C-Terminus Glycans with Critical Functional Role in the Maturation of Secretory Glycoproteins

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells

FUNCTIONAL FOOD BASED ON A COMBINATION OF POLYPHENOLS AND POLYSACCHARIDES WITH EMERGENT PROPERTIES FOR HYPOCALORIC DIET

The aim of this work was to develop a functional food used as an adjuvant for hypocaloric diet, based on a combination of polyphenols and polysaccharides extracted from berries pomace and chia seeds, presenting emergent properties. These bioactive compounds were encapsulated into liposomes before being added in the composition of a new protein bar. The antioxidant activity of polyphenols- polysaccharides combinations in different ratios was determined by TEAC and DPPH assays. It was observed that the mixture of polyphenols extracted from berries and polysaccharides extracted from chia seeds in a weight ratio of 5:1 had the highest antioxidant activity. A study of α-amylase inhibition in the presence of polyphenols, polysaccharides and their combinations was also performed to investigate the synergism of these compounds. A liposomal formulation of the selected synergistic combination was obtained in powder form by lyophilization. A final product including the synergistic combination was prepared in the form of a hypoglycemic food bar with amplified antioxidant effects. The product is further recommended for potential application as an adjuvant for a low-calorie diet