Peptide N-Glycosidase A (PNGase A)
Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase, Glycopeptidase from almonds, almond glycoamidase.
PNGase A from Almond cleaves N-Glycan chains linked to Asparagine from glycopeptides.
The enzyme hydrolysis an N(4)-(acetyl-beta-D-glucosaminyl) asparagine residue in which the glucosamine residue may be further glycosylated, to yield a (substituted) N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue.
The enzyme cleaves glycopeptides with or without alpha1,3-linked core fucose residues present in insect and plant glycoproteins.
N-glycosidase A requires glycopeptides as substrates. Glycopeptides can be obtained by digesting the glycoprotein with Trypsin as described by Kuester et al. (1997) or pepsin.
Does not act on (GlcNAc)Asn, because it requires the presence of more than two amino-acid residues in the substrate.
How to use PNGase A
PNGase A – in contrast to PNGase F - is able to cleave Asn-linked oligosaccharides (N-glycans) also if they contain fucose in α1,3-linkage to the innermost GlcNAc. It can, however, NOT act on glycoproteins, even if they have been denatured .
Therefore, glycoproteins must be proteolytically degraded before application of PNGase A
- Option I: PNGase A digestion of tryptic peptides. This is the safest procedure suitable also for sialylated glycans. Requires protein denaturation (e.g. by reducing SDS-PAGE or even S-alkylation).
- Option II: PNGase A digestion of peptidic peptides. Much simpler but may cause loss of acid labile substituents (sialic acids).
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 Tretter V, Altmann F, März L. (1991) Eur J Biochem. 199, 647-52.