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QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling.

Ai X, Do AT, Lozynska O, Kusche-Gullberg M, Lindahl U, Emerson CP - J. Cell Biol. (2003)

Bottom Line: In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus.QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling.CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

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QSulf1 regulates the interaction between HS and Wnt ligand to promote Wnt signaling. (A) 6-O– desulfated heparin does not inhibit Wnt signaling. C2C12 cells transfected with Tcf/LEF luciferase reporter to monitor Wnt signaling activity were stimulated by Wnt1-secreting cells in the presence of heparin or chemically 2-O–desulfated or 6-O–desulfated heparin. Luciferase activity was normalized to activities from cells cultured without heparin. 6-O–desulfated heparin had no effect on Wnt signaling, whereas heparin or 2-O– desulfated heparin completely inhibited Wnt signaling activity at a concentration of 10 μg/ml. (B) HS is required for Frizzled 3 receptor activation by Wnt1. Wild-type CHO cells or HS-deficient pgsd677 mutant CHO cells were transfected with a Frizzled 3 expression vector to initiate Wnt signaling. Expression of Frizzled 3 receptor alone did not activate Wnt signaling in wild-type CHO cells or mutant pgsd677 cells. Mutant pgsd677 cells are defective in Wnt1 signaling. (C) QSulf1 activity reduces the binding affinity between heparin and XWnt8. Heparin pretreated with QSulf1 or inactive QSulf1(C-A) mutant was incubated with HA–XWnt8 that was bound to heparin–agarose beads. The released HA–XWnt8 in the supernatant was measured by Western blot. Heparin treated with inactive QSulf1(C-A) mutant released HA–XWnt8 in a concentration-dependent manner, whereas QSulf1-desulfated heparin released much less HA–XWnt8. QSulf1 treatment reduced the competitive activity of heparin for HA–XWnt8 release consistently in four experiments, although the quantitative extent of research varied. (D) QSulf1 decreased XWnt8 binding to Glypican1. Glypican1–AP was treated by QSulf1, inactive QSulf1(C-A) mutant, or heparinase and then incubated with HA–XWnt8 to allow the binding. Glypican1-AP–HA-XWnt8 complex was separated by immunoprecipitation with AP antibodies and then analyzed by Western blot. QSulf1(C-A)-treated Glypican1 bound more HA–XWnt8 than QSulf1-treated Glypican1, and the binding was abolished by heparinase treatment. (E) QSulf1 decreased the binding of Glypican1 to HA–XWnt8-expressing cells. Glypican1–AP pretreated with either QSulf1 or QSulf1(C-A) was incubated with XWnt8-expressing cells. The bound Glypican1–AP on the cell surface was visualized by AP substrate staining.
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fig5: QSulf1 regulates the interaction between HS and Wnt ligand to promote Wnt signaling. (A) 6-O– desulfated heparin does not inhibit Wnt signaling. C2C12 cells transfected with Tcf/LEF luciferase reporter to monitor Wnt signaling activity were stimulated by Wnt1-secreting cells in the presence of heparin or chemically 2-O–desulfated or 6-O–desulfated heparin. Luciferase activity was normalized to activities from cells cultured without heparin. 6-O–desulfated heparin had no effect on Wnt signaling, whereas heparin or 2-O– desulfated heparin completely inhibited Wnt signaling activity at a concentration of 10 μg/ml. (B) HS is required for Frizzled 3 receptor activation by Wnt1. Wild-type CHO cells or HS-deficient pgsd677 mutant CHO cells were transfected with a Frizzled 3 expression vector to initiate Wnt signaling. Expression of Frizzled 3 receptor alone did not activate Wnt signaling in wild-type CHO cells or mutant pgsd677 cells. Mutant pgsd677 cells are defective in Wnt1 signaling. (C) QSulf1 activity reduces the binding affinity between heparin and XWnt8. Heparin pretreated with QSulf1 or inactive QSulf1(C-A) mutant was incubated with HA–XWnt8 that was bound to heparin–agarose beads. The released HA–XWnt8 in the supernatant was measured by Western blot. Heparin treated with inactive QSulf1(C-A) mutant released HA–XWnt8 in a concentration-dependent manner, whereas QSulf1-desulfated heparin released much less HA–XWnt8. QSulf1 treatment reduced the competitive activity of heparin for HA–XWnt8 release consistently in four experiments, although the quantitative extent of research varied. (D) QSulf1 decreased XWnt8 binding to Glypican1. Glypican1–AP was treated by QSulf1, inactive QSulf1(C-A) mutant, or heparinase and then incubated with HA–XWnt8 to allow the binding. Glypican1-AP–HA-XWnt8 complex was separated by immunoprecipitation with AP antibodies and then analyzed by Western blot. QSulf1(C-A)-treated Glypican1 bound more HA–XWnt8 than QSulf1-treated Glypican1, and the binding was abolished by heparinase treatment. (E) QSulf1 decreased the binding of Glypican1 to HA–XWnt8-expressing cells. Glypican1–AP pretreated with either QSulf1 or QSulf1(C-A) was incubated with XWnt8-expressing cells. The bound Glypican1–AP on the cell surface was visualized by AP substrate staining.

Mentions: To investigate the mechanisms by which QSulf1 6-O desulfation activity promotes Wnt signaling, we first tested the activity of soluble heparin and 6-O and 2-O chemically desulfated heparin on Wnt signal transduction in cells (Fig. 5 A). For these studies, Wnt1-expressing cells were cocultured with Wnt1-responsive C2C12 cells transfected with a TCF luciferase reporter to monitor Wnt signaling activity. At a concentration of 10 μg/ml, both soluble heparin and 2-O–desulfated heparin completely inhibit Wnt signaling activity, whereas 6-O–desulfated heparin has no inhibitory effect (Fig. 5 A). Therefore, soluble 6-O–sulfated HS could inhibit Wnt signaling by blocking the Frizzled receptor from binding the Wnt ligand. Alternatively, 6-O–sulfated HS could bind to Wnts and prevent Wnt presentation to its Frizzled receptor. To investigate whether HS blocks Frizzled receptor activity, we compared the Wnt1 signaling activity in wild-type CHO cells that express HSPGs and in pgsd677 mutant CHO cells that are defective in HS biosynthesis (Bai et al., 1999). Wnt1 signaling in CHO cells is dependent on expression of a transfected Frizzled 3 (mFZ3) receptor, which increases the response of these cells to Wnt1 by 12–15-fold in a coculture system with Wnt1-expressing cells, as assayed using a LEF/TCF luciferase reporter (unpublished data). CHO cells are dependent on transfected mFZ3 receptor for Wnt1 signal transduction, which makes it possible to directly monitor the requirement for HS in the response of Frizzled receptor to Wnt signal. In these mFZ3-dependent Wnt signaling assays, wild-type and pgsd677 mutant CHO cells were found to be inactive in Wnt1 signal transduction in the absence of Wnt1 ligand (Fig. 5 B), whereas in response to Wnt1 ligand, mFZ3-expressing wild-type CHO cells more actively respond to Wnt1 than do HS-deficient pgsd677 cells (which have only a slightly elevated response to Wnt1 compared with unstimulated control cells) (Fig. 5 B). These findings show that HS does not act as a general repressor of ligand-independent mFZ3 receptor activity. On the contrary, they provide evidence that HS is required for efficient mFZ3 receptor activation in response to Wnt1 ligand. Therefore, soluble sulfated heparin inhibits Wnt signaling probably by binding to Wnt ligand and subsequently preventing the release of the Wnt ligand to the receptor.


QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling.

Ai X, Do AT, Lozynska O, Kusche-Gullberg M, Lindahl U, Emerson CP - J. Cell Biol. (2003)

QSulf1 regulates the interaction between HS and Wnt ligand to promote Wnt signaling. (A) 6-O– desulfated heparin does not inhibit Wnt signaling. C2C12 cells transfected with Tcf/LEF luciferase reporter to monitor Wnt signaling activity were stimulated by Wnt1-secreting cells in the presence of heparin or chemically 2-O–desulfated or 6-O–desulfated heparin. Luciferase activity was normalized to activities from cells cultured without heparin. 6-O–desulfated heparin had no effect on Wnt signaling, whereas heparin or 2-O– desulfated heparin completely inhibited Wnt signaling activity at a concentration of 10 μg/ml. (B) HS is required for Frizzled 3 receptor activation by Wnt1. Wild-type CHO cells or HS-deficient pgsd677 mutant CHO cells were transfected with a Frizzled 3 expression vector to initiate Wnt signaling. Expression of Frizzled 3 receptor alone did not activate Wnt signaling in wild-type CHO cells or mutant pgsd677 cells. Mutant pgsd677 cells are defective in Wnt1 signaling. (C) QSulf1 activity reduces the binding affinity between heparin and XWnt8. Heparin pretreated with QSulf1 or inactive QSulf1(C-A) mutant was incubated with HA–XWnt8 that was bound to heparin–agarose beads. The released HA–XWnt8 in the supernatant was measured by Western blot. Heparin treated with inactive QSulf1(C-A) mutant released HA–XWnt8 in a concentration-dependent manner, whereas QSulf1-desulfated heparin released much less HA–XWnt8. QSulf1 treatment reduced the competitive activity of heparin for HA–XWnt8 release consistently in four experiments, although the quantitative extent of research varied. (D) QSulf1 decreased XWnt8 binding to Glypican1. Glypican1–AP was treated by QSulf1, inactive QSulf1(C-A) mutant, or heparinase and then incubated with HA–XWnt8 to allow the binding. Glypican1-AP–HA-XWnt8 complex was separated by immunoprecipitation with AP antibodies and then analyzed by Western blot. QSulf1(C-A)-treated Glypican1 bound more HA–XWnt8 than QSulf1-treated Glypican1, and the binding was abolished by heparinase treatment. (E) QSulf1 decreased the binding of Glypican1 to HA–XWnt8-expressing cells. Glypican1–AP pretreated with either QSulf1 or QSulf1(C-A) was incubated with XWnt8-expressing cells. The bound Glypican1–AP on the cell surface was visualized by AP substrate staining.
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Related In: Results  -  Collection

Show All Figures
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fig5: QSulf1 regulates the interaction between HS and Wnt ligand to promote Wnt signaling. (A) 6-O– desulfated heparin does not inhibit Wnt signaling. C2C12 cells transfected with Tcf/LEF luciferase reporter to monitor Wnt signaling activity were stimulated by Wnt1-secreting cells in the presence of heparin or chemically 2-O–desulfated or 6-O–desulfated heparin. Luciferase activity was normalized to activities from cells cultured without heparin. 6-O–desulfated heparin had no effect on Wnt signaling, whereas heparin or 2-O– desulfated heparin completely inhibited Wnt signaling activity at a concentration of 10 μg/ml. (B) HS is required for Frizzled 3 receptor activation by Wnt1. Wild-type CHO cells or HS-deficient pgsd677 mutant CHO cells were transfected with a Frizzled 3 expression vector to initiate Wnt signaling. Expression of Frizzled 3 receptor alone did not activate Wnt signaling in wild-type CHO cells or mutant pgsd677 cells. Mutant pgsd677 cells are defective in Wnt1 signaling. (C) QSulf1 activity reduces the binding affinity between heparin and XWnt8. Heparin pretreated with QSulf1 or inactive QSulf1(C-A) mutant was incubated with HA–XWnt8 that was bound to heparin–agarose beads. The released HA–XWnt8 in the supernatant was measured by Western blot. Heparin treated with inactive QSulf1(C-A) mutant released HA–XWnt8 in a concentration-dependent manner, whereas QSulf1-desulfated heparin released much less HA–XWnt8. QSulf1 treatment reduced the competitive activity of heparin for HA–XWnt8 release consistently in four experiments, although the quantitative extent of research varied. (D) QSulf1 decreased XWnt8 binding to Glypican1. Glypican1–AP was treated by QSulf1, inactive QSulf1(C-A) mutant, or heparinase and then incubated with HA–XWnt8 to allow the binding. Glypican1-AP–HA-XWnt8 complex was separated by immunoprecipitation with AP antibodies and then analyzed by Western blot. QSulf1(C-A)-treated Glypican1 bound more HA–XWnt8 than QSulf1-treated Glypican1, and the binding was abolished by heparinase treatment. (E) QSulf1 decreased the binding of Glypican1 to HA–XWnt8-expressing cells. Glypican1–AP pretreated with either QSulf1 or QSulf1(C-A) was incubated with XWnt8-expressing cells. The bound Glypican1–AP on the cell surface was visualized by AP substrate staining.
Mentions: To investigate the mechanisms by which QSulf1 6-O desulfation activity promotes Wnt signaling, we first tested the activity of soluble heparin and 6-O and 2-O chemically desulfated heparin on Wnt signal transduction in cells (Fig. 5 A). For these studies, Wnt1-expressing cells were cocultured with Wnt1-responsive C2C12 cells transfected with a TCF luciferase reporter to monitor Wnt signaling activity. At a concentration of 10 μg/ml, both soluble heparin and 2-O–desulfated heparin completely inhibit Wnt signaling activity, whereas 6-O–desulfated heparin has no inhibitory effect (Fig. 5 A). Therefore, soluble 6-O–sulfated HS could inhibit Wnt signaling by blocking the Frizzled receptor from binding the Wnt ligand. Alternatively, 6-O–sulfated HS could bind to Wnts and prevent Wnt presentation to its Frizzled receptor. To investigate whether HS blocks Frizzled receptor activity, we compared the Wnt1 signaling activity in wild-type CHO cells that express HSPGs and in pgsd677 mutant CHO cells that are defective in HS biosynthesis (Bai et al., 1999). Wnt1 signaling in CHO cells is dependent on expression of a transfected Frizzled 3 (mFZ3) receptor, which increases the response of these cells to Wnt1 by 12–15-fold in a coculture system with Wnt1-expressing cells, as assayed using a LEF/TCF luciferase reporter (unpublished data). CHO cells are dependent on transfected mFZ3 receptor for Wnt1 signal transduction, which makes it possible to directly monitor the requirement for HS in the response of Frizzled receptor to Wnt signal. In these mFZ3-dependent Wnt signaling assays, wild-type and pgsd677 mutant CHO cells were found to be inactive in Wnt1 signal transduction in the absence of Wnt1 ligand (Fig. 5 B), whereas in response to Wnt1 ligand, mFZ3-expressing wild-type CHO cells more actively respond to Wnt1 than do HS-deficient pgsd677 cells (which have only a slightly elevated response to Wnt1 compared with unstimulated control cells) (Fig. 5 B). These findings show that HS does not act as a general repressor of ligand-independent mFZ3 receptor activity. On the contrary, they provide evidence that HS is required for efficient mFZ3 receptor activation in response to Wnt1 ligand. Therefore, soluble sulfated heparin inhibits Wnt signaling probably by binding to Wnt ligand and subsequently preventing the release of the Wnt ligand to the receptor.

Bottom Line: In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus.QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling.CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

ABSTRACT
The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.

Show MeSH
Related in: MedlinePlus