Glycobiology Advance Access published online on September 3, 2009
Glycobiology, doi:10.1093/glycob/cwp132
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Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions
1 Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
2 Academic Unit of Molecular Medicine and Rheumatology, School of Medicine and Biomedical Science, University of Sheffield, Sheffield S10 2RX, United Kingdom
3 Department of Engineering Materials, University of Sheffield, Sheffield S1 3JD, United Kingdom
4 BD Biosciences – Discovery Labware, The Innovation Centre, Sheffield S1 4DP, United Kingdom
5 National Institute for Biological Standards and Control, Blanche Lane South Mimms, Potters Bar EN6 3QG, United Kingdom
6 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
* Corresponding author: tel: +44 161 2754195; fax: +44 161 2755082; email: anthony.day{at}manchester.ac.uk
Received on March 9, 2009; accepted on August 27, 2009
The interactions of glycosaminoglycans (GAGs) with proteins underlie a wide range of important biological processes. However, the study of such binding reactions has been hampered by the lack of a simple frontline analysis technique. Previously, we have reported that cold plasma polymerization can be used to coat microtiter plate surfaces with allyl amine to which GAGs (e.g. heparin) can be non-covalently immobilized retaining their ability to interact with proteins. Here we have assessed the capabilities of surface coats derived from different ratios of allyl amine and octadiene (100:0 to 0:100) to support the binding of diverse GAGs (e.g. chondroitin-4-sulfate, dermatan sulfate, heparin preparations and hyaluronan) in a functionally active state. The Link module from TSG-6 was used as a probe to determine the level of functional binding because of its broad (and unique) specificity for both sulfated and non-sulfated GAGs. All of the GAGs tested could bind this domain following their immobilization, although there were clear differences in their protein-binding activities depending on the surface chemistry to which they were adsorbed. On the basis of these experiments 100% allyl amine was chosen for the generation of a microtiter plate-based "sugar array"; X-ray photoelectron spectroscopy revealed that similar relative amounts of chondroitin-4-sulfate, dermatan sulfate and heparin (including two selectively de-sulfated derivatives) were immobilized onto this surface. Analysis of four unrelated proteins (i.e. TSG-6, complement factor H, fibrillin-1 and versican) illustrated the utility of this array to determine the GAG-binding profile and specificity for a particular target protein.
Key words: glycosaminoglycans / sugar array / glycosaminoglycan-protein interactions / microtiter plate-based assay
# Joint first authors
Current address: Mawson Institute, University of South Australia, Adelaide, SA 5001, Australia.