Keating Celina L, Kuhn Eric, Bals Julia, Cocco Alexandra R, Yousif Ashraf S, Matysiak Colette, Sangesland Maya, Ronsard Larance, Smoot Matthew, Moreno Thalia Bracamonte, Okonkwo Vintus, Setliff Ian, Georgiev Ivelin, Balazs Alejandro B, Carr Steven A, Lingwood Daniel
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.
The Broad Institute of The Massachusetts Institute of Technology and Harvard University , 415 Main Street , Cambridge , Massachusetts 02142 , United States of America.
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 -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.
在细胞中，天冬酰胺/N-连接聚糖在新生多肽共翻译过程中添加到糖蛋白上，这一附着过程有助于新生多肽的正确折叠。我们发现，在通过酰胺酶PNGase F修剪N-聚糖后，当酰胺酶从溶液中去除时，主要流感疫苗抗原和主要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-糖基化位点。通过在PNGase F处理之前、处理期间以及酰胺酶去除后对HA糖型进行质谱分析的位点特异性分析，证实了我们称之为N-糖基化的这一反应。去N-糖基化后再进行N-糖基化的迭代轮次至少可以重复三次，并且在其他病毒糖蛋白/疫苗抗原中也观察到了这种现象，包括来自HIV的包膜糖蛋白（Env）。共价N-聚糖重新附着是非酶促的，因为它发生在抑制PNGase F活性的金属离子存在的情况下。相反，N-糖基化依赖于蛋白质和聚糖之间的非共价组装，这种组装在酰胺酶存在的情况下形成，其中糖蛋白的线性化阻止了这种保留和随后的N-糖基化。这一反应表明，在某些实验条件下，一些糖蛋白可以组织自身的聚糖添加，突出了一种显著的自组装原理，这可能被证明对重新设计治疗性糖蛋白（如流感HA或HIV Env）很有用，其中聚糖序列和结构可以显著影响生物活性和疫苗效力。
