Spontaneous Glycan Reattachment Following N-Glycanase Treatment of Influenza and HIV Vaccine Antigens

J Proteome Res. 2020 Feb 7;19(2):733-743. doi: 10.1021/acs.jproteome.9b00620. Epub 2020 Jan 24.


In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. We found that following pruning of N-glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached N-glycan to its de-N-glycosylated positions when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation could be repeated at least three times and were observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was nonenzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a noncovalent assembly between protein and glycan, formed in the presence of the amidase, where linearization of the glycoprotein prevented this retention and subsequent N-glycanation. This reaction suggests that under certain experimental conditions, some glycoproteins can organize self-glycan addition, highlighting a remarkable self-assembly principle that may prove useful for re-engineering therapeutic glycoproteins such as influenza HA or HIV Env, where glycan sequence and structure can markedly affect bioactivity and vaccine efficacy.

Keywords: N-glycan; N-glycanation; NXS/T sequon; amidase; deglycosylation; glycoprotein; reattachment; self-organizing; spontaneous; test tube.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AIDS Vaccines*
  • HIV Antigens
  • Humans
  • Influenza Vaccines*
  • Influenza, Human*
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Polysaccharides


  • AIDS Vaccines
  • HIV Antigens
  • Influenza Vaccines
  • Polysaccharides
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase