Skip Navigation



Glycobiology Advance Access published online on September 29, 2008
Glycobiology, doi:10.1093/glycob/cwn097
This Article
Right arrow Advance Access manuscript (PDF) Freely available
Right arrow All Versions of this Article:
18/12/1119    most recent
cwn097v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Nielsen, T. C.
Right arrow Articles by Fuller, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nielsen, T. C.
Right arrow Articles by Fuller, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Minimum substrate requirements of endoglycosidase activities towards dermatan sulfate by electrospray ionization-tandem mass spectrometry

Timothy C. Nielsen, Peter J. Meikle1, John J. Hopwood and Maria Fuller{dagger}

Lysosomal Diseases Research Unit, Department of Genetic Medicine, Children, Youth and Women's Health Service, 72 King William Road, North Adelaide, South Australia, 5006
Discipline of Paediatrics, University of Adelaide, South Australia, 5005

* Corresponding author Phone: 61-8-8161-6741; Fax: 61-8-8161-7100; email: maria.fuller{at}adelaide.edu.au

Received on August 27, 2007; 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 non-reducing end of the substrate by ESI-MS/MS. We found that both activities preferentially degraded GlcA-rich substrate, with minor activity towards IdoA-rich substrate, and that a minimum of four and five monosaccharides was 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 non-reducing 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 sub-cellular 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 towards regions containing IdoA.

Key words: dermatan sulfate / endo-β-glucuronidase / endoglycosidase / Hyal-1 / mass spectrometry

1 Current address: Baker Heart Research Institute, 75 Commercial Road, Melbourne, Victoria, Australia, 3006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.