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. 1989 Dec;86(23):9188–9192. doi: 10.1073/pnas.86.23.9188

Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.

R König 1, G Ashwell 1, J A Hanover 1
PMCID: PMC298459  PMID: 2687874

Abstract

CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.

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Selected References

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

  1. Ahrens P. B., Ankel H. The role of asparagine-linked carbohydrate in natural killer cell-mediated cytolysis. J Biol Chem. 1987 Jun 5;262(16):7575–7579. [PubMed] [Google Scholar]
  2. Akiyama S., Fojo A., Hanover J. A., Pastan I., Gottesman M. M. Isolation and genetic characterization of human KB cell lines resistant to multiple drugs. Somat Cell Mol Genet. 1985 Mar;11(2):117–126. doi: 10.1007/BF01534700. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Chen C., Bonifacino J. S., Yuan L. C., Klausner R. D. Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J Cell Biol. 1988 Dec;107(6 Pt 1):2149–2161. doi: 10.1083/jcb.107.6.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  6. Herrmann B. G., Frischauf A. M. Isolation of genomic DNA. Methods Enzymol. 1987;152:180–183. doi: 10.1016/0076-6879(87)52018-3. [DOI] [PubMed] [Google Scholar]
  7. Kagawa Y., Takasaki S., Utsumi J., Hosoi K., Shimizu H., Kochibe N., Kobata A. Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J Biol Chem. 1988 Nov 25;263(33):17508–17515. [PubMed] [Google Scholar]
  8. Kane S. E., Troen B. R., Gal S., Ueda K., Pastan I., Gottesman M. M. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease. Mol Cell Biol. 1988 Aug;8(8):3316–3321. doi: 10.1128/mcb.8.8.3316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kirschmeier P. T., Housey G. M., Johnson M. D., Perkins A. S., Weinstein I. B. Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences. DNA. 1988 Apr;7(3):219–225. doi: 10.1089/dna.1988.7.219. [DOI] [PubMed] [Google Scholar]
  10. Kozutsumi Y., Segal M., Normington K., Gething M. J., Sambrook J. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature. 1988 Mar 31;332(6163):462–464. doi: 10.1038/332462a0. [DOI] [PubMed] [Google Scholar]
  11. König R., Ashwell G., Hanover J. A. Glycosylation of CD4. Tunicamycin inhibits surface expression. J Biol Chem. 1988 Jul 5;263(19):9502–9507. [PubMed] [Google Scholar]
  12. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  13. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  14. Stanley P. Selection of lectin-resistant mutants of animal cells. Methods Enzymol. 1983;96:157–184. doi: 10.1016/s0076-6879(83)96015-9. [DOI] [PubMed] [Google Scholar]
  15. Takeuchi M., Takasaki S., Miyazaki H., Kato T., Hoshi S., Kochibe N., Kobata A. Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 1988 Mar 15;263(8):3657–3663. [PubMed] [Google Scholar]
  16. Ueda K., Cardarelli C., Gottesman M. M., Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A. 1987 May;84(9):3004–3008. doi: 10.1073/pnas.84.9.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wei L., Gaillard I., Corvol P., Clauser E. Expression and characterization of recombinant human angiotensinogen in a heterologous eukaryotic cell line. Biochem Biophys Res Commun. 1988 Nov 15;156(3):1103–1110. doi: 10.1016/s0006-291x(88)80746-0. [DOI] [PubMed] [Google Scholar]

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