Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens.
Spontaneous glycan reattachment following N-glycanase treatment of influenza and HIV vaccine antigens.
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DOI：
10.1021/acs.jproteome.9b00620
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发表时间：
2020-01
影响因子：
4.4
通讯作者：
Celina Keating;E. Kuhn;Julia Bals;A. Cocco;A. Yousif;C. Matysiak;Maya Sangesland;Larance Ronsard;Matthew Smoot;Thalia Bracamonte Moreno;Vintus Okonkwo;Ian Setliff;I. Georgiev;A. Balazs;S. Carr;D. Lingwood
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作者：
Celina Keating;E. Kuhn;Julia Bals;A. Cocco;A. Yousif;C. Matysiak;Maya Sangesland;Larance Ronsard;Matthew Smoot;Thalia Bracamonte Moreno;Vintus Okonkwo;Ian Setliff;I. Georgiev;A. Balazs;S. Carr;D. Lingwood
在细胞中，天冬酰胺/ n -链聚糖被共翻译地添加到糖蛋白上，这一附着过程被认为是由新生多肽序列的折叠所支持的。我们发现，在氨基酶PNGase F对n -聚糖进行剪枝后，当氨基酶从溶液中移除时，主要的流感疫苗抗原和主要的病毒刺突蛋白血凝素（HA）会自发地将n -聚糖重新附着到其去n -糖基化的位置。这个反应，我们称之为n -糖基化，在暴露于PNGase F之前，暴露于PNGase F期间，以及在酰胺酶去除后，通过质谱法对HA糖型进行了位点特异性分析。在其他病毒糖蛋白/疫苗抗原（包括HIV的包膜糖蛋白（Env））中，去n -糖基化和n -糖基化的反复循环可以重复至少3次。共价n-聚糖的再附着是非酶促的，因为它发生在金属离子的存在下，抑制PNGase F的活性。相反，n-糖基化依赖于蛋白质和聚糖之间的非共价组装，在酰胺酶存在下形成，糖蛋白的线性化防止了这种保留和随后的n-糖基化。这一反应表明，在一定的实验条件下，一些糖蛋白可以组织自身的聚糖添加，并突出了一种显著的自组装原理，这可能有助于重组治疗性糖蛋白，如流感HA或HIV Env，其中聚糖的序列和结构可以显着影响生物活性和疫苗功效。
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.