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


Keating CL , Kuhn E , Bals J , Cocco AR , Yousif AS , Matysiak C , Sangesland M , Ronsard L , Smoot M , Moreno TB , Okonkwo V , Setliff I , Georgiev I , Balazs AB , Carr SA , Lingwood D , . Journal of proteome research. 2020 01 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 -glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached -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 -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.