A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator.
We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.
Chan
A L
AL
Department of Biochemistry, Imperial College of Science Technology and Medicine, London, UK.
Morris
H R
HR
Panico
M
M
Etienne
A T
AT
Rogers
M E
ME
Gaffney
P
P
Creighton-Kempsford
L
L
Dell
A
A
D000116:Acetylgalactosamine / Q000032:analysis*; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000201:enzymology*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D012794:Sialic Acids / Q000032:analysis*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics; D014162:Transfection
D006023:Glycoproteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D010959:Tissue Plasminogen Activator; D000116:Acetylgalactosamine