Biochim Biophys Acta. 2013 Nov;1830(11):5287-98. doi: 10.1016/j.bbagen.2013.07.014. Epub 2013 Jul 25.

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.

Biochimica et biophysica acta

Fabian Milz, Alexander Harder, Phillipp Neuhaus, Olga Breitkreuz-Korff, Volker Walhorn, Torben Lübke, Dario Anselmetti, Thomas Dierks

PMID: 23891937 DOI: 10.1016/j.bbagen.2013.07.014

Abstract

BACKGROUND: Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 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 Sulf1 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 Sulf1 bound significantly weaker to HS after enzymatic 6-O-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 6-O-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 Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance KD-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 Sulf1 along or across HS chains.

GENERAL SIGNIFICANCE: HD confers a novel and highly dynamic mode of protein interaction with HS.

© 2013.

Keywords: 1,9-dimethylmethylene blue; 2O-sulfate; 2S; 6-O-desulfated HS; 6O-sulfate; 6S; AFM; AFM-single-molecule force-spectroscopy; CS; DMMB; DS; DSMFS; ECM; FGF; GAG; GST; GlcA; GlcNAc; GlcNS; Glycosaminoglycans; HD; HRP; HS (HS+6S); HSPG; HS−6S; Heparan sulfate; IdoA; K5_NS; MBP; N-acetylglucosamine; N-sulfate; N-sulfated K5-polysaccharide; N-sulfoglucosamine; NS; Proteoglycans; SAX-HPLC; SDS; SMFS; SPR; Sulf1; Sulfatase; UA; atomic force microscopy; chondroitin sulfate; dermatan sulfate; dynamic SMFS; extracellular matrix; fibroblast growth factor; glucuronic acid; glutathione S-transferase; glycosaminoglycan; heparan sulfate; heparan sulfate proteoglycan; hexuronic acid; horseradish peroxidase; hydrophilic domain; iduronic acid; maltose binding protein; single molecule-force spectroscopy; sodium dodecyl sulfate; strong anion exchange-high performance liquid chromatography; surface plasmon resonance

Substances

• Glycosaminoglycans
• A73025
• Heparin
• Chondroitin Sulfates
• Heparitin Sulfate
• SULF1 protein, human
• Sulfotransferases

MeSH terms

• Binding Sites
• Chondroitin Sulfates / metabolism
• Glycosaminoglycans / metabolism
• Heparin / metabolism
• Heparitin Sulfate / metabolism
• Humans
• Hydrophobic and Hydrophilic Interactions
• Kinetics
• Protein Binding
• Sulfotransferases / metabolism

Publication Types

• Journal Article
• Research Support, Non-U.S. Gov't