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

M. D'Onofrio, C. M. Starr, M. K. Park, G. D. Holt, R. S. Haltiwanger, Gerald Warren Hart, J. A. Hanover

Research output: Contribution to journalArticle

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 λgt11 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 β-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.

Original languageEnglish (US)
Pages (from-to)9595-9599
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number24
StatePublished - 1988
Externally publishedYes

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Nuclear Pore
Porins
Acetylglucosamine
Nuclear Proteins
Complementary DNA
Glycoproteins
Open Reading Frames
Glycopeptides
Glycosylation
Galactosidases
Messenger RNA
Proteins
Oligonucleotide Probes
Liver
Nuclear Envelope
Gene Library
Base Pairing
Gel Chromatography
Electrophoresis
Thyroid Gland

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

D'Onofrio, M., Starr, C. M., Park, M. K., Holt, G. D., Haltiwanger, R. S., Hart, G. W., & Hanover, J. A. (1988). Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine. Proceedings of the National Academy of Sciences of the United States of America, 85(24), 9595-9599.

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine. / D'Onofrio, M.; Starr, C. M.; Park, M. K.; Holt, G. D.; Haltiwanger, R. S.; Hart, Gerald Warren; Hanover, J. A.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 24, 1988, p. 9595-9599.

Research output: Contribution to journalArticle

D'Onofrio, M, Starr, CM, Park, MK, Holt, GD, Haltiwanger, RS, Hart, GW & Hanover, JA 1988, 'Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine', Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 24, pp. 9595-9599.
D'Onofrio, M. ; Starr, C. M. ; Park, M. K. ; Holt, G. D. ; Haltiwanger, R. S. ; Hart, Gerald Warren ; Hanover, J. A. / Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine. In: Proceedings of the National Academy of Sciences of the United States of America. 1988 ; Vol. 85, No. 24. pp. 9595-9599.
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