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

Autor:	Keating CL; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Kuhn E; The Broad Institute of The Massachusetts Institute of Technology and Harvard University , 415 Main Street , Cambridge , Massachusetts 02142 , United States of America., Bals J; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Cocco AR; The Broad Institute of The Massachusetts Institute of Technology and Harvard University , 415 Main Street , Cambridge , Massachusetts 02142 , United States of America., Yousif AS; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Matysiak C; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Sangesland M; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Ronsard L; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Smoot M; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Moreno TB; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Okonkwo V; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Setliff I; Program in Chemical & Physical Biology , Vanderbilt University Medical Center , 340 Light Hall , Nashville 37232-0301 , United States of America.; Vanderbilt Vaccine Center , Vanderbilt University , 2213 Garland Avenue , Nashville , Tennessee 37232-0417 , United States of America., Georgiev I; Program in Chemical & Physical Biology , Vanderbilt University Medical Center , 340 Light Hall , Nashville 37232-0301 , United States of America.; Vanderbilt Vaccine Center , Vanderbilt University , 2213 Garland Avenue , Nashville , Tennessee 37232-0417 , United States of America.; Department of Pathology, Microbiology, and Immunology , Vanderbilt University Medical Center , C-3322 Medical Center North , Nashville , Tennessee 37232-2561 , United States of America.; Department of Electrical Engineering and Computer Science , Vanderbilt University , 2301 Vanderbilt Place , Nashville , Tennessee 37235-1826 , United States of America., Balazs AB; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America., Carr SA; The Broad Institute of The Massachusetts Institute of Technology and Harvard University , 415 Main Street , Cambridge , Massachusetts 02142 , United States of America., Lingwood D; The Ragon Institute of Massachusetts General Hospital , The Massachusetts Institute of Technology and Harvard University , 400 Technology Square , Cambridge , Massachusetts 02139 , United States of America.
Jazyk:	angličtina
Zdroj:	Journal of proteome research [J Proteome Res] 2020 Feb 07; Vol. 19 (2), pp. 733-743. Date of Electronic Publication: 2020 Jan 24.
DOI:	10.1021/acs.jproteome.9b00620
Abstrakt:	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.
Databáze:	MEDLINE
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