Role of cotranslational disulfide bond formation in the folding of the hemagglutinin-neuraminidase protein of Newcastle disease virus.
The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakman et al. (EMBO J. 11, 1717-1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, the t1/2 for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.
McGinnes
L W
LW
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655, USA.
Morrison
T G
TG
D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D015707:HN Protein / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D009522:Newcastle disease virus / Q000378:metabolism*; D014176:Protein Biosynthesis; D017510:Protein Folding; D011499:Protein Processing, Post-Translational
D000956:Antigens, Viral; D004220:Disulfides; D015707:HN Protein; D004229:Dithiothreitol