Evidence of random structural features in the heparin polymer

Linhardt, Robert J.; Merchant, Zohar M.; Rice, Kevin G.; Kim, Y. S.; Fitzgerald, Gerald L.; Grant, Arthur C.; Langer, Robert

doi:10.1021/bi00347a045

Cited by 25 publications

(15 citation statements)

References 25 publications

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“…5). Computer simulation studies (56,57) together with an improved understanding of the specificity of heparinase will be required to determine the precise number of possible nonreducing end terminal sequences present in bovine lung heparin. The saturated oligosaccharides observed correspond to 11.45% of the total oligosaccharides of dp Յ 10, identical to the mathematically predicted value of 10 -12% (55) and consistent with the known molecular weight of bovine lung heparin.…”

Section: Resultsmentioning

confidence: 99%

Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Thanawiroon

Rice

Toida

et al. 2004

Journal of Biological Chemistry

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145

152

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Liquid chromatography/mass spectrometry (LC/MS)is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversedphase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of ؊32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10,000 Da and a net charge of ؊60.The structural elucidation of complex carbohydrates remains one of the most difficult challenges for chemists, often requiring the application of multiple analytical approaches (1-5). Glycosaminoglycans (GAGs), 1 and heparin in particular, have proven to be extremely difficult to analyze because of high negative charge, polydispersity, and sequence heterogeneity (6, 7). Heparin and low molecular weight heparins, prepared through the controlled chemical or enzymatic fragmentation of heparin (8), are widely used as clinical anticoagulants. Despite their medical importance, these drugs are relatively uncharacterized in terms of their chemical structure. Moreover, heparin and the structurally related heparan sulfate exhibit many additional biological activities, making them of great interest in new drug discovery (9). Numerous challenges can arise from the structural investigation of biologically active heparin oligosaccharides, particularly those in recognition systems involving specific protein-carbohydrate interactions (10, 11). Such biologically important oligosaccharides often contain rare sequences (12, 13) and are present only in minute, often picomole quantities. New derivatization methods (14, 15), chromatography (15, 16), electrophoresis-ba...

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Section: Resultsmentioning

confidence: 99%

Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Thanawiroon

Rice

Toida

et al. 2004

Journal of Biological Chemistry

Self Cite

145

152

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“…It will be of interest to ascertain whether this is the sole non-random structural domain. It has been suggested that heparinase cleavage sites [GlcNSO3(6-OS03)al -4IdoA(2-OS03)] in heparin are randomly distributed (Linhardt et al, 1985). However, analyses of the less-commonly occurring heparin chains with strong anti-coagulant activity have indicated the existence of certain 'ordered' structural features.…”

Section: Discussionmentioning

confidence: 99%

Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate

Lyon¹,

Steward²,

Hampson³

et al. 1987

Biochemical Journal

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The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans.

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“…Several early attempts with mathematical modeling [51] and computer simulation [52]–[55] were focused on the structure of heparin. These methods were used in conjunction with cleavage experiments to test alternative hypotheses concerning the action pattern of heparin lyase I and the arrangement of specific oligosaccharides within the heparin chain.…”

Section: Discussionmentioning

confidence: 99%

A Computational Approach for Deciphering the Organization of Glycosaminoglycans

et al. 2010

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BackgroundIncreasing evidence has revealed important roles for complex glycans as mediators of normal and pathological processes. Glycosaminoglycans are a class of glycans that bind and regulate the function of a wide array of proteins at the cell-extracellular matrix interface. The specific sequence and chemical organization of these polymers likely define function; however, identification of the structure-function relationships of glycosaminoglycans has been met with challenges associated with the unique level of complexity and the nontemplate-driven biosynthesis of these biopolymers.Methodology/Principal FindingsTo address these challenges, we have devised a computational approach to predict fine structure and patterns of domain organization of the specific glycosaminoglycan, heparan sulfate (HS). Using chemical composition data obtained after complete and partial digestion of mixtures of HS chains with specific degradative enzymes, the computational analysis produces populations of theoretical HS chains with structures that meet both biosynthesis and enzyme degradation rules. The model performs these operations through a modular format consisting of input/output sections and three routines called chainmaker, chainbreaker, and chainsorter. We applied this methodology to analyze HS preparations isolated from pulmonary fibroblasts and epithelial cells. Significant differences in the general organization of these two HS preparations were observed, with HS from epithelial cells having a greater frequency of highly sulfated domains. Epithelial HS also showed a higher density of specific HS domains that have been associated with inhibition of neutrophil elastase. Experimental analysis of elastase inhibition was consistent with the model predictions and demonstrated that HS from epithelial cells had greater inhibitory activity than HS from fibroblasts.Conclusions/SignificanceThis model establishes the conceptual framework for a new class of computational tools to use to assess patterns of domain organization within glycosaminoglycans. These tools will provide a means to consider high-level chain organization in deciphering the structure-function relationships of polysaccharides in biology.

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Evidence of random structural features in the heparin polymer

Cited by 25 publications

References 25 publications

Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Liquid Chromatography/Mass Spectrometry Sequencing Approach for Highly Sulfated Heparin-derived Oligosaccharides

Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate

A Computational Approach for Deciphering the Organization of Glycosaminoglycans

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