Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Jun 1;276(Pt 2):511–518. doi: 10.1042/bj2760511

Natural human interferon-alpha 2 is O-glycosylated.

G R Adolf 1, I Kalsner 1, H Ahorn 1, I Maurer-Fogy 1, K Cantell 1
PMCID: PMC1151121  PMID: 2049076

Abstract

Natural human interferon alpha 2 (IFN-alpha 2) was isolated from a preparation of partially purified human leucocyte IFN by monoclonal-antibody immunoaffinity chromatography. The purified protein had a specific activity of 1.5 x 10(8) i.u./mg; it was estimated to constitute 10-20% of the total antiviral activity of leucocyte IFN. N-Terminal amino-acid-sequence analysis identified the subspecies IFN-alpha 2b and/or IFN-alpha 2c, whereas IFN-alpha 2a was not detectable. The structure of natural IFN-alpha 2 was found to differ from that of its recombinant (Escherichia coli-derived) equivalent. First, reverse-phase h.p.l.c. showed that natural IFN-alpha 2 was significantly more hydrophilic then expected. Secondly, the apparent molecular mass of the natural protein determined by SDS/PAGE was higher than that of recombinant IFN-alpha 2; incubation under mild alkaline conditions known to eliminate O-linked carbohydrates resulted in a reduction of the apparent molecular mass to that of the recombinant protein. On sequence analysis of proteolytic peptides, Thr-106 was found to be modified. These results suggested that Thr-106 of natural IFN-alpha 2 carries O-linked carbohydrates. Reverse-phase h.p.l.c. as well as SDS/PAGE of natural IFN-alpha 2 showed that glycosylation is heterogeneous. For characterization of the carbohydrate moieties, the protein was treated with neuraminidase and/or O-glycanase and analysed by gel electrophoresis; in addition, glycopeptides obtained by proteinase digestion and separated by h.p.l.c. were characterized by sequence analysis and m.s. Further information on the composition of the glycans was obtained by monosaccharide analysis. The results indicate that natural IFN-alpha 2 contains the disaccharide galactosyl-N-acetylgalactosamine (Gal-GalNAc) linked to Thr-106. In part of the molecules, this core carbohydrate carries (alpha-)N-acetylneuraminic acid, whereas a disaccharide, probably N-acetyl-lactosamine, is bound to Gal-GalNAc in another proportion of the protein. Further glycosylation isomers are present in small amounts. As IFN-alpha 2 is the only IFN-alpha species with a threonine residue at position 106, it may represent the only O-glycosylated human IFN-alpha protein.

Full text

PDF
515

Images in this article

515Fig. 4.
on p.515

Selected References

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

  1. Adolf G. R. Antigenic structure of human interferon omega 1 (interferon alpha II1): comparison with other human interferons. J Gen Virol. 1987 Jun;68(Pt 6):1669–1676. doi: 10.1099/0022-1317-68-6-1669. [DOI] [PubMed] [Google Scholar]
  2. Adolf G. R., Bodo G., Swetly P. Production of monoclonal antibodies to human IFN-alpha and their use for analysis of the antigenic composition of various natural interferons. J Cell Physiol Suppl. 1982;2:61–68. doi: 10.1002/jcp.1041130511. [DOI] [PubMed] [Google Scholar]
  3. Adolf G. R., Maurer-Fogy I., Kalsner I., Cantell K. Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase. J Biol Chem. 1990 Jun 5;265(16):9290–9295. [PubMed] [Google Scholar]
  4. Adolf G. R. Monoclonal antibodies and enzyme immunoassays specific for human interferon (IFN) omega 1: evidence that IFN-omega 1 is a component of human leukocyte IFN. Virology. 1990 Apr;175(2):410–417. doi: 10.1016/0042-6822(90)90425-q. [DOI] [PubMed] [Google Scholar]
  5. Allen G., Fantes K. H. A family of structural genes for human lymphoblastoid (leukocyte-type) interferon. Nature. 1980 Oct 2;287(5781):408–411. doi: 10.1038/287408a0. [DOI] [PubMed] [Google Scholar]
  6. Allen G. Structure and properties of human interferon-alpha from Namalwa lymphoblastoid cells. Biochem J. 1982 Dec 1;207(3):397–408. doi: 10.1042/bj2070397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bodo G. Procedures for large-scale production and partial purification of human interferon from lymphocyte (Namalva) cultures. Methods Enzymol. 1981;78(Pt A):69–75. doi: 10.1016/0076-6879(81)78099-6. [DOI] [PubMed] [Google Scholar]
  8. Cantell K., Hirvonen S., Kauppinen H. L., Myllylä G. Production of interferon in human leukocytes from normal donors with the use of Sendai virus. Methods Enzymol. 1981;78(Pt A):29–38. doi: 10.1016/0076-6879(81)78094-7. [DOI] [PubMed] [Google Scholar]
  9. Dworkin-Rastl E., Dworkin M. B., Swetly P. MOlecular cloning of human alpha and beta interferon genes from Namalwa cells. J Interferon Res. 1982;2(4):575–585. doi: 10.1089/jir.1982.2.575. [DOI] [PubMed] [Google Scholar]
  10. Figlin R. A., Itri L. M. Anti-interferon antibodies: a perspective. Semin Hematol. 1988 Jul;25(3 Suppl 3):9–15. [PubMed] [Google Scholar]
  11. Fish E. N., Banerjee K., Stebbing N. Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells. Biochem Biophys Res Commun. 1983 Apr 29;112(2):537–546. doi: 10.1016/0006-291x(83)91498-5. [DOI] [PubMed] [Google Scholar]
  12. Galton J. E., Bedford P., Scott J. E., Brand C. M., Nethersell A. B. Antibodies to lymphoblastoid interferon. Lancet. 1989 Sep 2;2(8662):572–573. doi: 10.1016/s0140-6736(89)90703-4. [DOI] [PubMed] [Google Scholar]
  13. Goeddel D. V., Leung D. W., Dull T. J., Gross M., Lawn R. M., McCandliss R., Seeburg P. H., Ullrich A., Yelverton E., Gray P. W. The structure of eight distinct cloned human leukocyte interferon cDNAs. Nature. 1981 Mar 5;290(5801):20–26. doi: 10.1038/290020a0. [DOI] [PubMed] [Google Scholar]
  14. Gribben J. G., Devereux S., Thomas N. S., Keim M., Jones H. M., Goldstone A. H., Linch D. C. Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF. Lancet. 1990 Feb 24;335(8687):434–437. doi: 10.1016/0140-6736(90)90665-r. [DOI] [PubMed] [Google Scholar]
  15. Henco K., Brosius J., Fujisawa A., Fujisawa J. I., Haynes J. R., Hochstadt J., Kovacic T., Pasek M., Schamböck A., Schmid J. Structural relationship of human interferon alpha genes and pseudogenes. J Mol Biol. 1985 Sep 20;185(2):227–260. doi: 10.1016/0022-2836(85)90401-2. [DOI] [PubMed] [Google Scholar]
  16. Hiscott J., Cantell K., Weissmann C. Differential expression of human interferon genes. Nucleic Acids Res. 1984 May 11;12(9):3727–3746. doi: 10.1093/nar/12.9.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hobbs D. S., Pestka S. Purification and characterization of interferons from a continuous myeloblastic cell line. J Biol Chem. 1982 Apr 25;257(8):4071–4076. [PubMed] [Google Scholar]
  18. Koeffler H. P., Golde D. W. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science. 1978 Jun 9;200(4346):1153–1154. doi: 10.1126/science.306682. [DOI] [PubMed] [Google Scholar]
  19. Labdon J. E., Gibson K. D., Sun S., Pestka S. Some species of human leukocyte interferon are glycosylated. Arch Biochem Biophys. 1984 Jul;232(1):422–426. doi: 10.1016/0003-9861(84)90558-7. [DOI] [PubMed] [Google Scholar]
  20. Levy W. P., Rubinstein M., Shively J., Del Valle U., Lai C. Y., Moschera J., Brink L., Gerber L., Stein S., Pestka S. Amino acid sequence of a human leukocyte interferon. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6186–6190. doi: 10.1073/pnas.78.10.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levy W. P., Shively J., Rubinstein M., Del Valle U., Pestka S. Amino-terminal amino acid sequence of human leukocyte interferon. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5102–5104. doi: 10.1073/pnas.77.9.5102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oh-eda M., Hasegawa M., Hattori K., Kuboniwa H., Kojima T., Orita T., Tomonou K., Yamazaki T., Ochi N. O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity. J Biol Chem. 1990 Jul 15;265(20):11432–11435. [PubMed] [Google Scholar]
  23. Ortaldo J. R., Herberman R. B., Harvey C., Osheroff P., Pan Y. C., Kelder B., Pestka S. A species of human alpha interferon that lacks the ability to boost human natural killer activity. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4926–4929. doi: 10.1073/pnas.81.15.4926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rubinstein M., Levy W. P., Moschera J. A., Lai C. Y., Hershberg R. D., Bartlett R. T., Pestka S. Human leukocyte interferon: isolation and characterization of several molecular forms. Arch Biochem Biophys. 1981 Aug;210(1):307–318. doi: 10.1016/0003-9861(81)90194-6. [DOI] [PubMed] [Google Scholar]
  25. Streuli M., Nagata S., Weissmann C. At least three human type alpha interferons: structure of alpha 2. Science. 1980 Sep 19;209(4463):1343–1347. doi: 10.1126/science.6158094. [DOI] [PubMed] [Google Scholar]
  26. Weck P. K., Apperson S., May L., Stebbing N. Comparison of the antiviral activities of various cloned human interferon-alpha subtypes in mammalian cell cultures. J Gen Virol. 1981 Nov;57(Pt 1):233–237. doi: 10.1099/0022-1317-57-1-233. [DOI] [PubMed] [Google Scholar]
  27. Zoon K. C., Smith M. E., Bridgen P. J., Anfinsen C. B., Hunkapiller M. W., Hood L. E. Amino terminal sequence of the major component of human lymphoblastoid interferon. Science. 1980 Feb 1;207(4430):527–528. doi: 10.1126/science.7352260. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES