Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway.

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration.

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

Immunoglobulins are the major glycoproteins involved in the modifications of total serum N-glycome in cirrhotic patients.

PURPOSE: N-glycosylation modifications in human serum glycoproteins have been described in hepatic cirrhosis. To identify the glycoproteins carrying these modifications and to determine their influences in the modification of the total serum N-glycome (TSNG) in cirrhotic patients, we have performed the glycosylation analysis of immunoglobulins, transferrin, 1 antitrypsin and haptoglobin of patients who have developed cirrhosis. EXPERIMENTAL DESIGN: The glycosylation analysis of immunoglobulins G, transferrin, 1 antitrypsin and haptoglobin of 14 patients who have developed cirrhosis and 11 healthy controls was performed using strategies based on MS, 2-DE and affinity chromatography. RESULTS: We demonstrated that the N-glycosylation of both hepatic and plasma cell secreted glycoproteins is modified, and that the major modifications of TSNG are carried by immunoglobulins A and G. CONCLUSIONS AND CLINICAL RELEVANCE: The search for glycomic biomarkers used as an alternative to liver biopsy for the assessment of fibrosis in chronic liver disease is extremely important. Variations in the glycosylation of immunoglobulins are responsible for the main modifications affecting the TSNG and effector properties of the Fc of these molecules, and certainly contribute to the pathophysiology of fibrosis.