Heparinase III (Hep III/Heparitinase) EC 18.104.22.168
Degrades heparan sulfate (HS)
Catalyses the eliminative scission of glycosidic linkages between N-sulfated or N-acetylated glucosamine (GlcNSO3 or GlcNAc) and glucuronic acid
Heparinase III (Heparitinase) acts on Heparan Sulphate in regions of low sulfation and it has little activity against heparin. The preferred substrates for heparinase III are N-Acetylated or N-sulfated glucosamines linked to glucuronic acid. (i.e. GlcNAc/ GlcNSO3 1 – 4 GlcA). In consequence it acts primarily in the non-sulfated domains (NAc domains) and transition zones of HS (see fig. 1) whereas the highly sulfated S-domains are resistant to Heparinase III. The enzyme tolerates C-6 sulfation of amino sugars. Heparinase III has weak activity with disaccharides containing Iduronic Acid (IdoA) and its action is blocked if the iduronate residue is sulfated at C-2 (IdoA, 2SO3).
The Heparinase III resistant S-domains in HS have a heparin-like structure being mainly composed of GlcNSO3 1 – 4 IdoA,2SO3 repeat units with variable O-sulfation at C-6 of the GlcNSO3 residue. They vary in length form 3 to 9 disaccharide units. The patterns and densities of 6-O-sulfate groups along S-domains are an important determinant of their protein binding specificities.
The selectivity of Heparinase III can be exploited for the preparation of S-domains from HS. High resolution gel filtration will separate the different size classes of S-domain and these can be further subfractionated by SAX-HPLC according to sulfate content and patterning. In this way a considerable amount of information can be acquired about the structure of HS. Isolated S-domains can be used for protein binding or bioactivity studies or for structural analysis by NMR. New developments in mass spectrometry are being applied to the analysis of composition and sequence of GAG fragments including HS S-domains and heparin oligomers. These methods hold great promise for thorough structural elucidation of HS/heparin and other GAGS.