Profiling of integrin α3 β1 N-linked oligosaccharides using DNA-sequencer.

A. Litynska, 2, P. Laidler, 1, E. Pochec, 3, N. Callewaert, 3 R. Contreras 3

1Institute of Zoology, Jagiellonian University, 2Institute of Medical Biochemistry, Medical College, Jagiellonian University, Kraków, Poland; 3Department of Molecular Biology, Ghent University and Flanders Interuniversity Institute for Biotechnology, Belgium

Aim: Integrins consist of noncovalently bound a and b type subunits that associate in various combinations to form heterodimers with a variety of ligand-binding specificities. Both of the subunits are heavily glycosylated. Cell adhesion regulated by integrins seems to be strongly modulated by glycosylation. The expression of a3b1 integrin very often increases in course of cancer progression. Therefore, we performed N-linked oligosaccharides profiling of this integrin expressed in human ureter epithelium cell lines: highly invasive T24 and the reference one (HCV29). Method: Integrin a3b1 (20 mg) purified via affinity chromatography on GD-6 peptide was deglycosylated with PNG-ase followed its SDS-PAGE and electroblotting onto PVDF membrane. After desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid and postderivatization cleanup the oligosaccharides were analysed using standard DNA-sequencing equipment. For further structural information exoglycosidase digestion was performed. Results: Both integrin subunits form HCV cell line showed great heterogeneity of glycan structures having bi- tri- and tetraantennary moieties. In addition, low amount of high mannose type glycans was also present. In contrast in highly invasive T24 cell line the major and dominating oligosaccharide was the tetraantennary complex type glycan terminated with sialic acid. Conclusion: The results allow to suggest that bladder cancer progression is accompanied by reprofiling of glycosylation of a3b1 integrin. The potential meaning of such changes are yet to be elucidated.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Pathology.