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Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells
Author(s) -
Jarvis Donald L.,
OkerBlom Christian,
Summers Max D.
Publication year - 1990
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240420402
Subject(s) - sf9 , glycosylation , glycoprotein , recombinant dna , spodoptera , tunicamycin , secretion , n linked glycosylation , biology , biochemistry , microbiology and biotechnology , gene , glycan , unfolded protein response
Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t‐PA) was expressed, N‐glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214–223, 1989). We also showed that t‐PA secretion was blocked by tunicamycin.(TM), an inhibitor of N‐glycosylation, but not by castanospermine (CS) or N‐methyldeoxynojirimycin, inhibitors of the initial steps in N‐linked oligosaccharide processing. This suggestedthat the addition, but not the processing, of N‐linked oligosaccharides is required for the secretion of recombinant t‐PA from baculovirus‐infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N‐glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane‐bound glycoproteins were expressed in control, TM‐treated, or CS‐treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N‐glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM‐treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM‐treated Sf9 cells by binding to them and producing transport‐incompetent heterooligomeric com‐plexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose‐regulated 78 kilodalton protein (BiP/GRP78).