Substrate specificity of 6-O-endosulfatase (Sulf-2) and its implications in synthesizing anticoagulant heparan sulfate.
Heparan sulfate (HS) 6-O-endosulfatase (Sulf) catalyzes the hydrolysis of 6-O-sulfo groups from HS polysaccharides. The resultant HS has reduced sulfation levels and displays altered biological activities. The Sulfs have been associated with several cancers and developmental problems and could function as a tool for editing specific HS structures. Here, we characterize the substrate specificity of human Sulf-2 using site-specifically radiolabeled synthetic polysaccharides. The enzyme was expressed and harvested from the conditioned medium of Chinese hamster ovary cells transfected with Sulf-2 expression plasmids. The uniquely [(35)S]sulfated polysaccharides were prepared using purified recombinant HS biosynthetic enzymes. We found that Sulf-2 is particularly effective in removing the 6-O-sulfo group residing in the trisulfated disaccharide repeating unit comprising 2-O-sulfated uronic acid and N-sulfated 6-O-sulfo glucosamine, but can also hydrolyze sulfo groups from N- and 6-O-sulfated disaccharides. In addition, we found that Sulf-2 treatment significantly decreases HS's ability to bind to platelet factor 4 (PF4), a chemokine, while binding to antithrombin is maintained. Because HS-PF4 complexes are the initiating cause of heparin-induced thrombocytopenia, this finding provides a promising strategy for developing heparin therapies with reduced side effects. Further understanding of Sulf-2 activity will help elucidate HS structure-function relationships and provide a valuable tool in tailoring HS-based anticoagulant drugs.
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, 27599, USA.
Elizabeth H
EH
Pempe
Tanya C
TC
Burch
Courtney J
CJ
Law
Jian
J
Liu
D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013429:Sulfatases; D015238:Sulfotransferases / Q000378:metabolism*
D000925:Anticoagulants; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D013429:Sulfatases; D015238:Sulfotransferases; C470792:SULF2 protein, human