Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

Liang Lin, Jianmin Shao, Maomao Sun, Jinxiu Liu, Gongjin Xu, Xumin Zhang, Ningzhi Xu, Rong Wang, Siqi Liu

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

Abstract

After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spot. These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKCα. The two truncated N proteins after incubation of PKCα exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKCα phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKCα phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and partially clarified the argument regarding the phosphorylation possibility of the N protein during the infection process of SARS-CoV to human host.

Original languageEnglish
Pages (from-to)296-303
Number of pages8
JournalInternational Journal of Mass Spectrometry
Volume268
Issue number2-3
DOIs
StatePublished - 1 Dec 2007

Keywords

  • Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry
  • N protein
  • Phosphorylation
  • Severe Acute Respiratory Syndrome (SARS)
  • Two-dimensional electrophoresis (2DE)

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