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Glycobiology Advance Access originally published online on November 20, 2008
Glycobiology 2009 19(4):337-343; doi:10.1093/glycob/cwn126
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Communication

A fucose-containing O-glycoepitope on bovine and human nucleolin

Silvia Aldi2, Cinzia Della Giovampaola2, Riccardo Focarelli2, Alessandro Armini3, Marina Ziche3, Federica Finetti3 and Floriana Rosati1,2

2 Department of Evolutionary Biology, University of Siena, Siena, Italy
3 Department of Molecular Biology, University of Siena, Siena, Italy

1 To whom correspondence should be addressed: Tel: +39-0577-234408; Fax: +39-0577-234476; e-mail: rosatif{at}unisi.it

Received on December 23, 2007; revised on October 26, 2008; accepted on November 3, 2008

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.

Key words: A431 human cancer cells / CVEC / glycoepitopes / nucleolin / RNA-interference


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