title: Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions.
abstract: 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 noncovalently 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 nonsulfated 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.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Marson
authors.forename: David E
authors.initials: DE
authors.lastname: Robinson
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Brookes
authors.forename: Barbara
authors.initials: B
authors.lastname: Mulloy
authors.forename: Michelle
authors.initials: M
authors.lastname: Wiles
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Clark
authors.forename: Helen L
authors.initials: HL
authors.lastname: Fielder
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Collinson
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Cain
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Kielty
authors.forename: Sally
authors.initials: S
authors.lastname: McArthur
authors.forename: David J
authors.initials: DJ
authors.lastname: Buttle
authors.forename: Robert D
authors.initials: RD
authors.lastname: Short
authors.forename: Jason D
authors.initials: JD
authors.lastname: Whittle
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
mesh_terms: D000499:Allylamine / Q000737:chemistry; D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D054794:Glycomics / Q000295:instrumentation* / Q000379:methods*; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D055616:Microtechnology / Q000295:instrumentation / Q000379:methods; D011485:Protein Binding; D013379:Substrate Specificity; D013499:Surface Properties; D013552:Swine; D053675:Versicans / Q000737:chemistry / Q000378:metabolism; D054392:Adipokines
chemical_list: D000499:Allylamine; D015815:Cell Adhesion Molecules; D017242:Complement Factor H; D000071838:Fibrillin-1; D000071837:Fibrillins; D006025:Glycosaminoglycans; D006493:Heparin; D037102:Lectins; D008840:Microfilament Proteins; D053675:Versicans; C000605722:FBN1 protein, human; D054392:Adipokines; C072273:TNFAIP6 protein, human; C508322:VCAN protein, human