Sialylation Shields Glycoproteins from Oxidative Stress: Mechanistic Insights into Sialic Acid Oxidation and Structural Stability

Sialylation, a crucial yet labile protein modification,
is increasingly
recognized for its role in modulating protein structure, function,
and stability. While the impact of oxidative stress on protein integrity
is well-established, the protective role of sialylation against such
damage remains poorly understood. This study employs a microscale
low-temperature plasma device to generate a controlled, deep radical
oxidation environment mimicking cellular oxidative stress. By subjecting
free sialic acids (Neu5Ac and Neu5Gc) to time-resolved deep radical
exposure, high-resolution mass spectrometry, and high-fidelity density
functional theory calculations, we establish an unprecedented oxidation
pathway, revealing unique stepwise side chain oxidation prior to ring
opening. Comprehensive radical oxidation maps comprising over 100
oxidative intermediates provide a molecular basis for the higher propensity
of Neu5Gc over Neu5Ac in resisting radical oxidation. Further, using
human transferrin as a model glycoprotein, we demonstrate the protective
role of sialylation against oxidative unfolding. Through a combination
of site mapping, enzymatic treatments, and all-ion unfolding ion mobility-mass
spectrometry, we identify specific protein sialylation patterns and
structural motifs that are crucial for maintaining structural stability
under oxidative stress. Our findings provide unprecedented insights
into the intricate interplay between sialylation and oxidative stress,
highlighting the importance of sialylation in stabilizing protein
conformations under various oxidative stresses.