Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)

Abstract

Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Δdisaccharides including ΔDiHA, ΔDi0S, ΔDi2S, ΔDi4S, ΔDi6S, ΔDi2,4S, ΔDi2,6S, ΔDi4,6S, and ΔDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4,6S-galNAc.