Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Dec;85(24):9595–9599. doi: 10.1073/pnas.85.24.9595

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.

M D'Onofrio 1, C M Starr 1, M K Park 1, G D Holt 1, R S Haltiwanger 1, G W Hart 1, J A Hanover 1
PMCID: PMC282808  PMID: 3200844

Abstract

The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a lambda gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a beta-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.

Full text

PDF
9595

Images in this article

Selected References

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

  1. Baglia F. A., Maul G. G. Nuclear ribonucleoprotein release and nucleoside triphosphatase activity are inhibited by antibodies directed against one nuclear matrix glycoprotein. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2285–2289. doi: 10.1073/pnas.80.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  3. Dabauvalle M. C., Schulz B., Scheer U., Peters R. Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin. Exp Cell Res. 1988 Jan;174(1):291–296. doi: 10.1016/0014-4827(88)90163-2. [DOI] [PubMed] [Google Scholar]
  4. Davis L. I., Blobel G. Identification and characterization of a nuclear pore complex protein. Cell. 1986 Jun 6;45(5):699–709. doi: 10.1016/0092-8674(86)90784-1. [DOI] [PubMed] [Google Scholar]
  5. Davis L. I., Blobel G. Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7552–7556. doi: 10.1073/pnas.84.21.7552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finlay D. R., Newmeyer D. D., Price T. M., Forbes D. J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. doi: 10.1083/jcb.104.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hanover J. A., Cohen C. K., Willingham M. C., Park M. K. O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J Biol Chem. 1987 Jul 15;262(20):9887–9894. [PubMed] [Google Scholar]
  9. Hanover J. A., Lennarz W. J., Young J. D. Synthesis of N- and O-linked glycopeptides in oviduct membrane preparations. J Biol Chem. 1980 Jul 25;255(14):6713–6716. [PubMed] [Google Scholar]
  10. Hart G. W., Brew K., Grant G. A., Bradshaw R. A., Lennarz W. J. Primary structural requirements for the enzymatic formation of the N-glycosidic bond in glycoproteins. Studies with natural and synthetic peptides. J Biol Chem. 1979 Oct 10;254(19):9747–9753. [PubMed] [Google Scholar]
  11. Holt G. D., Hart G. W. The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc. J Biol Chem. 1986 Jun 15;261(17):8049–8057. [PubMed] [Google Scholar]
  12. Holt G. D., Snow C. M., Senior A., Haltiwanger R. S., Gerace L., Hart G. W. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J Cell Biol. 1987 May;104(5):1157–1164. doi: 10.1083/jcb.104.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Houghten R. A., Li C. H. Reduction of sulfoxides in peptides and proteins. Methods Enzymol. 1983;91:549–559. doi: 10.1016/s0076-6879(83)91050-9. [DOI] [PubMed] [Google Scholar]
  14. Lathe R. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J Mol Biol. 1985 May 5;183(1):1–12. doi: 10.1016/0022-2836(85)90276-1. [DOI] [PubMed] [Google Scholar]
  15. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  16. Park M. K., D'Onofrio M., Willingham M. C., Hanover J. A. A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6462–6466. doi: 10.1073/pnas.84.18.6462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reinach F. C., Fischman D. A. Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes. The analysis of a conformation-specific antibody to myosin light chain 2. J Mol Biol. 1985 Feb 5;181(3):411–422. doi: 10.1016/0022-2836(85)90229-3. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Snow C. M., Senior A., Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. doi: 10.1083/jcb.104.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Swiedler S. J., Hart G. W., Tarentino A. L., Plummer T. H., Jr, Freed J. H. Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma. J Biol Chem. 1983 Oct 10;258(19):11515–11523. [PubMed] [Google Scholar]
  21. Unwin P. N., Milligan R. A. A large particle associated with the perimeter of the nuclear pore complex. J Cell Biol. 1982 Apr;93(1):63–75. doi: 10.1083/jcb.93.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wilbur W. J., Lipman D. J. Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci U S A. 1983 Feb;80(3):726–730. doi: 10.1073/pnas.80.3.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wolff B., Willingham M. C., Hanover J. A. Nuclear protein import: specificity for transport across the nuclear pore. Exp Cell Res. 1988 Oct;178(2):318–334. doi: 10.1016/0014-4827(88)90402-8. [DOI] [PubMed] [Google Scholar]
  24. Yoneda Y., Imamoto-Sonobe N., Yamaizumi M., Uchida T. Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells. Exp Cell Res. 1987 Dec;173(2):586–595. doi: 10.1016/0014-4827(87)90297-7. [DOI] [PubMed] [Google Scholar]
  25. Young J. D., Tsuchiya D., Sandlin D. E., Holroyde M. J. Enzymic O-glycosylation of synthetic peptides from sequences in basic myelin protein. Biochemistry. 1979 Oct 2;18(20):4444–4448. doi: 10.1021/bi00587a026. [DOI] [PubMed] [Google Scholar]
  26. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES