Legumains - a family of asparagine-specific cysteine endopeptidases involved in propolypeptide processing and protein breakdown in plants

Legumains are a recently discovered family of plant and animal cysteine endopeptidases with a cleavage specificity for Asn in the P1 position of peptide bonds. Asp-flanked peptide bonds also are cleaved, but with a much lower efficiency. Legumains evolved from GPI transamidase-like progenitors. Sequence analysis revealed three major groups of plant legumains corresponding to differences in the developmental and organ-specific gene expression. With the exception of a single cell wall specific representative, all legumains occur in the vacuolar compartment. Legumains are either involved in protein degradation or play a role in the processing of precursor proteins by Asn/Asp-specific limited proteolysis. Which function legumains perform depends on the conformational state of the substrate protein. A legumain acts as a vacuolar processing enzyme when it only has access to the regular processing sites of a precursor polypeptide, but it acts as a degradative enzyme when an altered conformation opens the substrate for unlimited proteolysis. The specificity of these interactions seems to be the result of a co-evolution of enzyme and substrate. The double function of legumains is particularly evident in the events of deposition and mobilisation of storage globulins during seed maturation and germination/seedling growth and in senescing and dying cells.     

New aspects of the molecular evolution of legumains, Asn-specific cysteine proteinases

The molecular evolution of asparagine-specific cysteine proteinases, called legumains, from plants and animals was analyzed using newly available related amino acid sequences from lower eukaryotes, bacteria and Archaea. The results suggest that genuine legumains originate from prokaryote pro-legumains. The evolutionary roots of genuine legumains from plants and animals descend from Parabasalia and Alveolata before developing into their respective separate branches headed by Chlorophyta and Placozoa. The branch of legumain-like plant/animal glycosylphosphatidyl inositol transamidases separated from the general evolutionary stem of legumains at the level of lower eukaryotes. Modeling of the 3D structure of a plant genuine legumain underlined the previously suggested similarity of the active site geometry of legumains with caspases, which are Asp-specific bacterial and eukaryote proteinases.     

Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases

The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.     

Participation of proteolytic enzymes in the interaction of plants with phytopathogenic microorganisms

Different forms of participation of proteolytic enzymes in pathogenesis and plant defense are reviewed. Together with extracellular proteinases, phytopathogenic microorganisms produce specific effectors with proteolytic activity and are able to act on proteins inside the plant cell. In turn, plants use both extracellular and intracellular proteinases for defense against phytopathogenic microorganisms. Among the latter, a special role belongs to vacuolar processing enzymes (legumains), which perform the function of caspases in the plant cell.     

Molecular cloning and functional expression of cDNA encoding the cysteine proteinase inhibitor with three cystatin domains from sunflower seeds

Two cysteine proteinase inhibitors, cystatins Sca and Scb, were previously isolated from sunflower seeds [Kouzuma et al. J. Biochem. 119 (1996) 1106-1113]. A cDNA clone encoding a novel phytocystatin with three repetitive cystatin domains was isolated from a cDNA library of sunflower seeds using the Sca cDNA fragment as a hybridization probe. The cDNA insert comprises 1,093 bp and encodes 282 amino acid residues. The deduced amino acid sequences of the domains are highly similar to each other (66-81%), sharing 65-90% identical residues with Sca. The cDNA was expressed in Escherichia coli cells, and then the recombinant sunflower multicystatin (SMC) was purified and its inhibitory activity toward papain was examined. SMC exhibited strong inhibitory activity toward papain, with a stoichiometry of 1:3, indicating that each cystatin domain independently functions as a potent cysteine proteinase inhibitor. Proteolysis of SMC with Asn-specific proteinase suggested that post-translational processing by an Asn-specific proteinase may give rise to mature Sca-like phytocystatins.     

Digestive proteases in bodies and faeces of the two-spotted spider mite,Tetranychus urticae

Digestive proteases of the phytophagous mite Tetranychus urticae have been characterised by comparing their activity in body and faecal extracts. Aspartyl, cathepsin B- and L-like and legumain activities were detected in both mite bodies and faeces, with a specific activity of aspartyl and cathepsin L-like proteases about 5- and 2-fold higher, respectively, in mite faeces than in bodies. In general, all these activities were maintained independently of the host plant where the mites were reared (bean, tomato or maize). Remarkably, this is the first report in a phytophagous mite of legumain-like activity, which was characterised for its ability to hydrolyse the specific substrate Z-VAN-AMC, its activation by DTT and inhibition by IAA but not by E-64. Gel free nanoLC–nanoESI-QTOF MS/MS proteomic analysis of mite faeces resulted in the identification of four cathepsins L and one aspartyl protease (from a total of the 29 cathepsins L, 27 cathepsins B, 19 legumains and two aspartyl protease genes identified the genome of this species). Gene expression analysis reveals that four cathepsins L and the aspartyl protease identified in the mite faeces, but also two cathepsins B and two legumains that were not detected in the faeces, were expressed at high levels in the spider mite feeding stages (larvae, nymphs and adults) relative to embryos. Taken together, these results indicate a digestive role for cysteine and aspartyl proteases in T. urticae. The expression of the cathepsins B and L, legumains and aspartyl protease genes analysed in our study increased in female adults after feeding on Arabidopsis plants over-expressing the HvCPI-6 cystatin, that specifically targets cathepsins B and L, or the CMe trypsin inhibitor that targets serine proteases. This unspecific response suggests that in addition to compensation for inhibitor-targeted enzymes, the increase in the expression of digestive proteases in T. urticae may act as a first barrier against ingested plant defensive proteins.     

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.     

Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases

Plant legumains are cysteine proteinases putatively involved in processing endogenous proteins. Phytocystatins (PhyCys) have been described as plant inhibitors of papain-like cysteine proteinases. Some PhyCys contain a carboxy terminal extension with an amino acid motif (SNSL) similar to that involved in the inhibition of legumain-like proteins by human cystatins. The role of these carboxy terminal extended PhyCys as inhibitors of legumain-like cysteine proteinases is here shown by in vitro inhibition of human legumain and legumain-like activities from barley extracts. Moreover, site-directed mutagenesis has demonstrated that the asparagine of the SNSL motif is essential in this inhibition. We prove for first time the existence of legumain inhibitors in plants.     

Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation,biotinylated protein

A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized. DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa. GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes. The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds. It degrades rapidly at the early stage of seed germination. These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.     

Identification and characterization of dehydrins in horse chestnut recalcitrant seeds

The fraction of heat-stable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stress-induced dehydrin-like polypeptides. In our experiments, in tissues of dormant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Low-molecular-weight heat-stable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not cross-react with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissue-specificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heat-stable proteins during the entire period of stratification and also radicle emersion. During radicle emergence, not only the fraction of heat-stable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cycloheximide, or α-amanitin, dehydrins did not disappeared from the fraction of heat-stable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could cross-react with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50-kD heat-stable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heat-sensitive proteins and was named as a dehydrin-like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heat-stable cytosolic proteins. The role and function of major heat-stable proteins in horse chestnut seeds are yet to be studied.     

Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds

Germination of cereals is accompanied by extensive change in the redox state of seed proteins. Proteins present in oxidized form in dry seeds are converted to the reduced state following imbibition. Thioredoxin (Trx) appears to play a role in this transition in cereals. It is not known, however, whether Trx-linked redox changes are restricted to cereals or whether they take place more broadly in germinating seeds. To gain information on this point, we have investigated a model legume, Medicago truncatula. Two complementary gel-based proteomic approaches were followed to identify Trx targets in seeds: Proteins were (1) labeled with a thiol-specific probe, monobromobimane (mBBr), following in vitro reduction by an NADP/Trx system, or (2) isolated on a mutant Trx affinity column. Altogether, 111 Trx-linked proteins were identified with few differences between axes and cotyledons. Fifty nine were new, 34 found previously in cereal or peanut seeds, and 18 in other plants or photosynthetic organisms. In parallel, the redox state of proteins assessed in germinating seeds using mBBr revealed that a substantial number of proteins that are oxidized or partly reduced in dry seeds became more reduced upon germination. The patterns were similar for proteins reduced in vivo during germination or in vitro by Trx. In contrast, glutathione and glutaredoxin were less effective as reductants in vitro. Overall, more than half of the potential targets identified with the mBBr labeling procedure were reduced during germination. The results provide evidence that Trx functions in the germination of seeds of dicotyledons as well as monocotyledons.     

Identification of a serodiagnostic antigen,legumain, by immunoproteomic analysis of excretory‐secretory products of Clonorchis sinensis adult worms

Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory‐secretory products (ESPs) of the parasites play important roles in host–parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2‐DE‐based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S‐transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno‐proteomic approaches in the identification of serodiagnostic antigens in the parasites.     

刺五加种子结构,后熟作用及其细胞化学研究

刺五加种子为扁肾形,种皮由一层细胞构成。种子脱落时,胚处于心形胚期,胚周围的胚乳细胞解体形成囊腔包囊胚,胚细胞原生质浓厚,胚乳细胞中贮存大量蛋白质和脂类,但两者均未见贮存多糖,有萌发潜能的种子只占全部种子的１２．８０％,种子经变温层积处理６个月即可完成后熟过程,其细胞化学特点是：处理１．５个月时胚细胞中开始积累多糖颗粒,至４个月时达最大量并一直保持至种子萌发。试验地种植条件下饱满种子经１８－２０个     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Classification of the caspase-hemoglobinase fold: detection of new families and implications for the origin of the eukaryotic separins

A comprehensive sequence and structural comparative analysis of the caspase-hemoglobinase protein fold resulted in the delineation of the minimal structural core of the protease domain and the identification of numerous, previously undetected members, including a new protease family typified by the HetF protein from the cyanobacterium Nostoc. The first bacterial homologs of legumains and hemoglobinases were also identified. Most proteins containing this fold are known or predicted to be active proteases, but multiple, independent inactivations were noticed in nearly all lineages. Together with the tendency of caspase-related proteases to form intramolecular or intermolecular dimers, this suggests a widespread regulatory role for the inactive forms. A classification of the caspase-hemoglobinase fold was developed to reflect the inferred evolutionary relationships between the constituent protein families. Proteins containing this domain were so far detected almost exclusively in bacteria and eukaryotes. This analysis indicates that caspase-hemoglobinase-fold proteases and their inactivated derivatives are widespread in diverse bacteria, particularly those with a complex development, such as Streptomyces, Anabaena, Mesorhizobium, and Myxococcus. The eukaryotic separin family was shown to be most closely related to the mainly prokaryotic HetF family. The phyletic patterns and evolutionary relationships between these proteins suggest that they probably were acquired by eukaryotes from bacteria during the primary, promitochondrial endosymbiosis. A similar scenario, supported by phylogenetic analysis, seems to apply to metacaspases and paracaspases, with the latter, perhaps, being acquired in an independent horizontal transfer to the eukaryotes. The acquisition of the caspase-hemoglobinase-fold domains by eukaryotes might have been critical in the evolution of important eukaryotic processes, such as mitosis and programmed cell death.     

The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice

Rice ( Oryza sativa L.) accumulates prolamines and glutelins as its major storage proteins. Glutelins are synthesized on rough endoplasmic reticulum as 57-kDa precursors; they are then sorted into protein storage vacuoles where they are processed into acidic and basic subunits. We report a novel rice glutelin mutant, W379 , which accumulates higher levels of the 57-kDa glutelin precursor. Genetic analysis revealed that the W379 phenotype is controlled by a single recessive nuclear gene. Using a map-based cloning strategy, we identified this gene, OsVPE1 , which is a homolog of the Arabidopsis βVPE gene. OsVPE1 encodes a 497-amino-acid polypeptide. Nucleotide sequence analysis revealed a missense mutation in W379 that changes Cys269 to Gly. Like the wild-type protein, the mutant protein is sorted into vacuoles; however, the enzymatic activity of the mutant OsVPE1 is almost completely eliminated. Further, we show that OsVPE1 is incorrectly cleaved, resulting in a mature protein that is smaller than the wild-type mature protein. Taken together, these results demonstrate that OsVPE1 is a cysteine protease that plays a crucial role in the maturation of rice glutelins. Further, OsVPE1 Cys269 is a key residue for maintaining the Asn-specific cleavage activity of OsVPE1.     

成熟脱水对种子发育和萌发的作用

成熟脱水是正常性种子发育的末端事件。种子在成熟时胚的脱水耐性增加;当种子萌发时胚变得不耐脱水。当种子获得脱水耐性时,糖、蛋白质和抗氧化防御系统等保护性物质积累;当脱水耐性丧失时,这些物质被降解。成熟脱水是种子从发育过程向萌发过程转变的“开关”,它降低发育的蛋白质和mRNA的合成,终止发育事件和促进萌发事件。顽拗性种子不经历成熟脱水的发育阶段,对脱水高度敏感。     

Carbonylated proteins accumulated as vitality decreases during long-term storage of beech (Fagus sylvatica L.) seeds

Key message

Carbonylation of proteins associated with a stress response may contribute to the lowered viability of naturally aged beech seeds, especially the desiccation tolerance-associated proteins and USP-like protein.

Abstract

Proteins are modified by a large number of reactions that involve reactive oxygen species-mediated oxidation. The direct oxidation of amino acids produces 2,4-dinitrophenylhydrazine-detectable protein products. Carbonylation is irreversible, and carbonylated proteins are marked for proteolysis or can escape degradation and form high molecular weight aggregates, which accumulate with age. Beech (Fagus sylvatica L.) seeds stored under optimal conditions for different periods of time, ranging from 2 to 13 years, were analyzed. Protein carbonylation was examined as a potential cause for the loss of viability of beech seeds, and the characteristic spots of protein carbonyls were identified. Here, we present and discuss the role of carbonylation in the proteome of beech seeds that contribute to the loss of seed viability during natural aging. The long-term storage of beech seeds is intricate because their germination capacity decreases with age and is negatively correlated with the level of protein carbonyls that accumulate in the seeds. We establish that protein synthesis, folding and degradation are the most affected biochemical traits in long-term stored beech seeds. In addition, we suggest that proteins associated with the stress response may have contributed to the lowered viability of beech seeds, especially the desiccation tolerance-associated proteins that include T-complex protein 1 and the universal stress protein (USP)-like protein, which is identified as carbonylated for first time here.