Biomedical subjects

Jelle Lahnstein

Publications and source records attributed to Jelle Lahnstein.

8 recordsLinked to original sources

PubMed2007

Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes.

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The Km and kcat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 microM and 120-130 s(-1), respectively, using NADH as the electron donor. The Km values were at least an order of magnitude higher for NADPH. The Km values for the MDHA radical were approximately 0.5-1.0 microM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a 'ping-pong' kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.

Amino Acid Sequence↗

PubMed2006

Hydrolysis of (1,4)-beta-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-beta-D-mannan endohydrolase and beta-D-mannosidase.

A family GH5 (family 5 glycoside hydrolase) (1,4)-beta-D-mannan endohydrolase or beta-D-mannanase (EC 3.2.1.78), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2-6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has K(m) constants of 0.16 mg x ml(-1) and 5.3 mM and kcat constants of 12.9 and 3.9 s(-1) respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-beta-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-beta-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-beta-D-manno-oligosaccharides to Man is a family GH1 beta-D-mannosidase, now designated HvbetaMANNOS1, but previously identified as a beta-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134-11143], which hydrolyses 4NP (4-nitrophenyl) beta-D-mannoside three times faster than 4NP beta-D-glucoside, and has an action pattern typical of a (1,4)-beta-D-mannan exohydrolase.

Amino Acid Sequence↗

PubMed2006

Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.).

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-beta-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The K(m) values were 40 microM for UDP-Gal and 55 muM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.

Amino Acid Sequence↗

PubMed2005

Characterization and expression patterns of UDP-D-glucuronate decarboxylase genes in barley.

UDP-D-glucuronate decarboxylase (EC 4.1.1.35) catalyzes the synthesis of UDP-D-xylose from UDP-D-glucuronate in an essentially irreversible reaction that is believed to commit glycosyl residues to heteroxylan and xyloglucan biosynthesis. Four members of the barley (Hordeum vulgare) UDP-D-glucuronate decarboxylase gene family, designated HvUXS1 to HvUXS4, have been cloned and characterized. Barley HvUXS1 appears to be a cytosolic enzyme, while the others are predicted to be membrane-bound proteins with single transmembrane helices. Heterologous expression of a barley HvUXS1 cDNA in Escherichia coli yields a soluble enzyme that converts UDP-d-glucuronate to UDP-D-xylose, is associated with a single molecule of bound NAD+, and is subject to feedback inhibition by UDP-D-xylose. Quantitative PCR shows that the HvUXS1 mRNA is most abundant among the 4 HvUXS genes, accounting for more than 80% of total HvUXS transcripts in most of the tissues examined. The abundance of HvUXS1 mRNA is 10-fold higher in mature roots and stems than in leaves, developing grains, or floral tissues. Transcriptional activities of HvUXS2 and HvUXS4 genes are relatively high in mature roots, coleoptiles, and stems compared with root tips, leaves, and floral tissues, while HvUXS3 mRNA is low in all tissues. In barley leaf sections, levels of the most abundant mRNA, encoding HvUXS1, reflect the amount of soluble enzymic protein and activity. In selected tissues where HvUXS1 transcript levels are high, cell walls have higher arabinoxylan contents.

Amino Acid Sequence↗

PubMed2005

Is the mature endotoxin Cry1Ac from Bacillus thuringiensis inactivated by a coagulation reaction in the gut lumen of resistant Helicoverpa armigera larvae?

Bacillus thuringiensis endotoxins (Bt-toxins) are the most important biopesticides used in controlling insect pests and vectors of diseases. The emergence of widespread resistance to Bt in some insect species is a serious threat to agricultural production. Analysis of Bt-resistant and susceptible laboratory strains of Helicoverpa armigera revealed elevated immune responses involving increased melanization and the presence of a soluble toxin-binding glycoprotein in the hemolymph and gut lumen of the resistant strain. We propose a resistance mechanism against toxins based on a systemic immune-induction that can be transmitted to the next generation by a maternal effect.

Amino Acid Sequence↗

PubMed2004

Post-translational modifications of arabinogalactan-peptides of Arabidopsis thaliana. Endoplasmic reticulum and glycosylphosphatidylinositol-anchor signal cleavage sites and hydroxylation of proline.

We have developed a method for separating the deglycosylated protein/peptide backbones of the small arabinogalactan (AG)-peptides from the larger classical arabinogalactan-proteins (AGPs). AGPs are an important class of plant proteoglycans implicated in plant growth and development. Separation of AG-peptides enabled us to identify eight of 12 AG-peptides from Arabidopsis thaliana predicted from genomic sequences. Of the remaining four, two have low abundance based on expressed sequence tag databases and the other two are only present in pollen (At3g20865) or flowers (At3g57690) and therefore would not be detected in our analysis. Characterization of AG-peptides was performed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry protein sequencing. These data provide (i) experimental evidence that AG-peptides are processed in vivo for the addition of a glycosylphosphatidylinositol (GPI) anchor, (ii) cleavage site information for both the endoplasmic reticulum secretion signal and the GPI-anchor signal for eight of the 12 AG-peptides, and (iii) experimental evidence that the Gly-Pro motif is hydroxylated in vivo. Furthermore, we show that AtAGP16 is GPI-anchored despite its unusually long hydrophobic C-terminal GPI-signal sequence. Prior to this work, the GPI-anchor cleavage site for only two plant proteins, NaAGP1 from Nicotiana alata and PcAGP1 from Pyrus communis, had been determined experimentally. Characterization of the post-translational modifications of AG-peptides contributes toward obtaining the complete primary structure of this class of biologically important plant proteoglycans and provides a greater understanding of post-translational modifications of plant proteins.

Amino Acid Sequence↗

PubMed2002

Bifunctional family 3 glycoside hydrolases from barley with alpha -L-arabinofuranosidase and beta -D-xylosidase activity. Characterization, primary structures, and COOH-terminal processing.

An alpha-l-arabinofuranosidase and a beta-d-xylosidase, designated ARA-I and XYL, respectively, have been purified about 1,000-fold from extracts of 5-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, ion exchange chromatography, chromatofocusing, and size-exclusion chromatography. The ARA-I has an apparent molecular mass of 67 kDa and an isoelectric point of 5.5, and its catalytic efficiency during hydrolysis of 4'-nitrophenyl alpha-l-arabinofuranoside is only slightly higher than during hydrolysis of 4'-nitrophenyl beta-d-xyloside. Thus, the enzyme is actually a bifunctional alpha-l-arabinofuranosidase/beta-d-xylosidase. In contrast, the XYL enzyme, which also has an apparent molecular mass of 67 kDa and an isoelectric point of 6.7, preferentially hydrolyzes 4'-nitrophenyl beta-d-xyloside, with a catalytic efficiency approximately 30-fold higher than with 4'-nitrophenyl alpha-l-arabinofuranoside. The enzymes hydrolyze wheat flour arabinoxylan slowly but rapidly hydrolyze oligosaccharide products released from this polysaccharide by (1 --> 4)-beta-d-xylan endohydrolase. Both enzymes hydrolyze (1 --> 4)-beta-d-xylopentaose, and ARA-I can also degrade (1 --> 5)-alpha-l-arabinofuranohexaose. ARA-I and XYL cDNAs encode mature proteins of 748 amino acid residues which have calculated molecular masses of 79.2 and 80.5 kDa, respectively. Both are family 3 glycoside hydrolases. The discrepancies between the apparent molecular masses obtained for the purified enzymes and those predicted from the cDNAs are attributable to COOH-terminal processing, through which about 130 amino acid residues are removed from the primary translation product. The genes encoding the ARA-I and XYL have been mapped to chromosomes 2H and 6H, respectively. ARA-I transcripts are most abundant in young roots, young leaves, and developing grain, whereas XYL mRNA is detected in most barley tissues.

Amino Acid Sequence↗

PubMed2002

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus.

The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration.

Amino Acids↗