Glycobiology Advance Access originally published online on September 29, 2008
Glycobiology 2008 18(12):1119-1128; doi:10.1093/glycob/cwn097
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Minimum substrate requirements of endoglycosidase activities toward dermatan sulfate by electrospray ionization-tandem mass spectrometry
Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women's Health Service, 72 King William Road, North Adelaide, SA 5006; and Discipline of Paediatrics, University of Adelaide, Adelaide, SA 5005, Australia
1 To whom correspondence should be addressed: Tel: +61-8-8161-6741; Fax: +61-8-8161-7100; e-mail: maria.fuller{at}adelaide.edu.au
Received on August 27, 2008; revised on September 1, 2008; accepted on September 24, 2008
The catabolism of dermatan sulfate (DS) commences with endohydrolysis of the polysaccharide to oligosaccharides by proposed endo-β-N-acetylhexosaminidase and endohexuronidase activities. To investigate the substrate specificities of these activities, we developed an assay to measure specific products of their action upon oligosaccharide substrates. Tetra- to tetradecasaccharides, rich in glucuronic acid (GlcA) or iduronic acid (IdoA), were obtained from chondroitinase ABC digests of chondroitin sulfate (CS)-A and DS, respectively, separated by gel-filtration chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Endo-β-N-acetylhexosaminidase and endohexuronidase cleavage of these oligosaccharides was then assessed by incubating with cell homogenate (source of endoglycosidase activity) and measuring di- to octasaccharide products derived from the nonreducing end of the substrate by ESI-MS/MS. We found that both activities preferentially degraded the GlcA-rich substrate, with minor activity toward the IdoA-rich substrate and that a minimum of four and five monosaccharides were required on the reducing side of the target glycosidic linkage for endo-β-N-acetylhexosaminidase and endohexuronidase cleavage, respectively. Thus, the minimum-sized substrates were a hexasaccharide for endo-β-N-acetylhexosaminidase and an octasaccharide for endohexuronidase. We observed that endo-β-N-acetylhexosaminidase sequentially removed tetrasaccharides from the nonreducing end of oligosaccharides when unrestricted by substrate length, whereas endohexuronidase activity was random and comparatively low. The activities displayed acidic pH optima and were shown by subcellular fractionation to reside in lysosomes and late endosomes. We suggest that these activities represent the known Hyal-1 and endo-β-glucuronidase enzymes and that these enzymes act in concert to degrade GlcA-rich domains of DS but are less active toward regions containing IdoA.
Key words: dermatan sulfate / endo-β-glucuronidase / endoglycosidase / Hyal-1 / mass spectrometry
2 Present address: Baker Heart Research Institute, 75 Commercial Road, Melbourne, Victoria 3006, Australia