Heparan Sulphate Oligosaccharides

Description

Heparan Sulphate (HS) is a near-universal co-receptor for many growth factors and morphogenic proteins; it has a complex domain organisation of alternating high and low sulphated regions (Int J Exp Pathol (2015). 96:203-231.) in which variations in polymer sulphation and epimerisation lead to considerable structural diversity designed for selective binding to its many client proteins. We take advantage of the substrate specificities of Heparinase I (Hep I) and Heparinase III (Hep III) for the preparation of HS-oligos from regions of low and high sulphation in HS.

Separation of HS-oligosaccharides (HS-oligos) in the enzyme digests is achieved by high resolution preparative gel filtration of the Hep I or Hep III enzyme digests. The separated HS-oligo fractions are then analysed for disaccharide composition and molecular size homogeneity. Although substantially homogeneous by SEC Superdex S-75, HS-oligos in each size class contain sequences that vary in pattern and density of sulphation and there may be some limited overlap with oligos in adjacent size groups.

HS-oligos. from both Hep I and Hep III digests are available in the size range from dp4 to dp20 where dp is degree of polymerisation, a dp4 is a tetrasaccharide, dp6 a hexasaccharide, dp8 an octasaccharide etc. up to dp20.

High mol wt fractions (>6000 daltons) are also available.

Heparinase I:  HS-Oligos:Hep I

Enzyme specificity is for the NS-amino sugar linkage to 2-O-sulphated iduronic acid;

GlcNS (+/-6S) – IdoA,2S

Hep I acts exclusively in the sulphated regions of HS, the S-domains, resulting in oligosaccharides of low/intermediate sulphation that resist the action of this enzyme. These regions comprise over 50% of the HS chain, and include the N- and 6-O sulphated transition zones, but there is a dearth of information on their conformational features and biological properties. A simplified, general formula for HS-Oligos produced by Hep I is:

^*UA,2S – ^**GlcNS, – [GlcA – GlcNAc]_n=0-to-8 – IdoA  – **GlcNS

^*UA (uronic acid) at the non-reducing end contains a C4/C5 double bond due the eliminative action of Hep I

^**amino sugars may be sulphated at C6

Heparinase III:  HS-Oligos:Hep III

Enzyme specificity is for the amino sugar linkage to glucuronic acid:

GlcNAc/GlcNS – GlcA

Hep III targets the low sulphated regions of HS; oligos excised by Hep III are enriched in sulphated sequences derived from the more highly sulphated S-domains of HS.  A simplified, general formula for HS-oligos produced by Hep III is:

^*UA – GlcNS - [IdoA (+/-2S) – **GlcNS]_n=0-to-8 – IdoA  – GlcNAc

^*UA (uronic acid) at the non-reducing end contains a C4/C5 double bond due the eliminative action of Hep III

^**amino sugars may be sulphated at C6

 Merry et al., (1999), J. Biol. Chem. 274, 18455-18462