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Mammalian prions (PrPSc) consist of misfolded, conformationally altered, self-replicating states of the sialoglycoprotein called prion protein or PrPC. Recent studies revealed that the sialylation status of PrPSc plays a major role in evading innate immunity and infecting a host. Establishing the type of linkage by which sialic acid residues are attached to galactose is important, as it helps to identify the sialyltransferases responsible for sialylating PrPC and outline strategies for manipulating the sialyation status of PrPSc. Using enzymatic treatment with sialidases and lectin blots, this study demonstrated that in N-linked glycans of PrPSc, the sialic acid residues are predominantly alpha 2,6-linked. High percentages of alpha 2,6-linked sialic acids were observed in PrPSc of three prion strains 22L, RML, and ME7, as well as PrPSc from brain, spleen, or N2a cells cultured in vitro. Moreover, the variation in the percentage of alpha 2,3- versus 2,6-linked sialic acid was found to be relatively minor between brain-, spleen-, or cell-derived PrPSc, suggesting that the type of linkage is independent of tissue type. Based on the current results, we propose that sialyltransferases of St6Gal family, which is responsible for attaching sialic acids via alpha 2,6-linkages to N-linked glycans, controls sialylation of PrPC and PrPSc. [ABSTRACT FROM AUTHOR]

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC) into the disease-associated, transmissible form (PrPSc). PrPC is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrPC and the strain-specific structure of PrPSc. The current work highlights the previously unappreciated role of sialylation of PrPC glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrPSc glycoform ratio. The current study demonstrates that undersialylated PrPC is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrPC. As a result, PMCAb-derived PrPSc was less sialylated than brain-derived PrPSc. A decrease in PrPSc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrPC using sialidase was found to increase the rate of PrPSc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrPC reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrPC was also found to control PrPSc glycoform ratio. A decrease in PrPC sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrPSc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrPC differed from that of spleen-derived PrPC. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrPC, suggesting that Neu1 is not responsible for desialylation of PrPC. The current work highlights previously unappreciated role of PrPC sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches. [ABSTRACT FROM AUTHOR]

A number of proteins associated with neurodegenerative disease undergo several types of posttranslational modifications. They include N-linked glycosylation of the prion protein and amyloid precursor protein, phosphorylation of tau and α-synuclein. Posttranslational modifications alter physical properties of proteins including their net and surface charges, affecting their processing, life-time and propensity to acquire misfolded, disease-associated states. As such, analysis of posttranslational modifications is important for understanding the mechanisms of pathogenesis. Recent studies documented that sialylation of the disease-associated form of the prion protein or PrP Sc controls the fate of prions in an organism and outcomes of prion infection. For assessing sialylation status of PrP Sc , we developed a reliable protocol that involves two-dimensional electrophoresis followed by Western blot (2D). The current chapter describes the procedure for the analysis of sialylation status of PrP Sc from various sources including central nervous system, secondary lymphoid organs, cultured cells, or PrP Sc produced in Protein Misfolding Cyclic Amplification.

Mammalian prions (PrP Sc ) consist of misfolded, conformationally altered, self-replicating states of the sialoglycoprotein called prion protein or PrP C . Recent studies revealed that the sialylation status of PrP Sc plays a major role in evading innate immunity and infecting a host. Establishing the type of linkage by which sialic acid residues are attached to galactose is important, as it helps to identify the sialyltransferases responsible for sialylating PrP C and outline strategies for manipulating the sialyation status of PrP Sc . Using enzymatic treatment with sialidases and lectin blots, this study demonstrated that in N-linked glycans of PrP Sc , the sialic acid residues are predominantly alpha 2,6-linked. High percentages of alpha 2,6-linked sialic acids were observed in PrP Sc of three prion strains 22L, RML, and ME7, as well as PrP Sc from brain, spleen, or N2a cells cultured in vitro. Moreover, the variation in the percentage of alpha 2,3- versus 2,6-linked sialic acid was found to be relatively minor between brain-, spleen-, or cell-derived PrP Sc , suggesting that the type of linkage is independent of tissue type. Based on the current results, we propose that sialyltransferases of St6Gal family, which is responsible for attaching sialic acids via alpha 2,6-linkages to N-linked glycans, controls sialylation of PrP C and PrP Sc ., (© 2017 Federation of European Biochemical Societies.)