Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.
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.
D'Onofrio
M
M
Laboratory of Biochemistry and Metabolism, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Starr
C M
CM
Park
M K
MK
Holt
G D
GD
Haltiwanger
R S
RS
Hart
G W
GW
Hanover
J A
JA
D000117:Acetylglucosamine / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D005944:Glucosamine / Q000031:analogs & derivatives*; D008099:Liver / Q000032:analysis; D008562:Membrane Glycoproteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000032:analysis*; D028861:Nuclear Pore Complex Proteins; D010449:Peptide Mapping; D051381:Rats
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; C049312:nuclear pore protein p62; D004247:DNA; D005944:Glucosamine; D000117:Acetylglucosamine