Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

Peptide: N-glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of beta-galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of beta-galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png(-)). The results show that the DdPNGase is an essential protein expressed throughout development and beta-galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png(-) cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

Normal pregnancy induced glucose metabolic stress in a longitudinal cohort of healthy women: Novel insights generated from a urine metabolomics study.

During normal pregnancy, mothers face a unique physiological challenge in the adaptation of glucose metabolism in preparation for the metabolic stress presented by fetal development. However, the responsible mechanism remains elusive. The purpose of this study is to investigate the mechanism of the metabolic stress of glucose metabolism in pregnant women using metabolomics method.A Ultra Performance Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometer-based untargeted metabolomics study was performed to investigate the dynamic urinary signature of the intermediates of glucose metabolism in a longitudinal cohort of 232 healthy pregnant women in their first, second, and third trimesters.Twelve glucose metabolic intermediates were screened out from hundreds of candidate metabolites using partial least squares discriminant analysis models. These 12 markers were mainly involved in the metabolic pathways of insulin resistance, glycolysis/gluconeogenesis, tricarboxylic acid cycle, nonabsorbable carbohydrate metabolism, and N-glycan biosynthesis. In particular, L-acetylcarnitine, a metabolite that is beneficial for the amelioration of insulin resistance, decreased in a time-dependent manner during normal pregnancy. Moreover, thiamine pyrophosphate, an intermediate product of glycolysis/gluconeogenesis, significantly increased in the second trimester, and argininosuccinic acid and oxalosuccinic acid, intermediates involved in the tricarboxylic acid cycle, significantly decreased in the third trimester, suggesting an increased glucose demand in the maternal body during fetal development.These findings provide novel insight into the normal pregnancy-induced elevation of insulin resistance and glycolysis/gluconeogenesis, as well as the observed reduction in the aerobic oxidation of glucose.

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System.

The peptide-N (4)-(N-acetyl-ß-D-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell.

[Expression, purification and characterization of N-glycanase from Schizosaccharomyces pombe in Escherichia coli].

One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b(+)/Png1p was transformed into E. coli BL21(DE3). The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Png1p in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30 degrees C; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100 degrees C.