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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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Golgi-targeted QSulf1 desulfates cell surface HSPGs and enhances Wnt1 signaling. (A) QSulf1 targeted to the Golgi apparatus or ER colocalizes with Golgi or ER markers. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were permeabilized and double stained with QSulf1 antibody and antibody against TGN 38 or ER resident protein PDI. QSulf1-Golgi and QSulf1-ER were localized in the Golgi apparatus and ER, respectively, as shown in overlay. (B) QSulf1 targeted to the Golgi apparatus or ER is enzymatically active on cellular HS substrate. QSulf1-Golgi and QSulf1-ER expressed in 293T cells by transient transfection were incubated with [35S]GAGs overnight (16 h) and released similar amounts of radioactivity as QSulf1. (C) Golgi-targeted, but not ER-targeted, QSulf1 decreases cell surface 10E4 IR. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were live cell stained with 10E4 antibody to assay cell surface 10E4 IR and then permeabilized and double stained with QSulf1 antibody. Expression of QSulf1-Golgi resulted in loss of 10E4 IR, whereas the expression of QSulf1-ER had no effect on 10E4 IR, as shown in overlay. An asterisk marks QSulf1-transfected cells. (D) QSulf1-Golgi expression decreases the percentage of cells with extracellular 10E4 IR. Cells stained with 10E4 were counted, and the percentage of 10E4 IR cells was calculated as the percent of total cells that were 10E4 stained in the control assay (Ctrl), or as the percent of transfected cells that expressed either QSulf1, QSulf1-Golgi, or QSulf1-ER. (E) QSulf1-Golgi enhances Wnt1 signaling activity. Lef/TCF luciferase reporter activity in C2C12 cells transfected with empty vector as control (Ctrl), QSulf1, QSulf1-Golgi, or QSulf1-ER. Luciferase activity was normalized to activities of extracts from control cells not induced by Wnt1.
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fig4: Golgi-targeted QSulf1 desulfates cell surface HSPGs and enhances Wnt1 signaling. (A) QSulf1 targeted to the Golgi apparatus or ER colocalizes with Golgi or ER markers. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were permeabilized and double stained with QSulf1 antibody and antibody against TGN 38 or ER resident protein PDI. QSulf1-Golgi and QSulf1-ER were localized in the Golgi apparatus and ER, respectively, as shown in overlay. (B) QSulf1 targeted to the Golgi apparatus or ER is enzymatically active on cellular HS substrate. QSulf1-Golgi and QSulf1-ER expressed in 293T cells by transient transfection were incubated with [35S]GAGs overnight (16 h) and released similar amounts of radioactivity as QSulf1. (C) Golgi-targeted, but not ER-targeted, QSulf1 decreases cell surface 10E4 IR. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were live cell stained with 10E4 antibody to assay cell surface 10E4 IR and then permeabilized and double stained with QSulf1 antibody. Expression of QSulf1-Golgi resulted in loss of 10E4 IR, whereas the expression of QSulf1-ER had no effect on 10E4 IR, as shown in overlay. An asterisk marks QSulf1-transfected cells. (D) QSulf1-Golgi expression decreases the percentage of cells with extracellular 10E4 IR. Cells stained with 10E4 were counted, and the percentage of 10E4 IR cells was calculated as the percent of total cells that were 10E4 stained in the control assay (Ctrl), or as the percent of transfected cells that expressed either QSulf1, QSulf1-Golgi, or QSulf1-ER. (E) QSulf1-Golgi enhances Wnt1 signaling activity. Lef/TCF luciferase reporter activity in C2C12 cells transfected with empty vector as control (Ctrl), QSulf1, QSulf1-Golgi, or QSulf1-ER. Luciferase activity was normalized to activities of extracts from control cells not induced by Wnt1.

Mentions: During biosynthesis, QSulf1 transits through the ER and Golgi apparatus, where HSPGs are assembled (Prydz and Dalen, 2000), and transports together with HSPGs to the surface of expressing cells (Dhoot et al., 2001; Ohto et al., 2002; unpublished data). To investigate whether QSulf1 localization on the cell surface is required for its activity, we expressed Golgi-tethered (QSulf1-Golgi) and ER-tethered (QSulf1-ER) forms of QSulf1 in 3T3 cells and C2C12 myoblasts and then assayed for their biochemical and cell biological activities in remodeling the sulfation states of HS and for Wnt induction activities. Neither QSulf1-Golgi nor QSulf1-ER is detected on the cell surfaces of expressing cells (unpublished data), but they colocalize with Golgi and ER markers, respectively (Fig. 4 A). ER- and Golgi-tethered QSulf1 isolated from expressing cells are enzymatically active in biochemical assays for sulfate release using [35S]GAG substrates (Fig. 4 B). QSulf1-ER protein is inactive in remodeling 10E4 IR and has little or no activity in Wnt signaling, whereas the QSulf1-Golgi protein is fully active in remodeling 10E4 IR as well as inducing Wnt signaling (Figs. 4, C–E). These findings establish that QSulf1 plays an indirect role in promoting Wnt signal transduction by remodeling the 6-O sulfation states of extracellular HSPGs, either through its activity on the cell surface or its activity in the Golgi apparatus during HS biosynthesis.


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)

Golgi-targeted QSulf1 desulfates cell surface HSPGs and enhances Wnt1 signaling. (A) QSulf1 targeted to the Golgi apparatus or ER colocalizes with Golgi or ER markers. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were permeabilized and double stained with QSulf1 antibody and antibody against TGN 38 or ER resident protein PDI. QSulf1-Golgi and QSulf1-ER were localized in the Golgi apparatus and ER, respectively, as shown in overlay. (B) QSulf1 targeted to the Golgi apparatus or ER is enzymatically active on cellular HS substrate. QSulf1-Golgi and QSulf1-ER expressed in 293T cells by transient transfection were incubated with [35S]GAGs overnight (16 h) and released similar amounts of radioactivity as QSulf1. (C) Golgi-targeted, but not ER-targeted, QSulf1 decreases cell surface 10E4 IR. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were live cell stained with 10E4 antibody to assay cell surface 10E4 IR and then permeabilized and double stained with QSulf1 antibody. Expression of QSulf1-Golgi resulted in loss of 10E4 IR, whereas the expression of QSulf1-ER had no effect on 10E4 IR, as shown in overlay. An asterisk marks QSulf1-transfected cells. (D) QSulf1-Golgi expression decreases the percentage of cells with extracellular 10E4 IR. Cells stained with 10E4 were counted, and the percentage of 10E4 IR cells was calculated as the percent of total cells that were 10E4 stained in the control assay (Ctrl), or as the percent of transfected cells that expressed either QSulf1, QSulf1-Golgi, or QSulf1-ER. (E) QSulf1-Golgi enhances Wnt1 signaling activity. Lef/TCF luciferase reporter activity in C2C12 cells transfected with empty vector as control (Ctrl), QSulf1, QSulf1-Golgi, or QSulf1-ER. Luciferase activity was normalized to activities of extracts from control cells not induced by Wnt1.
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Related In: Results  -  Collection

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fig4: Golgi-targeted QSulf1 desulfates cell surface HSPGs and enhances Wnt1 signaling. (A) QSulf1 targeted to the Golgi apparatus or ER colocalizes with Golgi or ER markers. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were permeabilized and double stained with QSulf1 antibody and antibody against TGN 38 or ER resident protein PDI. QSulf1-Golgi and QSulf1-ER were localized in the Golgi apparatus and ER, respectively, as shown in overlay. (B) QSulf1 targeted to the Golgi apparatus or ER is enzymatically active on cellular HS substrate. QSulf1-Golgi and QSulf1-ER expressed in 293T cells by transient transfection were incubated with [35S]GAGs overnight (16 h) and released similar amounts of radioactivity as QSulf1. (C) Golgi-targeted, but not ER-targeted, QSulf1 decreases cell surface 10E4 IR. 3T3 cells transfected with QSulf1-Golgi or QSulf1-ER were live cell stained with 10E4 antibody to assay cell surface 10E4 IR and then permeabilized and double stained with QSulf1 antibody. Expression of QSulf1-Golgi resulted in loss of 10E4 IR, whereas the expression of QSulf1-ER had no effect on 10E4 IR, as shown in overlay. An asterisk marks QSulf1-transfected cells. (D) QSulf1-Golgi expression decreases the percentage of cells with extracellular 10E4 IR. Cells stained with 10E4 were counted, and the percentage of 10E4 IR cells was calculated as the percent of total cells that were 10E4 stained in the control assay (Ctrl), or as the percent of transfected cells that expressed either QSulf1, QSulf1-Golgi, or QSulf1-ER. (E) QSulf1-Golgi enhances Wnt1 signaling activity. Lef/TCF luciferase reporter activity in C2C12 cells transfected with empty vector as control (Ctrl), QSulf1, QSulf1-Golgi, or QSulf1-ER. Luciferase activity was normalized to activities of extracts from control cells not induced by Wnt1.
Mentions: During biosynthesis, QSulf1 transits through the ER and Golgi apparatus, where HSPGs are assembled (Prydz and Dalen, 2000), and transports together with HSPGs to the surface of expressing cells (Dhoot et al., 2001; Ohto et al., 2002; unpublished data). To investigate whether QSulf1 localization on the cell surface is required for its activity, we expressed Golgi-tethered (QSulf1-Golgi) and ER-tethered (QSulf1-ER) forms of QSulf1 in 3T3 cells and C2C12 myoblasts and then assayed for their biochemical and cell biological activities in remodeling the sulfation states of HS and for Wnt induction activities. Neither QSulf1-Golgi nor QSulf1-ER is detected on the cell surfaces of expressing cells (unpublished data), but they colocalize with Golgi and ER markers, respectively (Fig. 4 A). ER- and Golgi-tethered QSulf1 isolated from expressing cells are enzymatically active in biochemical assays for sulfate release using [35S]GAG substrates (Fig. 4 B). QSulf1-ER protein is inactive in remodeling 10E4 IR and has little or no activity in Wnt signaling, whereas the QSulf1-Golgi protein is fully active in remodeling 10E4 IR as well as inducing Wnt signaling (Figs. 4, C–E). These findings establish that QSulf1 plays an indirect role in promoting Wnt signal transduction by remodeling the 6-O sulfation states of extracellular HSPGs, either through its activity on the cell surface or its activity in the Golgi apparatus during HS biosynthesis.

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