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

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.

SUBMITTER: Turnbull JE

PROVIDER: S-EPMC15832 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:The structure of heparin and heparan sulfate (Hep/HS) oligosaccharides, as determined by the length and the pattern of sulfation, acetylation, and uronic acid epimerization, dictates their biological function through modulating interactions with protein targets. But fine structural determination is a very challenging task due to the lability of the sulfate modifications and difficulties in separating isomeric HS chains. Previously, we reported a strategy for chemical derivatization involving permethylation, desulfation, and trideuteroperacetylation, combined with standard reverse phase LC-MS/MS that enables the structural sequencing for heparin/HS oligosaccharides of sizes up to dodecasaccharide by positionally replacing all sulfates with more stable trideuteroacetyl groups, allowing for robust MS/MS sequencing. However, isomeric oligosaccharides that contain both N-sulfation and N-acetylation become isotopomers after labeling, differing only in the sites of deuteration. This prevents chromatographic separation of these different mixed domain sequences post-derivatization, and makes sequencing by MS/MS difficult due to co-fragmentation of the isotopomers leading to chimeric product ion spectra. In order to improve chromatographic separation of mixed domain oligosaccharides, we have introduced a propionylation step in place of trideuteroacetylation for labeling of sites of sulfation. HS standard disaccharides have been used to evaluate the efficiency of this improved chemical derivatization. The results show that we can quantitatively replace sulfation with propionyl groups with the same high efficiency as the previously reported trideuteroacetylation. After derivatization, we demonstrate the ability to chromatographically separate two mixed domain tetrasaccharide isomers differing solely by the order of N-sulfation and N-acetylation, allowing for full sequencing of each by MS/MS. These results represent a marked improvement in the ability of our previously reported derivatization strategy to analyze complex mixtures of Hep/HS oligosaccharides without a decrease in sensitivity.

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

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