Structural and Substrate Specificity Analysis of 3-<i>O</i>-Sulfotransferase Isoform 5 to Synthesize Heparan Sulfate
Rylee Wander, Andrea M. Kaminski, Zhangjie Wang, Eduardo Stancanelli, Yongmei Xu, Vijayakanth Pagadala, Jine Li, J.M. Krahn, Truong Quang Pham, Jian Liu, Lars C. Pedersen
Abstract
Heparan sulfate 3-O-sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3-O-sulfated heparan sulfate. 3-O-sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST isoform 5 (3-OST-5) with 3′-phosphoadenosine-5′-phosphate (PAP) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3-O-sulfated 8-mers. Results from the structural analysis of the six 3-O-sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6-O-sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6-O-sulfo glucosamine saccharide that is surrounded by 2-O-sulfated iduronic acid. 3-OST-5-modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2-O-sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding of how to control the biosynthesis of 3-O-sulfated heparan sulfate with the desired biological activities.