N-linked glycosylation of the liver cancer biomarker GP73.
The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N-linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi-antennary with core fucose, with a smaller fraction of tri- and tetra-antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added.
Norton
Pamela A
PA
Department of Microbiology and Immunology, Drexel University College of Medicine, Doylestown, Pennsylvania 48902, USA. pamela.norton@drexelmed.edu
Comunale
Mary Ann
MA
Krakover
Jonathan
J
Rodemich
Lucy
L
Pirog
Natalie
N
D'Amelio
Anthony
A
Philip
Ramila
R
Mehta
Anand S
AS
Block
Timothy M
TM
D006528:Carcinoma, Hepatocellular / Q000737:chemistry*; D002846:Chromatography, Affinity; D005643:Fucose / Q000032:analysis; D006031:Glycosylation; D037102:Lectins; D008565:Membrane Proteins / Q000737:chemistry*; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry; D025942:Oligosaccharides, Branched-Chain / Q000032:analysis*; D014407:Tumor Cells, Cultured
C410824:GOLM1 protein, human; D037102:Lectins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D025942:Oligosaccharides, Branched-Chain; D005643:Fucose