SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans. (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2. (preprint)

Emergence of COVID-19 pandemic caused by SARS-CoV-2 demanded development of new therapeutic strategies and thus the understanding the mode of viral attachment, entry and replication has become key aspect for such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential in viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to the human angiotensin converting enzyme 2 (hACE2) for the entry and the serine protease TMPRSS2 for S protein priming. The heavily glycosylated S protein is comprised of two protein subunits (S1 and S2), and the receptor binding domain within S1 subunit binds with to the hACE2 receptor. Even though hACE2 has been known for two decades and has been recognized as the entry point of several human coronaviruses, no comprehensive glycosylation characterization of hACE2 has been reported. Herein, we describe the quantitative glycosylation mapping on hACE2 expressed in human cells by both glycoproteomics and glycomics. We observed heavy glycan occupancy at all the seven possible N-glycosylation sites and surprisingly, detected three novel O-glycosylation sites. In order to deduce the detailed structure of glycan epitopes on hACE2 involved with viral binding, we have characterized the terminal sialic acid linkages, presence of bisecting GlcNAc and also the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectra in addition to the use of bioinformatics tools to validate the hACE2 glycosylation. Elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor can aid in understanding the intriguing virus-receptor interactions and help in the development of novel therapeutics to circumvent the viral entry.

Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2 (preprint)

The current emergence of the novel coronavirus pandemic caused by SARS-CoV-2 demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor binding domain (RBD) of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation is strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research towards the development of a suitable vaccine candidate.