Immobilized peptide-N-glycosidase F onto magnetic nanoparticles: A biotechnological tool for protein deglycosylation under native conditions

The elucidation of glycans biological function is essential to
understand their role in biological processes, both normal and
pathological. Immobilized glycoenzymes are excellent tools for this purpose as they can selectively release glycans from glycoproteins without altering their backbone. They can be easily removed from the reaction mixture avoiding their
interference in subsequent experiments. Here, we describe the
immobilization of peptide-N-glycosidase F (PNGase F) onto
silica magnetic nanoparticles with immobilization yields of
86% and activity yields of 12%. Immobilized PNGase F showed
higher thermal stability than its soluble counterpart, and could
be reused for at least seven deglycosylation cycles. It was
efficient in the deglycosylation of several glycoproteins (ribonuclease B, bovine fetuin, and ovalbumin) and a protein lysate from the parasite Fasciola hepatica under nativeconditions, with similar performance to that of the solubleenzyme. Successful deglycosylation was evidenced by adecrease in specific lectin recognition of the glycoproteins (40%–80%). Moreover, deglycosylated F. hepatica lysate allowed us to confirm the role of parasite N-glycans in the inhibition of the lipopolysaccharide-induced maturation of dendritic cells. Immobilized PNGase F probed to be a robust biotechnological tool for deglycosylation of glycoproteins and complex biological samples under native conditions.

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