Viral infection. II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells.
The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia viral 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 hemin to the growth medium. Maximum enhancement was observed at 5 microM hemin. Data suggest that hemin 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.
Saha
D
D
Department of Chemistry, University of Nebraska, Lincoln 68588, USA.
Wu
S
S
Bose
A
A
Chatterjee
N
N
Chakraborty
A
A
Chatterjee
M
M
Gupta
N K
NK
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006427:Hemin / Q000494:pharmacology*; D017924:Nucleopolyhedroviruses / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D013696:Temperature; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000502:physiology
D006023:Glycoproteins; D011994:Recombinant Proteins; D006427:Hemin; C106539:p67 deglycosylase; D000118:Acetylglucosaminidase