Glycosylation Of Peanut (arachis Hypogaea L) Peroxidases

The cationic (C.PRX) and the anionic (A.PRX) peroxidase isozymes secreted into the medium of cell cultures are both 20% glycosylated proteins. They are stable and occur in high amounts (C.PRX, 5 mg and A.PRX 0.5 mg per litre of spent culture medium), and so provide an ideal system for the study of the effect of glycosylation on the structure and function of glycoproteins.;A novel method to detect glycoproteins and glycopeptides is proposed using periodic-acid Schiff\u27s reagent dot-blotting assay on nitrocellulose membrane. At least 0.1 ug of glycan can be detected within 40 minutes.;The amino acid sequence of C.PRX is confirmed to be the same as the base sequence of cDNA clone prxPNC1 by the amino acid sequencing of the smaller formic acid cleaved peptide fragment. Out of five potential N-glycosylation sites only three, Asn{dollar}\sb{lcub}60{rcub}{dollar}, Asn{dollar}\sb{lcub}144{rcub}{dollar} and Asn{dollar}\sb{lcub}185{rcub}{dollar} are used. Both Asn{dollar}\sb{lcub}209{rcub}{dollar} and Asn{dollar}\sb{lcub}275{rcub}{dollar} have a proline residue at the C-terminal of the consensus sequence and are not glycosylated. Computer modelling showed that Asn{dollar}\sb{lcub}60{rcub}{dollar} and Asn{dollar}\sb{lcub}185{rcub}{dollar} are located in hydrophilic {dollar}\beta{dollar}-turns, while Asn{dollar}\sb{lcub}144{rcub}{dollar} in a hydrophobic {dollar}\beta{dollar}-sheet. The glycosylated asparagines present a preference for {dollar}\beta{dollar}-turns.;On the basis of Concanavalin A binding, C.PRX is fractionated into two forms, either binding, (CP+), or not binding, (CP{dollar}-{dollar}), to Concanavalin A column. Further investigation showed that CP{dollar}-{dollar} and CP+ have the same peptide chain and differ in carbohydrate moiety. All the three glycans of CP{dollar}-{dollar} and CP+ probably have the same core structure but different lengths and terminal sugars. This microheterogeneity of glycans is caused by a co-secreted {dollar}\beta{dollar}-galactosidase, which has two isozymes with mass 60 kd, pl 7.3 and mass 66 kd, pl 7.6 individually.;The antisera against TPCK-tryptic glycopeptides of C.PRX and A.PRX were raised and the antibodies directed to glycans were purified. The epitope mapping showed that the immunogenicity of the three glycans of C.PRX is similar and is mainly directed towards the core structure (Xyl) (Man){dollar}\sb3{dollar}(Fuc) (GlcNAc){dollar}\sb2{dollar}. The carbohydrate moiety of many plant proteins may be the basis of often observed antigenic cross reaction among different glycoproteins that are distinct in both function and structure of peptide moiety.;PNGase eliminates C.PRX enzyme activity and this reaction is dose- and time-dependent. Antibodies directed towards glycans of C.PRX or A.PRX do not block enzyme activity, but the antibodies against whole C.PRX or A.PRX inhibit enzyme activity by 80% and 30% respectively. Glycans are probably not directly involved in the enzyme active centre but play a role in maintaining the conformation of the peptide. Removal of glycans may cause a change of the 3-D structure of the peptide and the loss of calcium and heme

Probing the endosperm gene expression landscape in Brassica napus

<p>Abstract</p> <p>Background</p> <p>In species with exalbuminous seeds, the endosperm is eventually consumed and its space occupied by the embryo during seed development. However, the main constituent of the early developing seed is the liquid endosperm, and a significant portion of the carbon resources for the ensuing stages of seed development arrive at the embryo through the endosperm. In contrast to the extensive study of species with persistent endosperm, little is known about the global gene expression pattern in the endosperm of exalbuminous seed species such as crucifer oilseeds.</p> <p>Results</p> <p>We took a multiparallel approach that combines ESTs, protein profiling and microarray analyses to look into the gene expression landscape in the endosperm of the oilseed crop <it>Brassica napus</it>. An EST collection of over 30,000 entries allowed us to detect close to 10,000 unisequences expressed in the endosperm. A protein profile analysis of more than 800 proteins corroborated several signature pathways uncovered by abundant ESTs. Using microarray analyses, we identified genes that are differentially or highly expressed across all developmental stages. These complementary analyses provided insight on several prominent metabolic pathways in the endosperm. We also discovered that a transcription factor <it>LEAFY COTYLEDON </it>(<it>LEC1</it>) was highly expressed in the endosperm and that the regulatory cascade downstream of <it>LEC1 </it>operates in the endosperm.</p> <p>Conclusion</p> <p>The endosperm EST collection and the microarray dataset provide a basic genomic resource for dissecting metabolic and developmental events important for oilseed improvement. Our findings on the featured metabolic processes and the <it>LEC1 </it>regulatory cascade offer new angles for investigation on the integration of endosperm gene expression with embryo development and storage product deposition in seed development.</p

Identification of the Mamestra configurata (Lepidoptera: Noctuidae) peritrophic matrix proteins and enzymes involved in peritrophic matrix chitin metabolism

The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens, and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and \u3b1-amylase) or other reactions (\u3b2-1,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a heterogenous group consisting of polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and 3 novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase-2 (McCHS-2), chitinase (McCHI), and \u3b2-N-acetylglucosaminidase (McNAG) enzymes, involved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI, and McNAG were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture.Peer reviewed: YesNRC publication: Ye

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma

ABSTRACTEarly detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfide-bond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation