Wheat Serine Research Articles

Proteomic identification of apoplastic proteins from rice, wheat, and barley after Magnaporthe oryzae infection

The fungal pathogen Magnaporthe oryzae causes devastating blast disease in various cereals, including rice (Oryza sativa), wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare). Despite previous reports on fungal host specificity, the mechanisms underlying differential host infection strategies remain unclear. This study aimed to identify differentially abundant proteins (DAPs) in the apoplast of rice, barley, and wheat following infection with two M. oryzae pathovars using liquid chromatography-tandem mass spectrometry (LC–MS/MS). LC–MS/MS analysis revealed an enrichment of both M. oryzae and host proteins in the apoplast during the compatible reaction compared to the incompatible reaction. DAPs from M. oryzae involved in the host interaction included secreted extracellular enzymes (e.g., hydrolases), which were significantly increased in the M. oryzae Oryzae (MoO)-infected rice apoplast. Among host proteins, the proportion of protein-modifying enzymes increased in the M. oryzae Triticum (MoT)-infected rice and MoO-infected wheat apoplastic fluids, particularly rice glycosidases, peroxidases, and serine proteases, as well as wheat serine proteases. Furthermore, DAPs from MoL-infected rice were enriched in carbohydrate metabolism, suggesting that carbohydrate metabolism-related proteins may play a vital role in rice resistance to MoL. Additionally, protein-modifying and cytoskeletal proteins, as well as stress-responsive proteins, were enriched in the MoO-infected wheat apoplastic fluid. Finally, DAPs from both MoO- and MoL-infected barley were enriched in hydrogen peroxide catabolism, suggesting that peroxidases may be vital for barley resistance to M. oryzae. The identification of DAPs from both M. oryzae strains and the three host plants offers valuable insights into the host specificity mechanisms of M. oryzae in cereal crops.

Reproductive sink enhanced drought induced senescence in wheat fertile line is associated with loss of antioxidant competence compared to its CMS line

Reproductive sinks regulate monocarpic senescence in wheat as desinking delayed flag leaf senescence under irrigated condition. In this study, wheat cv. HW 2041 and its isonuclear male sterile line (CMS) were subjected to post-anthesis water deficit stress to understand the association between sink strength, senescence and drought response in relation to oxidative stress and antioxidant defense at cellular and sub-cellular level. CMS plants maintained better water relations and exhibited delayed onset and progression of flag leaf senescence in terms of green leaf area, chlorophyll and protein content than fertile plants under water deficit stress (WDS). Delayed senescence in CMS plants under water deficit stress was associated with less reactive oxygen species generation, lower damage to membranes and better antioxidant defense both in terms of antioxidant enzyme activities and metabolite content compared to fertile plants. Expression of some senescence associated genes (SAGs) such as WRKY transcription factor (WRKY53), glutamine synthetase1 (GS1), wheat cysteine protease (WCP2) and wheat serine protease (WSP) was lower while catalse 2 (CAT2) transcript levels were higher in the CMS plants compared to HW2041 during senescence under water deficit stress. Antioxidant defense in chloroplasts was better in CMS line under water deficit stress compared to HW2041. This is the first report showing that reproductive sink enhanced drought induced senescence in flag leaf of wheat fertile line is associated with higher oxidative stress and damage and loss of antioxidant competence compared to its sterile line under water deficit stress. Higher expression of some SAGs and decline in superoxide dismutase and ascorbate peroxidase activity in the chloroplasts also contributed to the accelerated senescence in fertile line compared to its CMS line under WDS.

Molecular and Immunological Characterization of a Wheat Serine Proteinase Inhibitor as a Novel Allergen in Baker's Asthma

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.

Molecular and immunological characterization of a wheat serine proteinase-inhibitor as a novel allergen in baker's asthma

Molecular and immunological characterization of a wheat serine proteinase-inhibitor as a novel allergen in baker's asthma

Prediction of the mechanism of action of omuralide ( clasto-lactacystin β-lactone) on human cathepsin A based on a structural model of the yeast proteasome β5/PRE2-subunit/omuralide complex

Cathepsin A (CathA) is a lysosomal serine carboxypeptidase that exhibits homology and structural similarity to the yeast and wheat serine carboxypeptidases (CPY and CPW) belonging to the α/β-hydrolase fold family. Human CathA (hCathA) and CPW have been demonstrated to be inhibited by a proteasome (threonine protease) inhibitor, lactacystin, and its active derivative, omuralide ( clasto-lactacystin β-lactone), as well as chymostatin. A hCathA/omuralide complex model constructed on the basis of the X-ray crystal structures of the CPW/chymostatin complex and the yeast proteasome β-subunit (β5/PRE2)/omuralide one predicted that the conformation of omuralide in the active-site cleft of proteasome β5/PRE2 should be very similar to that of chymostatin at the S1 catalytic subsites in the hCathA- and CPW-complexes. The relative positions of the glycine residues, i.e., Gly57 in hCathA, Gly53 in CPW, and Gly47 in β5/PRE2, present in the oxyanion hole of each enzyme were also highly conserved. These results suggest that omuralide might inhibit hCathA and CPW at the S1 subsite in their active-site clefts through direct binding to the active serine residue.

Crystal Structure of Hydroxynitrile Lyase from Sorghum bicolor in Complex with the Inhibitor Benzoic Acid: A Novel Cyanogenic Enzyme,

The crystal structure of the hydroxynitrile lyase from Sorghum bicolor (SbHNL) in complex with the inhibitor benzoic acid has been determined at 2.3 A resolution and refined to a crystallographic R-factor of 16.5%. The SbHNL sequence places the enzyme in the alpha/beta hydrolase family where the active site nucleophile is predicted to be organized in a characteristic pentapeptide motif which is part of the active site strand-turn-helix motif. In SbHNL, however, a unique two-amino acid deletion is next to the putative active site Ser158, removing thereby the putative oxyanion hole-forming Tyr residue. The presented X-ray structure shows that the overall folding pattern of SbHNL is similar to that of the closely related wheat serine carboxypeptidase (CPD-WII); however, the deletion in SbHNL is forcing the putative active site residues away from the expected hydrolase binding site toward a small hydrophobic cleft, which also contains the inhibitor benzoic acid, defining thereby a completely different SbHNL active site architecture where the traditional view of a classic triad is not given any more. Rather, we propose a mechanism involving general base catalysis by the carboxy-terminal Trp270 carboxyl group and proton transfer toward the leaving nitrile group by an active site water molecule. The unexpected interactions of the inhibitor with the new SbHNL active site also reveal the structural basis for the enzyme's limited substrate specificity. The implications of this structure on the evolution of catalysis in the hydroxynitrile lyase superfamily are discussed.

Crystal structure of Kex1deltap, a prohormone-processing carboxypeptidase from Saccharomyces cerevisiae,.

Kex1p is a prohormone-processing serine carboxypeptidase found in Saccharomyces cerevisiae. In contrast to yeast serine carboxypeptidase (CPD-Y) and wheat serine carboxypeptidase II (CPDW-II), Kex1p displays a very narrow specificity for lysyl or arginyl residues at the C-terminus of the substrate. The structure of Kex1Deltap, an enzyme that lacks the acidic domain and membrane-spanning portion of Kex1p, has been solved by a combination of molecular replacement and multiple isomorphous replacement and refined to a resolution of 2.4 A. The S1' site of Kex1Deltap is sterically restricted compared to those from CPD-Y or CPDW-II; it also contains two acidic groups that are well positioned to interact with the basic group of a lysine or arginine side chain. The high specificity of Kex1p can therefore be explained by a combination of steric and electronic factors. The structure of the S1 site of Kex1Deltap is also well suited for binding of a lysine or arginine side chain, and the enzyme may therefore exhibit a preference for these residues at P1.

Structure determination of the human protective protein: twofold averaging reveals the three-dimensional structure of a domain which was entirely absent in the initial model.

Mutations in the human 'protective protein' result in the human lysosomal storage disease galactosialidosis. The structure of the human 'protective protein' has been determined using X-ray crystallography to a resolution of 2.2 A. Initial phases were obtained from molecular replacement calculations. A very partial search model comprising 30% of the scattering mass, was constructed from the atomic model of the wheat serine carboxypeptidase. This truncated probe was used to find the position of two monomers in the asymmetric unit. Subsequently, 'bootstrapping' cycles, consisting of twofold averaging and model expansion, retrieved the electron density for residues initially missing. In particular, it proved possible to add a domain (more than 110 residues) to the initial partial search model. In total, 314 residues per asymmetric unit were added to the 588 residues of the initial model. Factors contributing to our success are discussed.

Protease inhibitor studies and cloning of a serine carboxypeptidase cDNA from germinating seeds of pea (Pisum sativum L.).

The nature of the proteolytic activity found within the germinating pea (Pisum sativum) seed, 4 days from the initiation of imbibition, was determined by the use of specific protease inhibitors. These studies have shown most of the activity to belong to metallo or metal-activated and serine proteases. In order to investigate further the serine protease activity, a pea cotyledon germination cDNA library was, therefore, screened with a wheat cDNA (2437) [Baulcombe, D.C., Barker, R.F. & Jarvis, M.G. (1987) J. Biol. Chem. 262, 13726-13735] which had extensive similarity to the yeast serine carboxypeptidase Y gene. A positive cDNA clone (pNY551) was obtained which had extensive similarity to the four carboxypeptidases, Arabidopsis thaliana carboxypeptidase Y-like protein, rice serine carboxypeptidase III, barley serine carboxypeptidase III and wheat serine carboxypeptidase III precursor. Northern-blot analysis showed mRNA homologous to pNY551 to be expressed in late developmental pea seed and again during germination.

Crystallization of a soluble form of the Kex1p serine carboxypeptidase from Saccharomyces cerevisiae.

A soluble form of the killer factor and prohormone-processing carboxypeptidase, "Kex1 delta p," from Saccharomyces cerevisiae, has been crystallized in 17-22% poly(enthylene glycol) methyl ether (average M(r) = 5,000), 100 mM ammonium acetate, 5% glycerol, pH 6.5, at 20 degrees C. A native data set (2.8 A resolution) and four derivative data sets (3.0-3.2 A resolution) were collected at the Photon Factory (lambda = 1.0 A). The crystals belong to space group P2(1)2(1)2(1) with a =56.6 A, b = 84.0 A, c = 111.8 A. Freezing a Kex1 delta p crystal has facilitated the collection of a 2.4-A data set using a rotating anode source (lambda = 1.5418 A). Molecular replacement models have been built based on the structures of wheat serine carboxypeptidase (CPDW-II; Liao DI et al., 1992, Biochemistry 31:9796-9812) and yeast carboxypeptidase Y.

Peptide Aldehyde Complexes with Wheat Serine Carboxypeptidase II: Implications for the Catalytic Mechanism and Substrate Specificity

The structures of two ternary complexes of wheat serine carboxypeptidase II (CPD-WII), with a tetrapeptide aldehyde and a reaction product arginine, have been determined by X-ray crystallography at room temperature and −170°. The peptide aldehydes, antipain and chymostatin, form covalent adducts with the active-site serine 146. The CPD-WII antipain arginine model has a standard crystallographic R-factor of 0.162, with good geometry at 2.5 Å resolution for data collected at room temperature. The −170°C model of the chymostatin arginine complex has an R-factor of 0.174, with good geometry using data to 2.1 Å resolution. The structures suggest binding subsites N-terminal to the scissile bond. All four residues of chymostatin are well-localized in the putative S 1through S 4sites, while density is apparent only in S 1and S 2for antipain. In the S 1site, Val340 and 341, Phe215 and Leu216 form a hydrophobic binding surface, not a pocket, for the P 1phenylalanyl side-chain of chymostatin. The P 1arginyl of antipain also binds at this site, but the positive charge appears to be stabilized by additional solvent molecules. Thus, the hybrid nature of the S 1site accounts for the ability of CPD-WII to accept both hydrophobic and basic residues at P 1. Hydrogen bonds to the peptide substrate backbone are few and are made primarily with side-chains on the enzyme. Thus, substrate recognition by CPD-WII appears to have nothing in common with that of the other families of serine proteinases. The hemiacetal linkages to the essential Ser146 are of a single stereoisomer with tetrahedral geometry, with an oxygen atom occupying the "oxyanion hole" region of the enzyme. This atom accepts three hydrogen bonds, two from the polypeptide backbone and one from the positively-charged amino group of bound arginine, and must be negatively charged. Thus, the combination of ligands forms an excellent approxi mation to the oxyanion intermediate formed during peptide hydrolysis. Surprisingly, the (R) stereochemistry at the hemiacetal linkage is opposite to that expected by comparison to previously determined structures of peptide aldehydes complexed with Streptomyces griseusproteinase A. This is shown to be a consequence of the approximate mirror symmetry of the arrangement of catalytic groups in the two families of serine proteases and suggests that the stereochemical course of the two enzymatic reactions differ in handedness.

Three-dimensional structure of the human 'protective protein': structure of the precursor form suggests a complex activation mechanism.

The human 'protective protein' (HPP) forms a multi-enzyme complex with beta-galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis. Proteolytic cleavage of the precursor form of HPP involves removal of a 2 kDa excision peptide and results in a carboxypeptidase activity. The physiological relevance of this activity is, as yet, unknown. The crystal structure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer consists of a 'core' domain and a 'cap' domain. Comparison with the distantly related wheat serine carboxypeptidase dimer shows that the two subunits in the HPP dimer differ by 15 degrees in mutual orientation. Also, the helical subdomain forming part of the cap domains is very different. In addition, the HPP precursor cap domain contains a 'maturation' subdomain of 49 residues which fills the active-site cleft. Merely removing the 'excision' peptide located in the maturation subdomain does not render the catalytic triad solvent accessible. The activation mechanism of HPP is unique among proteases with known structure. It differs from the serine proteases in that the active site is performed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the chain segment physically rendering the catalytic triad solvent inaccessible in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptide and major conformational changes of the maturation subdomain, whereas the conformation of the enzymatic machinery is probably almost, or completely, unaffected.

Structure of the complex of L-benzylsuccinate with wheat serine carboxypeptidase II at 2.0-A resolution.

The structure of the complex of L-benzylsuccinate (Ki = 0.2 mM) bound to wheat serine carboxypeptidase II has been analyzed at 2.0-A resolution for native and inhibited crystals at -170 degrees C. The model has been refined and has a standard crystallographic R-factor of 0.176 for 57,734 reflections observed between 20.0- and 2.0-A resolution. The root mean square deviation from ideal bonds is 0.017 A and from ideal angles is 2.6 degrees. The model consists of 400 amino acids, 4 N-linked saccharide residues, and 430 water molecules. L-Benzylsuccinate occupies a narrow slot in the active site defined by Tyr 60, Tyr 239, and the polypeptide backbone. One carboxylate forms hydrogen bonds to Glu 145, Asn 51, the amide of Gly 52, and the catalytic His 397, suggestive of how the peptide C-terminal carboxylate is recognized by the enzyme. The phenyl ring stacks between Tyr 239 and Tyr 60, while the other carboxylate occupies the "oxyanion hole". One of the oxygens accepts hydrogen bonds from the amides of Tyr 147 and Gly 53, while the other forms a very close contact (2.3 A) with the O gamma of Ser 146, forcing the side chain into a conformation alternative to that found in the resting state of the enzyme. The inhibitor occupies the active site in a way that suggests that it can be regarded as a transition-state analogue of serine carboxypeptidases. The model suggests a novel enzymatic mechanism, involving substrate-assisted catalysis, that might account for the low pH optimum (4.0-5.5) of peptidase activity unique to this family of serine proteinases.

Refined atomic model of wheat serine carboxypeptidase II at 2.2-A resolution.

The crystal structure of the homodimeric serine carboxypeptidase II from wheat (CPDW-II, M(r) 120K) has been determined and fully refined at 2.2-A resolution to a standard crystallographic R factor of 16.9% using synchrotron data collected at the Brookhaven National Laboratory. The model has an rms deviation from ideal bond lengths of 0.018 A and from bond angles of 2.8 degrees. The model supports the general conclusions of an earlier study at 3.5-A resolution and will form the basis for investigation into substrate binding and mechanistic studies. The enzyme has an alpha + beta fold, consisting of a central 11-stranded beta-sheet with a total of 15 helices on either side. The enzyme, like other serine proteinases, contains a "catalytic triad" Ser146-His397-Asp338 and a presumed "oxyanion hole" consisting of the backbone amides of Tyr147 and Gly53. The carboxylate of Asp338 and imidazole of His397 are not coplanar in contrast to the other serine proteinases. A comparison of the active site features of the three families of serine proteinases suggests that the "catalytic triad" should actually be regarded as two diads, a His-Asp diad and a His-Ser diad, and that the relative orientation of one diad with respect to the other is not particularly important. Four active site residues (52, 53, 65, and 146) have unfavorable backbone conformations but have well-defined electron density, suggesting that there is some strain in the active site region. The binding of the free amino acid arginine has been analyzed by difference Fourier methods, locating the binding site for the C-terminal carboxylate of the leaving group. The carboxylate makes hydrogen bonds to Glu145, Asn51, and the amide of Gly52. The carboxylate of Glu145 also makes a hydrogen bond with that of Glu65, suggesting that one or both may be protonated. Thus, the loss of peptidase activity at pH > 7 may in part be due to deprotonation of Glu145. The active site does not reveal exposed peptide amides and carbonyl oxygen atoms that could interact with substrate in an extended beta-sheet fashion. The fold of the polypeptide backbone is completely different than that of trypsin or subtilisin, suggesting that this is a third example of convergent molecular evolution to a common enzymatic activity. Furthermore, it is suggested that the active site sequence motif "G-X-S-X-G/A", often considered the hallmark of serine peptidase or esterase activity, is fortuitous and not the result of divergent evolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase.

Structure of wheat serine carboxypeptidase II at 3.5-A resolution. A new class of serine proteinase.