Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.
The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.
Hegedus
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Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4, Canada.
Pfeifer
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Theilmann
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Kennard
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Gabathuler
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Jefferies
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Grigliatti
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D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D019327:Copper Sulfate / Q000494:pharmacology; D006224:Cricetinae; D004331:Drosophila melanogaster; D005822:Genetic Vectors; D006801:Humans; D008545:Melanoma; D058950:Melanoma-Specific Antigens; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D018411:Spodoptera; D014162:Transfection; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D019327:Copper Sulfate