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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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A two-state catch or presentation model of QSulf1 regulation of Wnt signaling. (A) In QSulf1-nonexpressing embryonic cells, HS chains on cell surface HSPGs are in a 6-O–sulfated state, which binds with high affinities to catch Wnt ligands, preventing functional interactions of bound Wnts with their Frizzled receptors. (B) In QSulf1-expressing cells, selective 6-O desulfation activity of QSulf1 removes 6-O sulfates from HS chains on cell surface HSPGs to convert HS to a low affinity binding state for Wnts. 6-O–desulfated HS then can present Wnt ligands to Frizzled receptor and can form functionally active Wnt–HS–Frizzled receptor complexes for initiation of Wnt signal transduction.
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fig6: A two-state catch or presentation model of QSulf1 regulation of Wnt signaling. (A) In QSulf1-nonexpressing embryonic cells, HS chains on cell surface HSPGs are in a 6-O–sulfated state, which binds with high affinities to catch Wnt ligands, preventing functional interactions of bound Wnts with their Frizzled receptors. (B) In QSulf1-expressing cells, selective 6-O desulfation activity of QSulf1 removes 6-O sulfates from HS chains on cell surface HSPGs to convert HS to a low affinity binding state for Wnts. 6-O–desulfated HS then can present Wnt ligands to Frizzled receptor and can form functionally active Wnt–HS–Frizzled receptor complexes for initiation of Wnt signal transduction.

Mentions: The activity of QSulf1 in Wnt binding to HS was also tested in XWnt8 binding to Glypican1, a cell surface HSPG that mediates Wnt signaling (Lin and Perrimon, 1999; Tsuda et al., 1999; Baeg et al., 2001). A soluble form of Glypican1 tagged with alkaline phosphatase (AP) was used in the binding assay to allow Glypican1 purification from the media of transfected cells (Chen and Lander, 2001). HA-tagged XWnt8 was incubated with the soluble Glypican1–AP, and complexes formed were immunoprecipitated by agarose beads coupled with monoclonal AP antibodies, followed by probing Western blots of protein complexes with HA-specific antibodies (Fig. 5 D). Pretreatment of Glypican1 with heparinase completely blocked its binding to XWnt8, establishing that XWnt8 binding to Glypican1 is mediated through the HS chains and not the protein core. QSulf1 treatment of Glypican1–AP significantly diminished, but did not completely block, formation of HA–XWnt8 binding, compared with treatment with C-A mutant QSulf1, which did not diminish complex formation (Fig. 5 D). QSulf1 treatment also blocked the binding of Glypican1–AP to XWnt8 expressed on the surface of living transfected 293T cells, as detected by immunohistochemical detection in a cell binding assay (Hsieh et al., 1999; Wu and Nusse, 2002), whereas QSulf1(C-A) treatment did not prevent binding (Fig. 5 E). Control and C-A mutant QSulf1–treated Glypican1–AP abundantly bound to the surfaces of HA–XWnt8-expressing cells, as detected by double immunostaining with HA antibodies (unpublished data). Together, these Wnt binding studies reveal that QSulf1 6-O sulfatase activity reduces the binding of Wnt to HS on Glypican1. The results of these binding studies are consistent with a two-state catch or presentation model (Fig. 6) in which 6-O–sulfated HS on cell surface HSPGs binds Wnts in a high affinity state to “catch” Wnt ligands and compete with the binding of Wnts to Frizzled receptor (Fig. 6 A). The HS 6-O desulfation activity of QSulf1 would convert the cell surface HS to a low affinity binding state for Wnts, which allows their HS-dependent “presentation” to the Frizzled receptors to initiate Wnt signal transduction (Fig. 6 B). By this mechanism, QSulf1, which is expressed in localized populations of somite and neural progenitor cells, would pattern Wnt signaling responses to localized populations of progenitors responding to widely dispersed Wnt signals.


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)

A two-state catch or presentation model of QSulf1 regulation of Wnt signaling. (A) In QSulf1-nonexpressing embryonic cells, HS chains on cell surface HSPGs are in a 6-O–sulfated state, which binds with high affinities to catch Wnt ligands, preventing functional interactions of bound Wnts with their Frizzled receptors. (B) In QSulf1-expressing cells, selective 6-O desulfation activity of QSulf1 removes 6-O sulfates from HS chains on cell surface HSPGs to convert HS to a low affinity binding state for Wnts. 6-O–desulfated HS then can present Wnt ligands to Frizzled receptor and can form functionally active Wnt–HS–Frizzled receptor complexes for initiation of Wnt signal transduction.
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Related In: Results  -  Collection

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fig6: A two-state catch or presentation model of QSulf1 regulation of Wnt signaling. (A) In QSulf1-nonexpressing embryonic cells, HS chains on cell surface HSPGs are in a 6-O–sulfated state, which binds with high affinities to catch Wnt ligands, preventing functional interactions of bound Wnts with their Frizzled receptors. (B) In QSulf1-expressing cells, selective 6-O desulfation activity of QSulf1 removes 6-O sulfates from HS chains on cell surface HSPGs to convert HS to a low affinity binding state for Wnts. 6-O–desulfated HS then can present Wnt ligands to Frizzled receptor and can form functionally active Wnt–HS–Frizzled receptor complexes for initiation of Wnt signal transduction.
Mentions: The activity of QSulf1 in Wnt binding to HS was also tested in XWnt8 binding to Glypican1, a cell surface HSPG that mediates Wnt signaling (Lin and Perrimon, 1999; Tsuda et al., 1999; Baeg et al., 2001). A soluble form of Glypican1 tagged with alkaline phosphatase (AP) was used in the binding assay to allow Glypican1 purification from the media of transfected cells (Chen and Lander, 2001). HA-tagged XWnt8 was incubated with the soluble Glypican1–AP, and complexes formed were immunoprecipitated by agarose beads coupled with monoclonal AP antibodies, followed by probing Western blots of protein complexes with HA-specific antibodies (Fig. 5 D). Pretreatment of Glypican1 with heparinase completely blocked its binding to XWnt8, establishing that XWnt8 binding to Glypican1 is mediated through the HS chains and not the protein core. QSulf1 treatment of Glypican1–AP significantly diminished, but did not completely block, formation of HA–XWnt8 binding, compared with treatment with C-A mutant QSulf1, which did not diminish complex formation (Fig. 5 D). QSulf1 treatment also blocked the binding of Glypican1–AP to XWnt8 expressed on the surface of living transfected 293T cells, as detected by immunohistochemical detection in a cell binding assay (Hsieh et al., 1999; Wu and Nusse, 2002), whereas QSulf1(C-A) treatment did not prevent binding (Fig. 5 E). Control and C-A mutant QSulf1–treated Glypican1–AP abundantly bound to the surfaces of HA–XWnt8-expressing cells, as detected by double immunostaining with HA antibodies (unpublished data). Together, these Wnt binding studies reveal that QSulf1 6-O sulfatase activity reduces the binding of Wnt to HS on Glypican1. The results of these binding studies are consistent with a two-state catch or presentation model (Fig. 6) in which 6-O–sulfated HS on cell surface HSPGs binds Wnts in a high affinity state to “catch” Wnt ligands and compete with the binding of Wnts to Frizzled receptor (Fig. 6 A). The HS 6-O desulfation activity of QSulf1 would convert the cell surface HS to a low affinity binding state for Wnts, which allows their HS-dependent “presentation” to the Frizzled receptors to initiate Wnt signal transduction (Fig. 6 B). By this mechanism, QSulf1, which is expressed in localized populations of somite and neural progenitor cells, would pattern Wnt signaling responses to localized populations of progenitors responding to widely dispersed Wnt signals.

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