tetano
Editor, Senior Moderator
J Proteome Res. 2020 Jan 8. doi: 10.1021/acs.jproteome.9b00620. [Epub ahead of print] [h=1]Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens.[/h]
Keating CL, Kuhn E, Bals J, Cocco AR, Yousif AS, Matysiak C, Sangesland M, Ronsard L, Smoot M, Barcamonte Moreno T, Okonkwo V, Setliff I, Georgiev I, Balazs AB, Carr SA, Lingwood D.
[h=3]Abstract[/h] In cells asparagine/N-linked glycans are added to glycoproteins co-translationally, in an attachment process that is thought to be supported by the folding of the nascent polypeptide sequence. We find that following pruning of N-glycan by the amidase PNGase F, the principle influenza vaccine antigen and major viral spike protein hemagglutinin (HA), spontaneously re-attached 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 3 times, and was observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was non-enzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a non-covalent 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 and highlights 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.
PMID: 31913636 DOI: 10.1021/acs.jproteome.9b00620
Keating CL, Kuhn E, Bals J, Cocco AR, Yousif AS, Matysiak C, Sangesland M, Ronsard L, Smoot M, Barcamonte Moreno T, Okonkwo V, Setliff I, Georgiev I, Balazs AB, Carr SA, Lingwood D.
[h=3]Abstract[/h] In cells asparagine/N-linked glycans are added to glycoproteins co-translationally, in an attachment process that is thought to be supported by the folding of the nascent polypeptide sequence. We find that following pruning of N-glycan by the amidase PNGase F, the principle influenza vaccine antigen and major viral spike protein hemagglutinin (HA), spontaneously re-attached 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 3 times, and was observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was non-enzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a non-covalent 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 and highlights 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.
PMID: 31913636 DOI: 10.1021/acs.jproteome.9b00620