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https://hdl.handle.net/2440/7840
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Type: | Journal article |
Title: | A strategy for rapid sequencing of heparan sulfate and heparin saccharides |
Author: | Turnbull, J. Hopwood, J. Gallagher, J. |
Citation: | Proceedings of the National Academy of Sciences of USA, 1999; 96(6):2698-2703 |
Publisher: | NATL ACAD SCIENCES |
Issue Date: | 1999 |
ISSN: | 0027-8424 1091-6490 |
Statement of Responsibility: | Jeremy E. Turnbull, John J. Hopwood, and John T. Gallagher |
Abstract: | Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and variable patterns of sulfation, especially in heparan sulfate (HS). HS and the structurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is accumulating that these interactions depend on specific saccharide sequences, but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress. We describe how HS and heparin saccharides can be sequenced rapidly by using an integrated strategy with chemical and enzymic steps. Attachment of a reducing-end fluorescent tag establishes a reading frame. Partial selective chemical cleavage at internal N-sulfoglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations of exosulfatases and exoglycosidases permits the selective removal of specific sulfates and monosaccharides from their nonreducing ends. PAGE of the products yields a pattern of fluorescent bands from which the saccharide sequence can be read directly. Data are presented on sequencing of heparin tetrasaccharides and hexasaccharides of known structure; these data show the accuracy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an HS decasaccharide of unknown structure. Application and further development of this sequencing strategy, called integral glycan sequencing, will accelerate progress in defining the structure-activity relationships of these complex GAGs and lead to important insights into their biological functions. |
Keywords: | Heparin Heparitin Sulfate Sequence Analysis Carbohydrate Sequence Molecular Sequence Data |
DOI: | 10.1073/pnas.96.6.2698 |
Published version: | http://dx.doi.org/10.1073/pnas.96.6.2698 |
Appears in Collections: | Aurora harvest 4 Paediatrics publications |
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