Biophysical investigation of human heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A: a mutual effect of enzyme oligomerisation and glycosaminoglycan ligand binding.
3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfate d-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.
Wille
Iris
I
Institute of Pharmaceutical Sciences, University of Graz, Universitätsplatz 1, A-8010 Graz, Austria.
Rek
Angelika
A
Krenn
Evelyn
E
Kungl
Andreas J
AJ
D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D006025:Glycosaminoglycans; D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; D015238:Sulfotransferases; C102180:heparan sulfate D-glucosaminyl 3-O-sulfotransferase