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Glycobiology Advance Access originally published online on May 11, 2009
Glycobiology 2009 19(8):899-909; doi:10.1093/glycob/cwp065
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Published by Oxford University Press 2009.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Targeted glycoproteomic identification of cancer cell glycosylation

Alex S Powlesland3, Paul G Hitchen3, Simon Parry2,3, Sarah A Graham3, Maria Marcela Barrio4, Maria Teresa Elola5, José Mordoh4,5, Anne Dell3, Kurt Drickamer3 and Maureen E Taylor1,3

3 Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London SW7 2AZ, UK
4 Centro de Investigaciones Oncológicas FUCA, Zabala 2836, 1426 Buenos Aires, Argentina
5 Fundación Instituto Leloir-IIBBA CONICET, Patricias Argentinas 435, 1405 Buenos Aires, Argentina

1 To whom correspondence should be addressed: Tel: +44-207-594-5281; Fax: +44-207-594-3057; e-mail: m.taylor{at}imperial.ac.uk

Received on March 3, 2009; revised on May 6, 2009; accepted on May 6, 2009

GalMBP is a fragment of serum mannose-binding protein that has been modified to create a probe for galactose-containing ligands. Glycan array screening demonstrated that the carbohydrate-recognition domain of GalMBP selectively binds common groups of tumor-associated glycans, including Lewis-type structures and T antigen, suggesting that engineered glycan-binding proteins such as GalMBP represent novel tools for the characterization of glycoproteins bearing tumor-associated glycans. Blotting of cell extracts and membranes from MCF7 breast cancer cells with radiolabeled GalMBP was used to demonstrate that it binds to a selected set of high molecular weight glycoproteins that could be purified from MCF7 cells on an affinity column constructed with GalMBP. Proteomic and glycomic analysis of these glycoproteins by mass spectrometry showed that they are forms of CD98hc that bear glycans displaying heavily fucosylated termini, including Lewisx and Lewisy structures. The pool of ligands was found to include the target ligands for anti-CD15 antibodies, which are commonly used to detect Lewisx antigen on tumors, and for the endothelial scavenger receptor C-type lectin, which may be involved in tumor metastasis through interactions with this antigen. A survey of additional breast cancer cell lines reveals that there is wide variation in the types of glycosylation that lead to binding of GalMBP. Higher levels of binding are associated either with the presence of outer-arm fucosylated structures carried on a variety of different cell surface glycoproteins or with the presence of high levels of the mucin MUC1 bearing T antigen.

Key words: breast cancer cells / lectin / proteomics / protein engineering / tumor glycosylation

2 Current address: AgResearch Limited, Ruakura Research Centre, East Street, Private Bag 3123, Hamilton 3240, New Zealand.


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