Resistance to an herbivore through engineered cyanogenic glucoside synthesis.

The entire pathway for synthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been transferred from Sorghum bicolor to Arabidopsis thaliana. Here, we document that genetically engineered plants are able to synthesize and store large amounts of new natural products. The presence of dhurrin in the transgenic A. thaliana plants confers resistance to the flea beetle Phyllotreta nemorum, which is a natural pest of other members of the crucifer group, demonstrating the potential utility of cyanogenic glucosides in plant defense.

Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability.

The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable than the (1-->3,1-->4)-beta-glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes showed increased thermostability compared with the wild-type (1-->3,1-->4)-beta-glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.

Use of electrospray mass spectrometry for mass determination of grape (Vitis vinifera) juice pathogenesis-related proteins: a potential tool for varietal differentiation.

Methods based on liquid chromatography-mass spectrometry (LC-MS) and protein trap mass spectrometry (trap-MS) were developed to determine the complement of pathogenesis-related (PR) proteins in grape juice. Trap-MS was superior to LC-MS in terms of simplicity, rapidity, and sensitivity. Proteins with a wide range of masses (13--33 kDa) were found in the juices of 19 different varieties of grape (Vitis vinifera) and were identified as mostly PR-5 type (thaumatin-like) and PR-3 type (chitinases) proteins. Although the PR proteins in juices of grapes are highly conserved, small consistent differences in molecular masses were noted when otherwise identical proteins were compared from different varieties. These differences persisted through different harvest years and in fruits grown in different Australian locations. With the definition of four different masses for PR-5 proteins (range = 21,239--21,272 Da) and nine different masses of PR-3 proteins (range = 25,330--25,631 Da) and using statistical analysis, the methods developed could be used for varietal differentiation of grapes grown in several South Australian locations on the basis of the PR protein composition of the juice. It remains to be seen whether this technology can be extended to grapes grown worldwide and to wine and other fruit-derived products to assist with label integrity to the benefit of consumers.

The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity.

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with p-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

Proline accumulation in developing grapevine fruit occurs independently of changes in the levels of delta1-pyrroline-5-carboxylate synthetase mRNA or protein.

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 micromol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Delta1-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.

Characterisation of several Hsp70 interacting proteins from mammalian organelles.

Since both the spectrum and characteristics of in vivo substrates with affinity for Hsp70 members are largely unknown, we have investigated the range and type of mammalian organellar proteins which selectively interact with immobilised Escherichia coli Hsp70 (DnaK). Amongst a subset of organellar proteins selectively retained on DnaK, the major constituents represent unstable proteins and subunits of oligomeric proteins. The interactions with DnaK were diminished in the presence of mt-Hsp70 and BiP, while the complexes formed with DnaK were dissociated in the presence of K+ and GrpE-like co-chaperones, suggesting that these organellar proteins constitute general Hsp70 substrates. Protein sequence analysis identified the major DnaK interacting constituents as the mitochondrial transcription factor A, the alpha- (but not the beta-) subunit of succinyl CoA synthetase, mitochondrial 2,4-dienoyl CoA reductase, endoplasmic reticulum cyclophilin-B, peroxisomal multifunctional enzyme and a previously undescribed peroxisomal protein suspected to represent an isoform of 2,4-dienoyl CoA reductase. The selective retention of these fully synthesised proteins on Hsp70 most likely reflects the function of this molecular chaperone in protein biogenesis, but additionally, could extend the known functions of Hsp70 to include modulating the activities of certain proteins or enzymes which are important in cellular homeostasis.

Crystallization and preliminary X-ray analysis of beta-glucan exohydrolase isoenzyme ExoI from barley (Hordeum vulgare).

Crystals of a beta-glucan exohydrolase purified from extracts of young barley seedlings have been obtained by vapour diffusion in the presence of ammonium sulfate and polyethylene glycol. The enzyme exhibits broad substrate specificity against (1,3)-, (1,3;1,4)- and (1,3;1,6)-beta-glucans, and related oligosaccharides. Crystal dimensions of up to 0.8 x 0.4 x 0.6 mm have been observed. The crystals belong to the tetragonal space group P41212 or P43212. Cell parameters are a = b = 102.1 and c = 184.5 A, and there appear to be eight molecules in the asymmetric unit. The crystals diffract to at least 2.2 A resolution using X-rays from a rotating-anode generator.

Evidence for the existence of distinct mammalian cytosolic, microsomal, and two mitochondrial GrpE-like proteins, the Co-chaperones of specific Hsp70 members.

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein ( approximately 21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic ( approximately 40 kDa), microsomal ( approximately 50 kDa), and additional mitochondrial ( approximately 22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed approximately 47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3'-untranslated region of the mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo.

We report here the cloning and optimized expression at 16 degrees C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas Km values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

The genes encoding mammalian chaperonin 60 and chaperonin 10 are linked head-to-head and share a bidirectional promoter.

Chaperonins are a class of stress-inducible molecular chaperones involved in protein folding. We report the cloning, sequencing and characterisation of the rat mitochondrial chaperonin 60 and chaperonin 10 genes. The two genes are arranged in a head-to-head configuration and together comprise 14 kb and contain 14 introns. The genes are linked together by a region of approximately 280 bp, which constitutes a bidirectional promoter and includes a common heat-shock element. Insertion of the shared promoter region between two reporter genes is sufficient to drive their expression under both constitutive and heat-shock conditions. The arrangement of the mammalian chaperonin genes suggests the potential to provide the coordinated regulation of their products in a manner that is mechanistically distinct from, yet conceptually similar to, that employed by the bacterial chaperonin (groE) operon.

Polysaccharide hydrolases in germinated barley and their role in the depolymerization of plant and fungal cell walls.

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (1 --> 3,1 --> 4)-beta-glucans which comprise in excess of 90% by weight of these walls. The (1 --> 3,1 --> 4)-beta-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (1 --> 3,1 --> 4)-beta-glucans, but beta-glucan exohydrolases and beta-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (1 --> 3,1 --> 4)-beta-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 --> 3)-beta-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 --> 4)-beta-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 --> 4)-beta-xylan exohydrolases and alpha-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed.

Identification and characterization of a fruit-specific, thaumatin-like protein that accumulates at very high levels in conjunction with the onset of sugar accumulation and berry softening in grapes.

The protein composition of the grape (Vitis vinifera cv Muscat of Alexandria) berry was examined from flowering to ripeness by gel electrophoresis. A protein with an apparent molecular mass of 24 kD, which was one of the most abundant proteins in extracts of mature berries, was purified and identified by amino acid sequence to be a thaumatin-like protein. Combined cDNA sequence analysis and electrospray mass spectrometry revealed that this protein, VVTL1 (for V. vinifera thaumatin-like protein 1), is synthesized with a transient signal peptide as seen for apoplastic preproteins. Apart from the removal of the targeting signal and the formation of eight disulfide bonds, VVTL1 undergoes no other posttranslational modification. Southern, northern, and western analyses revealed that VVTL1 is found in the berry only and is encoded by a single gene that is expressed in conjunction with the onset of sugar accumulation and softening. The exact role of VVTL1 is unknown, but the timing of its accumulation correlates with the inability of the fungal pathogen powdery mildew (Uncinula necator) to initiate new infections of the berry. Western analysis revealed that the presence of thaumatin-like proteins in ripening fruit might be a widespread phenomenon.

The role of molecular chaperones in mitochondrial protein import and folding.

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperons in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular to mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

Cellobiohydrolase I from Trichoderma reesei: identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein.

(R,S)-3,4-Epoxybutyl beta-cellobioside, but not the corresponding propyl and pentyl derivatives, inactivates specifically and irreversibly cellobiohydrolase I from Trichoderma reesei by covalent modification of Glu212, the putative active-site nucleophile. The position and identity of the modified amino acid residue were determined using a combination of comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry, tandem mass spectrometry and microsequencing. It was found that the core protein corresponds to the N-terminal sequence pyrGlu1-Gly434 (Gly435) of intact cellobiohydrolase I. In the particular enzyme samples investigated, the asparagine residues in positions 45, 270 and 384 are each linked to a single 2-acetamido-2-deoxy-D-glucopyranose residue.

Isolation and characterisation of a cDNA encoding rat mitochondrial GrpE, a stress-inducible nucleotide-exchange factor of ubiquitous appearance in mammalian organs.

In contrast to the E. coli chaperones DnaK, GroEL and GroES, cDNAs encoding mitochondrial homologues of DnaJ and GrpE from higher eukaryotes have yet to be reported. Based on peptide sequences, we have isolated a cDNA encoding a 217 residue nuclear encoded precursor of rat mitochondrial GrpE (mt-GrpE) including a typical mitochondrial presequence of 27 residues. Western blotting revealed that the 21 kDa GrpE homologue is present exclusively in the mitochondrial fraction where it comprises only approximately 0.03% of the total soluble protein, while Northern blotting showed that the mt-GrpE transcript is present in most if not all organs. By contrast to other mitochondrial chaperones, the levels of mt-GrpE and its transcript in cultured cells are only marginally increased in response to the proline analog L-azetidine 2-carboxylic acid but not by heat shock. Furthermore, members of the GrpE family exhibit a much lower degree of sequence identity than do the well studied members of the Hsp70, Hsp60 and Hsp10 families.

Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome.

Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclear-encoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (rho 0) rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in rho 0 cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the rho 0 state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles.

A new approach to fecal occult blood testing based on the detection of haptoglobin.

BACKGROUND: Colorectal carcinoma is a common disease, occurring in 1 in 20 adults in Western society, and there is a compelling need for an effective early diagnostic test. Several serum tests, including carcinoembryonic antigen have been used, but none are sufficiently sensitive for the early diagnosis of the disease. METHODS: In a novel approach using fecal extracts from patients with colorectal cancer as the antigen for immunization, several MoAbs were produced. One (FE14.1) was found to react with the feces from patients with colon cancer, but not with those from normal subjects. A sandwich enzyme-linked immunoadsorbent assay was developed, and its ability to diagnose colorectal carcinoma evaluated. RESULTS: Of the patients with colorectal carcinoma, 91.5% (43/46) were positive compared with 1.9% of normal individuals (4/211). Analysis of the N-terminal amino acid sequence of a subunit of the molecule detected by FE14.1 shows it to be the beta chain of haptoglobin. CONCLUSIONS: The assay developed in this study has several advantages compared with current fecal occult blood tests, including no requirement for dietary restriction and the ability to distinguish between upper and lower gastrointestinal bleeding, while retaining the sensitivity and specificity of the current tests. Furthermore, the sensitivity of the tests increases to 100% if the FE14.1 and HemeSelect are combined. In addition, the study shows the potential to produce anticancer agents by immunizing with fecal material.

Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone.

Two beta-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positional identity. Both enzymes hydrolyze the (1,3)-beta-glucan, laminarin, but also hydrolyze (1,3;1,4)-beta-glucan and 4-nitrophenyl beta-D-glucoside. The complete sequence of 602 amino acid residues of the mature beta-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated betaI and betaII, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as beta-glucosidases and although they hydrolyze 4-nitrophenyl beta-glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the (1,4)-beta-glucan glucohydrolase type. Both the beta-glucan exohydrolase isoenzyme ExoI and the beta-glucosidase isoenzyme betaII release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain.

Stereochemical course of glucan hydrolysis by barley (1-->3)- and (1-->3, 1-->4)-beta-glucanases.

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1-->3, 1-->4)-beta-glucan by barley (1-->3)-beta-glucanase (E.C. 3.2.1.39) isoenzyme GII and (1-->3, 1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by 1H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e-->e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of beta-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation.

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.