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

Abstract

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 native conditions, with similar performance to that of the soluble enzyme. Successful deglycosylation was evidenced by a decrease 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.

Spontaneous and site-specific immobilization of PNGase F via spy chemistry

“…16 To efficiently immobilize PNGase F, affinity interaction of the special tag coexpressed from fused protein and reactive groups modified on the surface of solid materials, including the hexahistidine – nickel, 9 GST – GSH, 17 and CBM – cellulose 18 is the traditional way to immobilize PNGase F. However, the affinity interaction is derived from noncovalent forces such as hydrogen bonds, van der Waals forces, electrostatic bonds, and hydrophobic interactions, and the partial interactions may dissociate in the harsh deglycosylation environment. To construct more stable PNGase F conjugates, covalent linkages were reacted on functional groups from protein with modified reactive groups on the surface of solid phases, including amine, 19 carboxyl, 20,21 epoxy, 22 cyanate ester, 23 vinyl azlactone, 24 and thiol–ene groups. 25 Although the covalent immobilization of PNGase F can be achieved by the above reaction, the protein active domain and biofunction may be compromised and even inactivated after immobilization by these nonspecific reactions.…”