Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Jan;3(1):30–38. doi: 10.1002/pro.5560030105

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

M Manneberg 1, A Friedlein 1, H Kurth 1, H W Lahm 1, M Fountoulakis 1
PMCID: PMC2142480  PMID: 8142896

Abstract

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Aeed P. A., Guido D. M., Mathews W. R., Elhammer A. P. Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells. Biochemistry. 1992 Aug 4;31(30):6951–6961. doi: 10.1021/bi00145a013. [DOI] [PubMed] [Google Scholar]
  2. Arakawa T., Yphantis D. A., Lary J. W., Narhi L. O., Lu H. S., Prestrelski S. J., Clogston C. L., Zsebo K. M., Mendiaz E. A., Wypych J. Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure. J Biol Chem. 1991 Oct 5;266(28):18942–18948. [PubMed] [Google Scholar]
  3. Baldwin M. A., Stahl N., Reinders L. G., Gibson B. W., Prusiner S. B., Burlingame A. L. Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein. Anal Biochem. 1990 Nov 15;191(1):174–182. doi: 10.1016/0003-2697(90)90405-x. [DOI] [PubMed] [Google Scholar]
  4. Bazan J. F. Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6934–6938. doi: 10.1073/pnas.87.18.6934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Edge C. J., Rademacher T. W., Wormald M. R., Parekh R. B., Butters T. D., Wing D. R., Dwek R. A. Fast sequencing of oligosaccharides: the reagent-array analysis method. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6338–6342. doi: 10.1073/pnas.89.14.6338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fischer T., Thoma B., Scheurich P., Pfizenmaier K. Glycosylation of the human interferon-gamma receptor. N-linked carbohydrates contribute to structural heterogeneity and are required for ligand binding. J Biol Chem. 1990 Jan 25;265(3):1710–1717. [PubMed] [Google Scholar]
  7. Fountoulakis M., Gentz R. Effect of glycosylation on properties of soluble interferon gamma receptors produced in prokaryotic and eukaryotic expression systems. Biotechnology (N Y) 1992 Oct;10(10):1143–1147. doi: 10.1038/nbt1092-1143. [DOI] [PubMed] [Google Scholar]
  8. Fountoulakis M., Juranville J. F., Manneberg M. Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins. J Biochem Biophys Methods. 1992 Jun;24(3-4):265–274. doi: 10.1016/0165-022x(94)90078-7. [DOI] [PubMed] [Google Scholar]
  9. Fountoulakis M., Juranville J. F. Reduced binding capacity of concanavalin A-sepharose after treatment with chaotropic agents. J Biochem Biophys Methods. 1993 Sep;27(2):127–132. doi: 10.1016/0165-022x(93)90056-t. [DOI] [PubMed] [Google Scholar]
  10. Fountoulakis M., Juranville J. F., Stüber D., Weibel E. K., Garotta G. Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli. J Biol Chem. 1990 Aug 5;265(22):13268–13275. [PubMed] [Google Scholar]
  11. Fountoulakis M., Kania M., Ozmen L., Loetscher H. R., Garotta G., van Loon A. P. Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins. J Immunol. 1989 Nov 15;143(10):3266–3276. [PubMed] [Google Scholar]
  12. Fountoulakis M., Schlaeger E. J., Gentz R., Juranville J. F., Manneberg M., Ozmen L., Garotta G. Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells. Eur J Biochem. 1991 Jun 1;198(2):441–450. doi: 10.1111/j.1432-1033.1991.tb16034.x. [DOI] [PubMed] [Google Scholar]
  13. Fountoulakis M., Zulauf M., Lustig A., Garotta G. Stoichiometry of interaction between interferon gamma and its receptor. Eur J Biochem. 1992 Sep 15;208(3):781–787. doi: 10.1111/j.1432-1033.1992.tb17248.x. [DOI] [PubMed] [Google Scholar]
  14. Fu D., van Halbeek H. N-glycosylation site mapping of human serotransferrin by serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Anal Biochem. 1992 Oct;206(1):53–63. doi: 10.1016/s0003-2697(05)80010-7. [DOI] [PubMed] [Google Scholar]
  15. Garotta G., Ozmen L., Fountoulakis M. Development of interferon-gamma antagonists as an example of biotechnology application to approach new immunomodulators. Pharmacol Res. 1989 Nov-Dec;21 (Suppl 2):5–17. doi: 10.1016/s1043-6618(89)80013-1. [DOI] [PubMed] [Google Scholar]
  16. Gentz R., Hayes A., Grau N., Fountoulakis M., Lahm H. W., Ozmen L., Garotta G. Analysis of soluble human and mouse interferon-gamma receptors expressed in eukaryotic cells. Eur J Biochem. 1992 Dec 1;210(2):545–554. doi: 10.1111/j.1432-1033.1992.tb17453.x. [DOI] [PubMed] [Google Scholar]
  17. Goochee C. F., Gramer M. J., Andersen D. C., Bahr J. B., Rasmussen J. R. The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Biotechnology (N Y) 1991 Dec;9(12):1347–1355. doi: 10.1038/nbt1291-1347. [DOI] [PubMed] [Google Scholar]
  18. Grabenhorst E., Hofer B., Nimtz M., Jäger V., Conradt H. S. Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications. Eur J Biochem. 1993 Jul 1;215(1):189–197. doi: 10.1111/j.1432-1033.1993.tb18022.x. [DOI] [PubMed] [Google Scholar]
  19. Hardy M. R., Townsend R. R. Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection. Proc Natl Acad Sci U S A. 1988 May;85(10):3289–3293. doi: 10.1073/pnas.85.10.3289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  21. Hotchkiss A., Refino C. J., Leonard C. K., O'Connor J. V., Crowley C., McCabe J., Tate K., Nakamura G., Powers D., Levinson A. The influence of carbohydrate structure on the clearance of recombinant tissue-type plasminogen activator. Thromb Haemost. 1988 Oct 31;60(2):255–261. [PubMed] [Google Scholar]
  22. Knepper T. P., Arbogast B., Schreurs J., Deinzer M. L. Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry. Biochemistry. 1992 Nov 24;31(46):11651–11659. doi: 10.1021/bi00161a053. [DOI] [PubMed] [Google Scholar]
  23. Kobata A. Structures and functions of the sugar chains of glycoproteins. Eur J Biochem. 1992 Oct 15;209(2):483–501. doi: 10.1111/j.1432-1033.1992.tb17313.x. [DOI] [PubMed] [Google Scholar]
  24. Kuroda K., Geyer H., Geyer R., Doerfler W., Klenk H. D. The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology. 1990 Feb;174(2):418–429. doi: 10.1016/0042-6822(90)90095-9. [DOI] [PubMed] [Google Scholar]
  25. Lee Y. C. High-performance anion-exchange chromatography for carbohydrate analysis. Anal Biochem. 1990 Sep;189(2):151–162. doi: 10.1016/0003-2697(90)90099-u. [DOI] [PubMed] [Google Scholar]
  26. Narhi L. O., Arakawa T., Aoki K. H., Elmore R., Rohde M. F., Boone T., Strickland T. W. The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem. 1991 Dec 5;266(34):23022–23026. [PubMed] [Google Scholar]
  27. Ozmen L., Gribaudo G., Fountoulakis M., Gentz R., Landolfo S., Garotta G. Mouse soluble IFN gamma receptor as IFN gamma inhibitor. Distribution, antigenicity, and activity after injection in mice. J Immunol. 1993 Apr 1;150(7):2698–2705. [PubMed] [Google Scholar]
  28. Paulson J. C. Glycoproteins: what are the sugar chains for? Trends Biochem Sci. 1989 Jul;14(7):272–276. doi: 10.1016/0968-0004(89)90062-5. [DOI] [PubMed] [Google Scholar]
  29. Sharon N., Lis H. Carbohydrates in cell recognition. Sci Am. 1993 Jan;268(1):82–89. doi: 10.1038/scientificamerican0193-82. [DOI] [PubMed] [Google Scholar]
  30. Stüber D., Friedlein A., Fountoulakis M., Lahm H. W., Garotta G. Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity. Biochemistry. 1993 Mar 9;32(9):2423–2430. doi: 10.1021/bi00060a038. [DOI] [PubMed] [Google Scholar]
  31. Takeuchi M., Takasaki S., Shimada M., Kobata A. Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J Biol Chem. 1990 Jul 25;265(21):12127–12130. [PubMed] [Google Scholar]
  32. Townsend R. R., Hardy M. R., Hindsgaul O., Lee Y. C. High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection. Anal Biochem. 1988 Nov 1;174(2):459–470. doi: 10.1016/0003-2697(88)90044-9. [DOI] [PubMed] [Google Scholar]
  33. Velardo M. A., Bretthauer R. K., Boutaud A., Reinhold B., Reinhold V. N., Castellino F. J. The presence of UDP-N-acetylglucosamine:alpha-3-D-mannoside beta 1,2-N-acetylglucosaminyltransferase I activity in Spodoptera frugiperda cells (IPLB-SF-21AE) and its enhancement as a result of baculovirus infection. J Biol Chem. 1993 Aug 25;268(24):17902–17907. [PubMed] [Google Scholar]
  34. van Loon A. P., Ozmen L., Fountoulakis M., Kania M., Haiker M., Garotta G. High-affinity receptor for interferon-gamma (IFN-gamma), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-gamma receptor protein. J Leukoc Biol. 1991 May;49(5):462–473. doi: 10.1002/jlb.49.5.462. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES