posted on 2021-11-30, 21:05authored byRylee Wander, Andrea M. Kaminski, Zhangjie Wang, Eduardo Stancanelli, Yongmei Xu, Vijayakanth Pagadala, Jine Li, Juno M. Krahn, Truong Quang Pham, Jian Liu, Lars C. Pedersen
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.