Glycosylation is among the most abundant of all post-translational modifications (PTM) (1). A highly complex PTM, glycosylation has diverse roles that affect both protein function in cellular processes as well as disease manifestation (2). Several large glycoproteomics studies published recently illustrate how PNGase F can be used in combination with mass spectrometry to identify hundreds to thousands of N-linked glycosylation sites in complex biological samples (2).
Furthermore, glycosylation has been shown to dramatically affect properties of biotherapeutics. For example, the pharmacokinetics and potency of recombinant erythropoietin are critically affected by its glycosylation status, while the ability of monoclonal antibodies to mediate efficacy via ADCC is affected by the fucose content of the Fc region glycan (3). Clearly, with glycosylation having such prominent roles in biology, disease progression and therapeutic protein function, as well as serving as a biomarker in diseases like cancer, tools that facilitate glycoprotein characterization are extremely important.
One such tool is the glycosidase PNGase F, which is used extensively for studying glycoproteins. First isolated from Elizabethkingia miricola (formerly Flavobacterium meningosepticum), PNGase F catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins (4). Despite being active against most N-linked glycans, PNGase F cannot cleave when the innermost GlcNAc is linked to an α-1,3-fucose, most commonly found in plant glycoproteins and in some insect glycoproteins (5). During glycan removal, the modified asparagine residue is converted to aspartic acid, which imparts a +1Da mass change that can be detected by mass-spectrometry workflows utilizing trypsin or alternative proteases.
These PNGase F properties make it a valuable scientific tool for use in conjunction with downstream analytical techniques, both to identify specific Asn residues that undergo N-linked glycosylation as well as to characterize the chemical structure(s) of the released oligosaccharide chains.