Protein glycosylation in extracellular vesicles: Structural characterization and biological functions.

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Mutations of Cys and Ser residues in the α5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan.

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.