Variable posttranslational modifications of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), started in 2019 in China and quickly spread into a global pandemic. Nucleocapsid protein (N protein) is highly conserved and is the most abundant protein in coronaviruses and is thus a potential target for both vaccine and point-of-care diagnostics. N Protein has been suggested in the literature as having posttranslational modifications (PTMs), and accurately defining these PTMs is critical for its potential use in medicine. Reports of phosphorylation of N protein have failed to provide detailed site-specific information. We have performed comprehensive glycomics, glycoproteomics and proteomics experiments on two different N protein preparations. Both were expressed in HEK293 cells; one was in-house expressed and purified without a signal peptide (SP) sequence, and the other was commercially produced with a SP channeling it through the secretory pathway. Our results show completely different PTMs on the two N protein preparations. The commercial product contained extensive N- and O-linked glycosylation as well as O-phosphorylation on site Thr393. Conversely, the native N Protein model had O-phosphorylation at Ser176 and no glycosylation, highlighting the importance of knowing the provenance of any commercial protein to be used for scientific or clinical studies. Recent studies have indicated that N protein can serve as an important diagnostic marker for COVID-19 and as a major immunogen by priming protective immune responses. Thus, detailed structural characterization of N protein may provide useful insights for understanding the roles of PTMs on viral pathogenesis, vaccine design and development of point-of-care diagnostics.

Keywords: N protein phosphorylation; N protein site-mapping; SARS-CoV-2 nucleocapsid posttranslational modifications; SARS-CoV-2 phosphoproteomics; glycosylation of SARS-CoV-2 N protein.

Fig. 1

Structural proteins of SARS-CoV-2. (A) Structure of SARS-CoV-2 showing key proteins and structure of N protein. (B) Representation of N protein expression without SP, N(SP-) and with SP, N(SP+). (C) Western blot evaluation of MagneHis™ Protein Purified N(SP-).

Fig. 2

SDS PAGE evaluation of N protein. (A) N protein expressed with SP, N(SP+) (60 kDa). (B) N protein expressed without SP, N(SP-) (47 kDa).

Fig. 3

Domain structure and PTMs of the commercial N protein, produced with SP N(SP+) (top) and the N protein, expressed in-house, without the SP N(SP-) (bottom).

Fig. 4

SARS-CoV-2 N protein sequence coverage and detected PTM sites for N(SP+) and N(SP-).

Fig. 5

Site-specific relative quantification of N- and O-linked glycosylation of N(SP+). (A) Relative percentage of N-glycans at sites N47 (complex-type glycans only) and N269 (mainly high-mannose type glycans, Man5 structure as the most abundant). (B) Relative percentage of O-glycans at sites T148, T165, T166, T205, S206, T391 and S404. Phosphorylation is detected on T393. These sites showed major glycan occupancy ranging from 47–99%. Minor O-glycosylated site distribution is shown in the SI. Minor glycoforms with less than 2% abundance are referred to as “other.”

Fig. 6

PTM analysis of N(SP+) and N(SP-). (A) CID MS2 of N(SP+) phosphorylated O-glycopeptide ³⁸⁸KQQTVTLLPA³⁹⁷ indicating phosphate at Thr393. (B) HCD MS2 of N(SP-) phosphorylated peptide ¹⁷⁰GFYAEGSR¹⁷⁷ showing phosphate at Ser176.