys₅₁) in the C-terminal motif (C-X₂-C-X₈-C-X₃-C). By molecular modelling, it was predicted that these amino acid residues form a loop, which does not alter the relative positions of the neighbouring cysteines. Deletion of this loop resulted in anR. meliloti FdxN mutant protein, which exhibited almost 70% wild-type activity, indicating that the predicted loop does not affect Fe-S cluster binding and plays no crucial role in activity of the FdxN protein.