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. 1988 Oct;8(10):4063–4070. doi: 10.1128/mcb.8.10.4063

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells.

A J Dorner 1, M G Krane 1, R J Kaufman 1
PMCID: PMC365475  PMID: 2460739

Abstract

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonthron D. T., Handin R. I., Kaufman R. J., Wasley L. C., Orr E. C., Mitsock L. M., Ewenstein B., Loscalzo J., Ginsburg D., Orkin S. H. Structure of pre-pro-von Willebrand factor and its expression in heterologous cells. Nature. 1986 Nov 20;324(6094):270–273. doi: 10.1038/324270a0. [DOI] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Dorner A. J., Bole D. G., Kaufman R. J. The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins. J Cell Biol. 1987 Dec;105(6 Pt 1):2665–2674. doi: 10.1083/jcb.105.6.2665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  6. Gibson R., Kornfeld S., Schlesinger S. The effect of oligosaccharide chains of different sizes on the maturation and physical properties of the G protein of vesicular stomatitis virus. J Biol Chem. 1981 Jan 10;256(1):456–462. [PubMed] [Google Scholar]
  7. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hendershot L., Bole D., Köhler G., Kearney J. F. Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein. J Cell Biol. 1987 Mar;104(3):761–767. doi: 10.1083/jcb.104.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hunt C., Morimoto R. I. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6455–6459. doi: 10.1073/pnas.82.19.6455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaufman R. J., Murtha P., Ingolia D. E., Yeung C. Y., Kellems R. E. Selection and amplification of heterologous genes encoding adenosine deaminase in mammalian cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3136–3140. doi: 10.1073/pnas.83.10.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaufman R. J., Wasley L. C., Spiliotes A. J., Gossels S. D., Latt S. A., Larsen G. R., Kay R. M. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol. 1985 Jul;5(7):1750–1759. doi: 10.1128/mcb.5.7.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kim S. K., Wold B. J. Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA. Cell. 1985 Aug;42(1):129–138. doi: 10.1016/s0092-8674(85)80108-2. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Leavitt R., Schlesinger S., Kornfeld S. Impaired intracellular migration and altered solubility of nonglycosylated glycoproteins of vesicular stomatitis virus and Sindbis virus. J Biol Chem. 1977 Dec 25;252(24):9018–9023. [PubMed] [Google Scholar]
  15. Lee A. S. The accumulation of three specific proteins related to glucose-regulated proteins in a temperature-sensitive hamster mutant cell line K12. J Cell Physiol. 1981 Jan;106(1):119–125. doi: 10.1002/jcp.1041060113. [DOI] [PubMed] [Google Scholar]
  16. Lodish H. F., Kong N., Snider M., Strous G. J. Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature. 1983 Jul 7;304(5921):80–83. doi: 10.1038/304080a0. [DOI] [PubMed] [Google Scholar]
  17. Mazzarella R. A., Green M. ERp99, an abundant, conserved glycoprotein of the endoplasmic reticulum, is homologous to the 90-kDa heat shock protein (hsp90) and the 94-kDa glucose regulated protein (GRP94). J Biol Chem. 1987 Jun 25;262(18):8875–8883. [PubMed] [Google Scholar]
  18. McGarry T. J., Lindquist S. Inhibition of heat shock protein synthesis by heat-inducible antisense RNA. Proc Natl Acad Sci U S A. 1986 Jan;83(2):399–403. doi: 10.1073/pnas.83.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Munro S., Pelham H. R. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. doi: 10.1016/0092-8674(86)90746-4. [DOI] [PubMed] [Google Scholar]
  20. Olden K., Pratt R. M., Jaworski C., Yamada K. M. Evidence for role of glycoprotein carbohydrates in membrane transport: specific inhibition by tunicamycin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):791–795. doi: 10.1073/pnas.76.2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pelham H. R. Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell. 1986 Sep 26;46(7):959–961. doi: 10.1016/0092-8674(86)90693-8. [DOI] [PubMed] [Google Scholar]
  22. Pennica D., Holmes W. E., Kohr W. J., Harkins R. N., Vehar G. A., Ward C. A., Bennett W. F., Yelverton E., Seeburg P. H., Heyneker H. L. Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature. 1983 Jan 20;301(5897):214–221. doi: 10.1038/301214a0. [DOI] [PubMed] [Google Scholar]
  23. Pohl G., Källström M., Bergsdorf N., Wallén P., Jörnvall H. Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences. Biochemistry. 1984 Jul 31;23(16):3701–3707. doi: 10.1021/bi00311a020. [DOI] [PubMed] [Google Scholar]
  24. Pollok B. A., Anker R., Eldridge P., Hendershot L., Levitt D. Molecular basis of the cell-surface expression of immunoglobulin mu chain without light chain in human B lymphocytes. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9199–9203. doi: 10.1073/pnas.84.24.9199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sharma S., Rodgers L., Brandsma J., Gething M. J., Sambrook J. SV40 T antigen and the exocytotic pathway. EMBO J. 1985 Jun;4(6):1479–1489. doi: 10.1002/j.1460-2075.1985.tb03806.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shiu R. P., Pouyssegur J., Pastan I. Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3840–3844. doi: 10.1073/pnas.74.9.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sorger P. K., Pelham H. R. The glucose-regulated protein grp94 is related to heat shock protein hsp90. J Mol Biol. 1987 Mar 20;194(2):341–344. doi: 10.1016/0022-2836(87)90380-9. [DOI] [PubMed] [Google Scholar]
  28. Ting J., Wooden S. K., Kriz R., Kelleher K., Kaufman R. J., Lee A. S. The nucleotide sequence encoding the hamster 78-kDa glucose-regulated protein (GRP78) and its conservation between hamster and rat. Gene. 1987;55(1):147–152. doi: 10.1016/0378-1119(87)90258-7. [DOI] [PubMed] [Google Scholar]
  29. To R. Y., Booth S. C., Neiman P. E. Inhibition of retroviral replication by anti-sense RNA. Mol Cell Biol. 1986 Dec;6(12):4758–4762. doi: 10.1128/mcb.6.12.4758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Urlaub G., Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]

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