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Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin.

ABSTRACT: Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (?30%) than in control MCs where ?95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.

SUBMITTER: Dagalv A

PROVIDER: S-EPMC3248000 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin.

Dagälv Anders A Holmborn Katarina K Kjellén Lena L Abrink Magnus M

The Journal of biological chemistry 20111102 52

Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in th ...[more]

PMID: 22049073

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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.

Dataset Information

Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin.

Publications

Lowered expression of heparan sulfate/heparin biosynthesis enzyme N-deacetylase/n-sulfotransferase 1 results in increased sulfation of mast cell heparin.

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