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M J McPherson

Publications and source records attributed to M J McPherson.

At least 19 recordsLinked to original sources
PubMed1998

Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs.

Plant defence strategies usually involve the action of several gene products. Transgenic resistance strategies are likely to have enhanced efficacy when they involve more than one transgene. Here we explore possible mechanisms for the co-delivery of multiple effectors via a single transgene. As an example we report the co-delivery of two distinct proteinase inhibitors in Arabidopsis thaliana (L.) Heynh. to examine resistance against plant parasitic nematodes. A cysteine and serine proteinase inhibitor have been joined as translational fusions by one of two peptide linkers. One linker, part of the spacer region of a plant metallothionein-like protein (PsMTa), was selected to be cleaved in planta. A second linker, derived from the fungal enzyme galactose oxidase (GO) was chosen to be refractory to cleavage in planta. Western blot analysis of cell extracts confirmed the expected pattern of predominantly single inhibitors derived from the PsMTa construct and a primarily dual inhibitor from the GO construct. Analysis of cyst and root-knot nematodes recovered from transgenic Arabidopsis expressing inhibitors as single or dual molecules revealed the uptake of inhibitors with the exception of those linked by the PsMTa linker. This unexpected result may be due to residues of the PsMTa linker interacting with cell membranes. Despite lack of ingestion, PsMTa-linked cowpea trypsin inhibitor (CpTI) affected the sexual development of the cyst nematodes, indicating an external site of action. The engineered cystatin (Oc-I delta D86) component from the PsMTa constuct had no effect, indicating that ingestion is necessary for the cystatin to be effective. The delivery of dual inhibitors linked by the GO linker showed a clear additive effect over either inhibitor delivered singly. The application of this technology to other plant pathogens is discussed.

Animals↗
PubMed1998

Developmental expression and biochemical analysis of the Arabidopsis atao1 gene encoding an H2O2-generating diamine oxidase.

A copper amine oxidase encoding gene, atao1, has been isolated and characterized from Arabidopsis thaliana. Sequence analysis reveals that atao1 encodes a 668 amino acid polypeptide (ATAO1) with 48% identity to copper amine oxidases from pea and lentil. The promoter region of atao1 was transcriptionally fused with the reporter genes encoding beta-glucuronidase and modified green fluorescent protein. Analysis of transgenic Arabidopsis together with in situ hybridization of wild-type plants reveals temporally and spatially discrete patterns of gene expression in lateral root cap cells, vascular tissue of roots, developing leaves, the hypocotyl, and in the style/stigmatal tissue. Enzyme activity assays show that ATAO1 preferentially oxidizes the aliphatic diamine putrescine with production of the corresponding aldehyde, ammonia and hydrogen peroxide, a recognized plant signal molecule and substrate for peroxidases. Histochemical analysis reveals that atao1 expression in developing tracheary elements precedes and overlaps with lignification and therefore is a good marker for vascular development. In both vascular tissue and the root cap, atao1 expression occurs in cells destined to undergo programmed cell death.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction.

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.

Amine Oxidase (Copper-Containing)↗
PubMed1997

Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines.

Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

Amino Acid Sequence↗
PubMed1997

Resistance to both cyst and root-knot nematodes conferred by transgenic Arabidopsis expressing a modified plant cystatin.

Plant nematodes are major pests of agriculture. Transgenic plant technology has been developed based on the use of proteinase inhibitors as nematode anti-feedants. The approach offers prospects for novel plant resistance and reduced use of environmentally damaging nematicides. A modified rice cystatin, Oc-I delta D86, expressed as a transgene in Arabidopsis thaliana, has a profound effect on the size and fecundity of females for both Heterodera schachtii (beet-cyst nematode) and Meloidogyne incognita (root-knot nematode). No females of either species achieved the minimum size they require for egg production. Ingestion of Oc-I delta D86 from the plant was correlated with loss of cysteine proteinase activity in the intestine thereby suppressing normal growth, as required of an effective antifeedant plant defence.

Animals↗
PubMed1997

Continual green-fluorescent protein monitoring of cauliflower mosaic virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana.

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressive from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.

Animals↗
PubMed1997

Characterization of two cDNAs encoding cysteine proteinases from the soybean cyst nematode Heterodera glycines.

Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long propeptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-I delta D86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.

Amino Acid Sequence↗
PubMed1996

Characterization of intestinally active proteinases of cyst-nematodes.

Cryostat sections of juvenile and adult female stages of the soybean cyst-nematode, Heterodera glycines, were incubated with 4 different naphthylamide-linked peptide substrates to localize and characterize proteinase activity within the animal. Detected activity was restricted to the intestine and 2 distinct classes of proteinase were identified on the basis of substrate specificity and sensitivity to plant proteinase inhibitors. A cathepsin L-like cysteine proteinase activity capable of hydrolysing the synthetic substrates Z-Ala-Arg-Arg-MNA and Z-Phe-Arg-MNA but not Z-Arg-Arg-MNA or L-Arg-NA was inhibited by an engineered variant of a cysteine proteinase inhibitor from rice (Oc-I delta D86). The cleavage of Z-Phe-Arg-MNA was sensitive to inhibition by a combination of Oc-I delta D86 and cowpea trypsin inhibitor (CpTI). Degenerate oligonucleotide primers were used to amplify fragments of cysteine proteinase genes from 2 cyst-nematodes, H. glycines and Globodera pallida. Comparison of the H. glycines fragment with known genes established highest homology to cathepsin L-like genes. In contrast, the amplified G. pallida fragment displayed greatest homology to cathepsin B-like genes from Caenorhabditis elegans.

Amino Acid Sequence↗
PubMed1996

AIDS-associated infections in salivary glands: autopsy survey of 60 cases.

We reviewed the autopsy findings for the submandibular glands of 60 patients with AIDS who were autopsied at the George Washington University Medical Center (Washington, DC) from 1982 to 1992. AIDS-associated infections in the submandibular glands were compared with those in the pancreas and lung. Cytomegalovirus intranuclear inclusions were found in 10 cases, and Pneumocystis carinii infection was found in one case. Disseminated mycobacterial and fungal infections were not identified in the submandibular gland, even in the presence of documented pancreatic and pulmonary infection (P < .05). Overall, the major salivary glands of patients with AIDS are less frequently involved with disseminated opportunistic infections than is either the lung or the pancreas (P < .01 and P < .001, respectively).

AIDS-Related Opportunistic Infections↗
PubMed1995

Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution.

BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ). RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms. CONCLUSIONS: The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Cloning and molecular analysis of the pea seedling copper amine oxidase.

A pea seedling amine oxidase cDNA has been isolated and sequenced. A single long open reading frame has amino acid sequences corresponding to those determined from active site peptide (Janes, S.M., Palcic, M.M., Scaman, C.H., Smith, A.J., Brown, D.E., Dooley, D.M., Mure, M., and Klinman, J.P. (1992) Biochemistry 31, 12147-12154) and N-terminal sequencing experiments. The latter reveals the protein to have a 25-amino acid leader sequence with characteristics of a secretion signal peptide, as expected for this extracellular enzyme. Comparisons of the amino acid sequence of the mature pea enzyme (649 amino acids) with that of the mature lentil enzyme (569 amino acids; Rossi, A., Petruzzelli, R., and Finazzi-Agrò, A. (1992) FEBS Lett. 301, 253-257) reveal important and unexpected differences particularly with regard to protein length. Sequencing of part of the lentil gene identified several frameshift differences within the coding region resulting in a mature lentil protein of exactly the same length, 649 amino acids, as the pea enzyme. Multiple alignments of 10 copper amine oxidase sequences reveal 33 completely conserved residues of which 10 are found within 41 aligned residues at the C-terminal tails, the region missing from the original lentil sequence. One of only four conserved histidines is found in this region and may represent the third ligand to the copper. The pea enzyme contains around 3-4% carbohydrate as judged by deglycosylation experiments. We have also demonstrated by hybridization analysis that copper amine oxidase genes are present in a range of mono- and dicotyledonous plants.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida.

The cysteine proteinase inhibitor, oryzacystatin-I (Oc-I), and several engineered Oc-I variants have been tested for efficacy in inhibiting growth and development of both the free-living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc-I, an alignment of 28 cystatins and a molecular model of Oc-I were generated. Inhibitory activities (Ki) of wild-type and variant forms of Oc-I against both papain and the C. elegans cysteine proteinase, gcp-1, were measured. For one variant, in which residue Asp86 was deleted (Oc-I deltaD86), the Ki was reduced by 13- to 14-fold. LD50 studies to test the effect of Oc-I and Oc-I delta D86 against C. elegans showed the relative median potency of Oc-I delta D86 to be 0.76 that of wild-type Oc-I. When expressed in tomato hairy roots both Oc-I and Oc-I delta D86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc-I deltaD86-expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.

Amino Acid Sequence↗
PubMed1995

Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition.

Cystatins are small protein inhibitors of cysteine proteinases. The relative importance of the N-terminal region of cystatins, and of a conserved glycine within this region, remains unclear despite several studies. It was found that deletion of the N-terminal 21 amino acids abolishes the inhibitory capacity of oryzacystatin-I. The importance of a conserved glycine residue (Gly10) was also examined by replacing it with 11 other amino acids. Three further glycine residues (Gly5, -6 and -11) in this N-terminal region of oryzacystatin-I were similarly mutated. Only those variants in which Gly10 was substituted show any significant change in inhibitory capacity compared with wild-type oryzacystatin-I. The inhibitory characteristics of hybrid cystatin molecules comprising regions of chicken egg white cystatin and oryzacystatin were also examined. It is suggested that in common with animal cystatins, the N-terminal region of the plant cystatin, oryzacystatin-I, and in particular the highly conserved Gly10 residue are important for effective inhibition of papain.

Amino Acid Sequence↗
PubMed1994

Structure and mechanism of galactose oxidase. The free radical site.

Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.

Base Sequence↗
PubMed1994

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli.

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

Amino Acid Sequence↗
PubMed1993

Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli.

The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.

Bacterial Proteins↗