Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase.
A new post-translational modification site in the growth factor domain of urinary type plasminogen activator has been identified. A glycopeptide containing the monosaccharide, fucose, covalently linked directly to the peptide backbone has been isolated from the tryptic digest of pro-urokinase expressed in a mouse hybridoma cell line Sp 2/0 Ag 14. The glycopeptide was isolated by semi-preparative reversed phase high performance liquid chromatography. The identity of a fucose containing peptide was confirmed by carbohydrate analysis, amino acid analysis and plasma desorption mass spectrometry (PDMS). A combination of these methodologies showed an equimolar ratio of peptide and fucose in the glycopeptide. This modification is not detected without mass spectrometry because the fucose residue is hydrolyzed under standard acidic conditions of amino acid composition and N-terminal sequence analysis. The site of attachment of fucose to the peptide has been localized towards the N-terminus (within first 23 amino acids) of the protein. Also, the carbohydrate composition of recombinant pro-urokinase is reported.
Kentzer
E J
EJ
PPD Analytical Research Department, Abbott Laboratories, North Chicago, Illinois 60064.
Buko
A
A
Menon
G
G
Sarin
V K
VK
D000595:Amino Acid Sequence; D005643:Fucose / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D006601:Hexoses / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D006601:Hexoses; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005643:Fucose; D014357:Trypsin; D014568:Urokinase-Type Plasminogen Activator