Peptide‐<i>N</i>⁴‐(<i>N</i>‐acetyl‐β‐glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1 → 3 to the asparagine‐linked <i>N</i>‐acetylglucosamine residue

TLDR

The authors compared PNGase F from *Flavobacterium meningosepticum* with PNGase A from sweet almonds for deglycosylating plant N‑glycopeptides and N‑glycoproteins, using bromelain glycopeptide and horseradish peroxidase‑C glycoprotein substrates that bear xylose β1‑2‑linked to β‑mannose and fucose α1‑3‑linked to the innermost N‑acetylglucosamine. PNGase F from *F.

Abstract

The ability of peptide‐ N 4 ‐( N ‐acetyl‐β‐glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N‐glycopeptides and N‐glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase‐C glycoprotein, which contain xylose linked β1 → 2 to β‐mannose and fucose linked α1 → 3 to the innermost N ‐acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100‐fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of α1 → 3‐linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that α1 → 3 fucosylation of the inner N ‐acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.