A strategy for rapid sequencing of heparan sulfate and heparin saccharides.

2.50
Hdl Handle:
http://hdl.handle.net/10541/91415
Title:
A strategy for rapid sequencing of heparan sulfate and heparin saccharides.
Authors:
Turnbull, Jeremy E; Hopwood, John J; Gallagher, John T
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.
Affiliation:
School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, England. j.e.turnbull@bham.ac.uk
Citation:
A strategy for rapid sequencing of heparan sulfate and heparin saccharides. 1999, 96 (6):2698-703 Proc. Natl. Acad. Sci. U.S.A.
Journal:
Proceedings of the National Academy of Sciences of the United States of America
Issue Date:
16-Mar-1999
URI:
http://hdl.handle.net/10541/91415
PubMed ID:
10077574
Type:
Article
Language:
en
ISSN:
0027-8424
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorTurnbull, Jeremy Een
dc.contributor.authorHopwood, John Jen
dc.contributor.authorGallagher, John Ten
dc.date.accessioned2010-02-08T15:30:06Z-
dc.date.available2010-02-08T15:30:06Z-
dc.date.issued1999-03-16-
dc.identifier.citationA strategy for rapid sequencing of heparan sulfate and heparin saccharides. 1999, 96 (6):2698-703 Proc. Natl. Acad. Sci. U.S.A.en
dc.identifier.issn0027-8424-
dc.identifier.pmid10077574-
dc.identifier.urihttp://hdl.handle.net/10541/91415-
dc.description.abstractSulfated 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.en
dc.language.isoenen
dc.subject.meshCarbohydrate Sequence-
dc.subject.meshHeparin-
dc.subject.meshHeparitin Sulfate-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshSequence Analysis-
dc.titleA strategy for rapid sequencing of heparan sulfate and heparin saccharides.en
dc.typeArticleen
dc.contributor.departmentSchool of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT, England. j.e.turnbull@bham.ac.uken
dc.identifier.journalProceedings of the National Academy of Sciences of the United States of Americaen

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