Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background: Sulfl is a cell-surface sulfatase removing internal 6-0-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulfl employs a unique hydrophilic domain (HD). Methods: Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction. Results: Full-length Sulfl interacts broadly with sulfated glycosaminoglycans (GAGS) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulfl bound significantly weaker to HS after enzymatic 6-0-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 60-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., -500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force-distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD. Conclusions: Specific substrate binding of Sal is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K-D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell-surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulfl along or across HS chains. General significance: HD confers a novel and highly dynamic mode of protein interaction with HS. (C) 2013 Elsevier B.V. All rights reserved.