Viral infection: II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells

Debabrata Saha, Shiyong Wu, Avirup Bose, Nabendu Chatterjee, Arup Chakraborty, Madhumita Chatterjee, Naba K. Gupta

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia vital infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of heroin to the growth medium. Maximum enhancement was observed at 5 μM heroin. Data suggest that heroin prevents the activation of latent p67- DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus- infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells). In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells. Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin- deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells. We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG.

Original languageEnglish (US)
Pages (from-to)373-382
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume342
Issue number2
DOIs
StatePublished - Jun 15 1997

Keywords

  • Baculovirus expression system
  • Deglycosylation
  • Glycoproteins
  • Heroin
  • p67
  • p67- DG

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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