Heparan Sulphate - Guidance notes
Heparan sulphate (HS) is widely recognised as the major glycosaminoglycan on cell surfaces; it is an essential co-receptor for many growth factors, morphogenic proteins and signalling structural elements in the extracellular matrix such as fibronectin and various types of collagen. It has a distinctive molecular organisation in which sulphated domains are spaced along the polymer chain separated by regions that are largely devoid of sulphate groups. Despite sharing a common design the HS fine structure is often typical of the cell of origin due to variability of sulphation patterns in the sulphated regions. This variability probably underpins specific protein binding activities at cell surfaces.
Kjellen, L. and Lindahl, U. (2018) Specificity of glycosaminoglycan-protein interactions. Current Opinion in Structural Biology 50:101–108
Gallagher J. (2015) Fell-Muir Lecture: Heparan Sulphate and the art of cell regulation: a polymer chain conducts the protein orchestra. Int J Exp Pathol. 96:203-231.
Xu D. and Esko JD: (2014) Demystifying heparan sulfate-protein interactions. Annu Rev Biochem . 83:129-157.
To assist investigations of this most intriguing of glycosaminoglycans we offer several preparations of Heparan Sulphates that vary in molecular size and sulphation including a unique set of selectively-desulphated Heparan Sulphates. Please also see our heparin oligosaccharides that mimic some of the sulphation variability seen in HS.
1. Heparan Sulphate fraction I: Mol wt approx. 40kDa
Relatively low in Sulphation (when compared to parent GAG-HS01)/High Molecular Weight HS.
40 N-sulphate groups per 100 disaccharides 35 O-sulphate groups per 100 disaccharides *Polymer sulphation (N- and O-sulphate groups) is 75 per 100 disaccharides. The domain structure of this preparation is broadly similar to the most common form of heparan sulphate present on cell surfaces
2. Heparan Sulphate fraction II
Intermediate level of Sulphation HS.
55 N-sulphate groups per 100 disaccharides 70 O-sulphate groups per 100 disaccharides *Polymer sulphation (N- and O-sulphate groups) is 125 per 100 disaccharides
3. Heparan Sulphate fraction III: Mol wt approx. 9kDa
High Sulphate/Low Molecular Weight HS.
65 N-sulphate groups per 100 disaccharides 110 O-sulphate groups per 100 disaccharides *Polymer sulphation (N- and O-sulphate groups) is 175 per 100 disaccharide
4. Heparan Sulphate
Heparan Sulphate isolated from porcine mucosa. This material
contains both high and low sulphated species of HS and gives a bimodal
molecular weight profile with fractions of approx. 40kDa and 9kDa; the smaller
of the two fractions is the more sulphated component. This preparation has been
separated by preparative SAX-hplc to give the three HS fractions listed above.
Composition of HS Fractions
Heparan Sulphates Modified by Specific Chemical Desulphation
These heparan sulphates (HS) were prepared by targeted desulphation of a highly-purified preparation of porcine heparan sulphate derived from intestinal mucosa. This material also has a domain structure like that of cell surface HS. However note that this HS is a different preparation from the GAG-H01 that was used to prepare the HS fractions described above.
Standard chemical methods were used to selectively remove sulphate groups from C2 of iduronic acid (de2S HS), C6 of glucosamine (de6S HS) or the N-sulphate from N-sulphated glucosamine (deNS HS); the deNS HS contains free amino groups due to the loss of N-sulphates. In deNS/Ac HS the free amino groups have been acetylated.
Gel filtration profiles of the modified HS on Superdex S-75 hplc revealed only very minor reductions in molecular size compared to the parental material probably due to the loss of sulphate groups.
With the exception of 6-O desulphated HS the procedures lead to the loss of over 95% of the targeted sulphate groups.
6-O desulphated HS; in this preparation the reduction in 6-O-sulphate groups was 70%. 6-O-sulphates were completely removed from the trisulphated disaccharide (UA,2S – GlcNS,6S) but the targeted desulphation of UA – GlcNAc,6S and UA – GlcNS,6S was incomplete (50% and 70% loss of 6-O-sulphates respectively).
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Disaccharide analysis of selectively desulphated Heparan Sulphates: comparison of parental material with the desulphated preparations.