Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion.
The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.
Allen
S
S
University of Manchester, School of Biological Sciences, United Kingdom.
Naim
H Y
HY
Bulleid
N J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D017510:Protein Folding; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D010959:Tissue Plasminogen Activator; D004229:Dithiothreitol