Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications

Abstract

The devastating coronavirus disease 2019 (COVID-19) pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its viral components. Compared to the Spike protein, which is the primary target for currently available vaccines or antibodies, knowledge about other virion structural components is incomplete. Using high-resolution mass spectrometry, we report a comprehensive post-translational modification (PTM) analysis of nucleocapsid phosphoprotein (NCP), the most abundant structural component of the SARS-CoV-2 virion. In addition to phosphoryl groups, we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs, including N-glycans and ubiquitin. Based on newly identified PTMs, refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs. These data can facilitate the design of novel vaccines or therapeutics targeting NCP, as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

Keywords: SARS-CoV-2; glycosylation; mass spectrometry; nucleocapsid protein; post-translational modification.

Figure 1

Overall workflow for comprehensive PTM survey of SARS-CoV-2 NCP using multi-enzyme digestion and enrichment techniques. DTT: dithiothreitol; FASP: filter aided sample preparation; HILIC: hydrophilic interaction liquid chromatography; IAA: iodacetamide; NCP: nucleocapsid phosphoprotein; PTM: post-translational modification; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SIMAC: sequential elution from IMAC (immobilized metal ion affinity based chromatography).

Figure 2

Summary of PTMs in SARS-CoV-2 NCP. A: Distribution of PTMs identified in this study related to functional subunits and domains of SARS-CoV-2 NCP. PTM notation can be found in right lower legend. Predominant N-glycan forms were directly marked on both glycosylation sites. B: Venn diagram summary of SARS-CoV-2 NCP phosphorylation sites identified in our and previous studies. C: Lysates from SARS-CoV-2 NCP expressed 293T cells were subjected to N and IgG pull-down followed by immunoblotting analyses. CTD: C terminal domain; NCP: nucleocapsid phosphoprotein; NTD: N terminal domain; PTM: post-translational modification; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Figure 3

SARS-CoV-2 NCP decorated with glycans. A: Relative LC-MS intensity of four major N-glycan categories on both sites. B: The most likely glycan structure on site N77 was added to the NTD RNA-binding domain SARS-CoV-2 NCP based on NTD binding model with dsRNA (Protein Data Bank ID: 7ACS, sequence 44-180). C: The most likely glycan structure on site N269 was added to the CTD dimerization domain SARS-CoV-2 NCP based on model of four CTD subunits polymerized (Protein Data Bank ID: 6WZQ, sequence 247-364), with each subunit colored differently. All NCP sequences were represented in cartoon mode while glycans were represented in sphere mode. CTD: C terminal domain; dsRNA: double-stranded RNA; LC-MS: liquid chromatography with mass spectrometry; NCP: nucleocapsid phosphoprotein; NTD: N terminal domain; PTM: post-translational modification; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.