Abstract
Heparan sulfate (HS) polysaccharides are ubiquitous in animal tissues as components of proteoglycans, and they participate in many important biological processes. HS carbohydrate chains are complex and can contain rare structural components such as N-unsubstituted glucosamine (GlcN). Commercially available HS preparations have been invaluable in many types of research activities. In the course of preparing microarrays to include probes derived from HS oligosaccharides, we found an unusually high content of GlcN residue in a recently purchased batch of porcine intestinal mucosal HS. Composition and sequence analysis by mass spectrometry of the oligosaccharides obtained after heparin lyase III digestion of the polysaccharide indicated two and three GlcN in the tetrasaccharide and hexasaccharide fractions, respectively. 1H NMR of the intact polysaccharide showed that this unusual batch differed strikingly from other HS preparations obtained from bovine kidney and porcine intestine. The very high content of GlcN (30%) and low content of GlcNAc (4.2%) determined by disaccharide composition analysis indicated that N-deacetylation and/or N-desulfation may have taken place. HS is widely used by the scientific community to investigate HS structures and activities. Great care has to be taken in drawing conclusions from investigations of structural features of HS and specificities of HS interaction with proteins when commercial HS is used without further analysis. Pending the availability of a validated commercial HS reference preparation, our data may be useful to members of the scientific community who have used the present preparation in their studies.
Highlights
Letter aRelative intensity (% relative to the base peak) is shown in parentheses; when the ion is the base peak, relative intensity (100%) was not shown. bAlthough both ΔUA-glucosamine fully N-sulfated (GlcNS)-UA-GlcN and ΔUA-GlcN-UA-GlcNS are possible, lyase III does not favor GlcN-UA for cleavage,[12] and ΔUA-GlcNS-UA-GlcN is not proposed as the main tetrasaccharide product
10E4 antibody is closely associated with prion lesions in the brain of mice infected with scrapie.[20]
Heparan sulfate (HS) isolated from bovine kidney is available from Sigma but is expensive
Summary
10E4 antibody is closely associated with prion lesions in the brain of mice infected with scrapie.[20]. Among the identified main ions in the di- to deca-saccharide fractions, only one component, m/z 715, present in fraction F3, contains a single N-acetylglucosamine residue with the sequence of ΔUA-GlcN-UA-GlcNAc (Table 1 and Supplemental Figure S2b). 1H NMR spectra for three preparations of porcine mucosal HS-1C, prepared as described by Casu et al.,[21] and HO-10697 and HO-10095, very old batches from Celsus, are shown in Supplemental Figure S3; all of them contain the same prominent acetyl methyl signal at 2.04 ppm. Full experimental procedure; figures of gel filtration chromatography of HS oligosaccharides, negative-ion ESI-CID-MS/MS spectra of hexa- and tetrasaccharide components in F3, 1H NMR spectra of porcine intestinal HS, and anomeric region of 13C-1H HSQC NMR spectra; and table of integrated volumes of anomeric cross-peaks in the 13C-1H HSQC NMR spectra (PDF).
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