Substrate Specificity and Domain Functions of Extracellular Heparan Sulfate 6-O-Endosulfatases, QSulf1 and QSulf2

Xingbin Ai,Anh-Tri Do,Marion Kusche-Gullberg,Ulf Lindahl,Ke Lu,Charles P Emerson

doi:10.1074/jbc.m511902200

Abstract

The extracellular sulfatases (Sulfs) are an evolutionally conserved family of heparan sulfate (HS)-specific 6-O-endosulfatases. These enzymes remodel the 6-O-sulfation of cell surface HS chains to promote Wnt signaling and inhibit growth factor signaling for embryonic tissue patterning and control of tumor growth. In this study we demonstrate that the avian HS endosulfatases, QSulf1 and QSulf2, exhibit the same substrate specificity toward a subset of trisulfated disaccharides internal to HS chains. Further, we show that both QSulfs associate exclusively with cell membrane and are enzymatically active on the cell surface to desulfate both cell surface and cell matrix HS. Mutagenesis studies reveal that conserved amino acid regions in the hydrophilic domains of QSulf1 and QSulf2 have multiple functions, to anchor Sulf to the cell surface, bind to HS substrates, and to mediate HS 6-O-endosulfatase enzymatic activity. Results of our current studies establish the hydrophilic domain (HD) of Sulf enzymes as an essential multifunctional domain for their unique endosulfatase activities and also demonstrate the extracellular activity of Sulfs for desulfation of cell surface and cell matrix HS in the control of extracellular signaling for embryonic development and tumor progression.

Highlights

We have identified a novel family of extracellular heparan sulfate (HS) 6-O-sulfatases (Sulfs) that modify the 6-O-sulfation states of cell surface Heparan sulfate proteoglycans (HSPGs) to function as extracellular signaling regulators [10]
We have identified a novel family of extracellular HS 6-O-sulfatases (Sulfs) that modify the 6-O-sulfation states of cell surface HSPGs to
Similar results were obtained with comparable QSulf1HD deletions. These results indicate that hydrophilic domain (HD) domain is multifunctional, with conserved sequences required for cell surface association and GAG binding, as well as the enzyme activity of Sulfs

Summary

EXPERIMENTAL PROCEDURES

Cloning of QSulf cDNA—A stage 12 quail cDNA library was screened under low stringency conditions using a cDNA probe of the QSulf enzymatic domain. Cells were incubated with the appropriate secondary antibody in antibody dilution buffer (PBS, 0.1% Triton X-100, 20% goat serum) for 1 h at room temperature. Western Blot Analysis—Proteins in cell extracts (20 ␮g), concentrated condition medium, or on anti-Myc-agarose beads were separated on 10% SDS-polyacrylamide gel and transferred to a Hybond-P membrane. Heparan Sulfate Binding Assays—The QSulf2HD, QSulf2HDC, QSulf2HDS, and QSulf2HD⌬C14 was transiently expressed in 293T cells and subsequently purified from cell lysates (500 ␮l) with 25 ␮l of anti-Myc beads. After washing three times with PBS, the anti-Myc beads with bound QSulf2HD fragments were aliquotted into 10 ␮l and incubated with 5 ␮l of [35S]HS (ϳ2 ϫ 104 cpm) at room temperature for 1 h. The anti-Myc beads were subsequently washed with ice-cold PBS, and the 35S radioactivity retained on the beads was quantified with Beckman scintillation counter. Cells were cultured overnight in the ECM-coated wells, and the medium was quantified for released 35S radioactivity from ECM

RESULTS

Chondroitin sulfate

DISCUSSION