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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 desulfates cell surface HSPGs in living cells without affecting the stability of HSPG core proteins. (A) QSulf1 and chlorate desulfate cell surface HS, as assayed by IR to 10E4 antibody. Control 3T3 cells were transfected with empty vector, cultured with or without 25 mM chlorate to block sulfation, and then live cell stained with 10E4 antibody, and antibody reactivity was assayed by fluorescence microscopy. A majority of untransfected cells (Ctrl) express 10E4 IR on the cell surface, and chlorate treatment removes 10E4 IR. Cells transfected with QSulf1–MycHis or catalytic mutant QSulf1(C-A)–MycHis were live stained for extracellular 10E4 IR and then permeabilized to assay QSulf1 or QSulf1(C-A) with a His antibody. QSulf1-expressing cells (QSulf1) lose cell surface 10E4 IR, whereas QSulf1(C-A)-expressing cells remain immunoreactive, as shown in the overlay. Note that QSulf1 expression does not alter 10E4 IR on adjacent cells. An asterisk marks transfected cells and an arrow marks cells adjacent to QSulf1-expressing cells. (B) Quantitative analysis of the effects of QSulf1 expression and chlorate treatment on 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 or QSulf1(C-A). (C) QSulf1 expression does not alter the sulfation of cell surface CS. Extracellular CS was visualized with CS56 antibody in untransfected control (Ctrl) cultures treated with or without chlorate or in QSulf1-transfected cultures. An asterisk marks the cells transfected with QSulf1. Assays were conducted in duplicate in three independent experiments, counting >100 cells in each assay. (D) The protein core of Glypican1 remains on the cell surface of QSulf1-expressing cells and chlorate-treated cells. 3T3 cells cotransfected with Glypican1–Myc and untagged QSulf1 were live cell stained with Myc antibody to detect cell surface Glypican1–Myc. Cells were then permeabilized and immunostained for QSulf1 expression with QSulf1 antibody. Control cells were cotransfected with Glypican1–Myc and pAG empty vector plasmids, with or without chlorate treatment, followed by live cell staining to assay extracellular Glypican1–Myc. Similar Glypican1 staining was detected in control and QSulf1-transfected cells. (E) QSulf1 expression and chlorate treatment do not alter the stability or gel mobility of Glypican1. Western blot assays of cell extracts from 293 cells cotransfected with Glypican1–Myc, with pAG empty vector (Ctrl), QSulf1, or QSulf1(C-A) plasmids. Western blots were probed with anti-Myc and anti-QSulf1 antibodies.
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fig2: QSulf1 desulfates cell surface HSPGs in living cells without affecting the stability of HSPG core proteins. (A) QSulf1 and chlorate desulfate cell surface HS, as assayed by IR to 10E4 antibody. Control 3T3 cells were transfected with empty vector, cultured with or without 25 mM chlorate to block sulfation, and then live cell stained with 10E4 antibody, and antibody reactivity was assayed by fluorescence microscopy. A majority of untransfected cells (Ctrl) express 10E4 IR on the cell surface, and chlorate treatment removes 10E4 IR. Cells transfected with QSulf1–MycHis or catalytic mutant QSulf1(C-A)–MycHis were live stained for extracellular 10E4 IR and then permeabilized to assay QSulf1 or QSulf1(C-A) with a His antibody. QSulf1-expressing cells (QSulf1) lose cell surface 10E4 IR, whereas QSulf1(C-A)-expressing cells remain immunoreactive, as shown in the overlay. Note that QSulf1 expression does not alter 10E4 IR on adjacent cells. An asterisk marks transfected cells and an arrow marks cells adjacent to QSulf1-expressing cells. (B) Quantitative analysis of the effects of QSulf1 expression and chlorate treatment on 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 or QSulf1(C-A). (C) QSulf1 expression does not alter the sulfation of cell surface CS. Extracellular CS was visualized with CS56 antibody in untransfected control (Ctrl) cultures treated with or without chlorate or in QSulf1-transfected cultures. An asterisk marks the cells transfected with QSulf1. Assays were conducted in duplicate in three independent experiments, counting >100 cells in each assay. (D) The protein core of Glypican1 remains on the cell surface of QSulf1-expressing cells and chlorate-treated cells. 3T3 cells cotransfected with Glypican1–Myc and untagged QSulf1 were live cell stained with Myc antibody to detect cell surface Glypican1–Myc. Cells were then permeabilized and immunostained for QSulf1 expression with QSulf1 antibody. Control cells were cotransfected with Glypican1–Myc and pAG empty vector plasmids, with or without chlorate treatment, followed by live cell staining to assay extracellular Glypican1–Myc. Similar Glypican1 staining was detected in control and QSulf1-transfected cells. (E) QSulf1 expression and chlorate treatment do not alter the stability or gel mobility of Glypican1. Western blot assays of cell extracts from 293 cells cotransfected with Glypican1–Myc, with pAG empty vector (Ctrl), QSulf1, or QSulf1(C-A) plasmids. Western blots were probed with anti-Myc and anti-QSulf1 antibodies.

Mentions: To investigate whether QSulf1 is enzymatically active in vivo, we tested the effects of QSulf1 expression on 10E4 antibody immunoreactivity (IR) of cell surface HSPGs. 10E4 antibody specifically recognizes sulfated N-acetylglucosamine residues (David et al., 1992; Yip et al., 2002), and 10E4 IR to cell surface HSPGs is sensitive to chlorate treatment at concentrations that preferentially block 6-O sulfation (Safaiyan et al., 1999; Yip et al., 2002). 80% of 3T3 cells have cell surface 10E4 IR, as determined using a live cell staining assay, and chlorate treatment reduces 10E4 IR to <20% of cells (Fig. 2, A and B). Transfected QSulf1 also reduces 10E4 IR to a similar extent, whereas enzymatically inactive QSulf1(C-A) has no effect. Significantly, QSulf1 does not disrupt 10E4 IR on immediately adjacent cells (Fig. 2 A), even though QSulf1 is abundant on the surface of expressing cells. These observations indicate that QSulf1 functions cell autonomously to remodel the sulfation states of HSPGs on expressing cells. QSulf1 expression does not disrupt the sulfation state of CS on the cell surface, as assayed by live cell staining of QSulf1-transfected cells with a CS-specific antibody, CS56 (Avnur and Geiger, 1984), which recognizes sulfated epitopes also removed by chlorate treatment (Yip et al., 2002) (Fig. 2 C). These findings support the specificity of QSulf1 for HS, as observed in biochemical studies (Figs. 1, B and C).


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 desulfates cell surface HSPGs in living cells without affecting the stability of HSPG core proteins. (A) QSulf1 and chlorate desulfate cell surface HS, as assayed by IR to 10E4 antibody. Control 3T3 cells were transfected with empty vector, cultured with or without 25 mM chlorate to block sulfation, and then live cell stained with 10E4 antibody, and antibody reactivity was assayed by fluorescence microscopy. A majority of untransfected cells (Ctrl) express 10E4 IR on the cell surface, and chlorate treatment removes 10E4 IR. Cells transfected with QSulf1–MycHis or catalytic mutant QSulf1(C-A)–MycHis were live stained for extracellular 10E4 IR and then permeabilized to assay QSulf1 or QSulf1(C-A) with a His antibody. QSulf1-expressing cells (QSulf1) lose cell surface 10E4 IR, whereas QSulf1(C-A)-expressing cells remain immunoreactive, as shown in the overlay. Note that QSulf1 expression does not alter 10E4 IR on adjacent cells. An asterisk marks transfected cells and an arrow marks cells adjacent to QSulf1-expressing cells. (B) Quantitative analysis of the effects of QSulf1 expression and chlorate treatment on 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 or QSulf1(C-A). (C) QSulf1 expression does not alter the sulfation of cell surface CS. Extracellular CS was visualized with CS56 antibody in untransfected control (Ctrl) cultures treated with or without chlorate or in QSulf1-transfected cultures. An asterisk marks the cells transfected with QSulf1. Assays were conducted in duplicate in three independent experiments, counting >100 cells in each assay. (D) The protein core of Glypican1 remains on the cell surface of QSulf1-expressing cells and chlorate-treated cells. 3T3 cells cotransfected with Glypican1–Myc and untagged QSulf1 were live cell stained with Myc antibody to detect cell surface Glypican1–Myc. Cells were then permeabilized and immunostained for QSulf1 expression with QSulf1 antibody. Control cells were cotransfected with Glypican1–Myc and pAG empty vector plasmids, with or without chlorate treatment, followed by live cell staining to assay extracellular Glypican1–Myc. Similar Glypican1 staining was detected in control and QSulf1-transfected cells. (E) QSulf1 expression and chlorate treatment do not alter the stability or gel mobility of Glypican1. Western blot assays of cell extracts from 293 cells cotransfected with Glypican1–Myc, with pAG empty vector (Ctrl), QSulf1, or QSulf1(C-A) plasmids. Western blots were probed with anti-Myc and anti-QSulf1 antibodies.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172803&req=5

fig2: QSulf1 desulfates cell surface HSPGs in living cells without affecting the stability of HSPG core proteins. (A) QSulf1 and chlorate desulfate cell surface HS, as assayed by IR to 10E4 antibody. Control 3T3 cells were transfected with empty vector, cultured with or without 25 mM chlorate to block sulfation, and then live cell stained with 10E4 antibody, and antibody reactivity was assayed by fluorescence microscopy. A majority of untransfected cells (Ctrl) express 10E4 IR on the cell surface, and chlorate treatment removes 10E4 IR. Cells transfected with QSulf1–MycHis or catalytic mutant QSulf1(C-A)–MycHis were live stained for extracellular 10E4 IR and then permeabilized to assay QSulf1 or QSulf1(C-A) with a His antibody. QSulf1-expressing cells (QSulf1) lose cell surface 10E4 IR, whereas QSulf1(C-A)-expressing cells remain immunoreactive, as shown in the overlay. Note that QSulf1 expression does not alter 10E4 IR on adjacent cells. An asterisk marks transfected cells and an arrow marks cells adjacent to QSulf1-expressing cells. (B) Quantitative analysis of the effects of QSulf1 expression and chlorate treatment on 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 or QSulf1(C-A). (C) QSulf1 expression does not alter the sulfation of cell surface CS. Extracellular CS was visualized with CS56 antibody in untransfected control (Ctrl) cultures treated with or without chlorate or in QSulf1-transfected cultures. An asterisk marks the cells transfected with QSulf1. Assays were conducted in duplicate in three independent experiments, counting >100 cells in each assay. (D) The protein core of Glypican1 remains on the cell surface of QSulf1-expressing cells and chlorate-treated cells. 3T3 cells cotransfected with Glypican1–Myc and untagged QSulf1 were live cell stained with Myc antibody to detect cell surface Glypican1–Myc. Cells were then permeabilized and immunostained for QSulf1 expression with QSulf1 antibody. Control cells were cotransfected with Glypican1–Myc and pAG empty vector plasmids, with or without chlorate treatment, followed by live cell staining to assay extracellular Glypican1–Myc. Similar Glypican1 staining was detected in control and QSulf1-transfected cells. (E) QSulf1 expression and chlorate treatment do not alter the stability or gel mobility of Glypican1. Western blot assays of cell extracts from 293 cells cotransfected with Glypican1–Myc, with pAG empty vector (Ctrl), QSulf1, or QSulf1(C-A) plasmids. Western blots were probed with anti-Myc and anti-QSulf1 antibodies.
Mentions: To investigate whether QSulf1 is enzymatically active in vivo, we tested the effects of QSulf1 expression on 10E4 antibody immunoreactivity (IR) of cell surface HSPGs. 10E4 antibody specifically recognizes sulfated N-acetylglucosamine residues (David et al., 1992; Yip et al., 2002), and 10E4 IR to cell surface HSPGs is sensitive to chlorate treatment at concentrations that preferentially block 6-O sulfation (Safaiyan et al., 1999; Yip et al., 2002). 80% of 3T3 cells have cell surface 10E4 IR, as determined using a live cell staining assay, and chlorate treatment reduces 10E4 IR to <20% of cells (Fig. 2, A and B). Transfected QSulf1 also reduces 10E4 IR to a similar extent, whereas enzymatically inactive QSulf1(C-A) has no effect. Significantly, QSulf1 does not disrupt 10E4 IR on immediately adjacent cells (Fig. 2 A), even though QSulf1 is abundant on the surface of expressing cells. These observations indicate that QSulf1 functions cell autonomously to remodel the sulfation states of HSPGs on expressing cells. QSulf1 expression does not disrupt the sulfation state of CS on the cell surface, as assayed by live cell staining of QSulf1-transfected cells with a CS-specific antibody, CS56 (Avnur and Geiger, 1984), which recognizes sulfated epitopes also removed by chlorate treatment (Yip et al., 2002) (Fig. 2 C). These findings support the specificity of QSulf1 for HS, as observed in biochemical studies (Figs. 1, B and C).

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