Glycosylation States on Intact Proteins Determined by NMR Spectroscopy

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B

Reversible O -Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition

-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. -Acetyl migration within the exocyclic glycerol-like side chain of mono- -acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8- -acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9- -acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that -acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the -acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of -acetyl migration is extremely low), reversible -acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9- -acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9- -acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid -acetylation and -acetyl migration to host-microbe interactions and potential application of the more stable synthetic -acetyl mimics