A fucose-containing O-glycoepitope on bovine and human nucleolin.
In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.
Aldi
Silvia
S
Department of Evolutionary Biology, University of Siena, Siena, Italy.
Della Giovampaola
Cinzia
C
Focarelli
Riccardo
R
Armini
Alessandro
A
Ziche
Marina
M
Finetti
Federica
F
Rosati
Floriana
F
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D005643:Fucose / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D010750:Phosphoproteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D049881:Unio; D000097796:Nucleolin
D000939:Epitopes; D005643:Fucose; D006023:Glycoproteins; D007074:Immunoglobulin G; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin