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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 selectively removes 6-O sulfates from HS in vivo. [35S]HS prepared from metabolically labeled stable 293T cell lines that expressed wild-type QSulf1 and enzymatically inactive QSulf1(C-A) mutant was prepared for disaccharide analysis. The resulting disaccharide fractions were resolved by HPLC anion exchange chromatography and analyzed as described previously. The arrows correspond to the elution positions of disaccharide components. (A) The disaccharide components of 293T cells that expressed inactive QSulf1(C-A) mutant protein. (B) The disaccharide components of QSulf1-expressing stable 293T cell lines. GMS, GlcA-GlcNS6S; IMS, IdoA-GlcNS6S; ISM, IdoA2S-GlcNS; ISMS, IdoA2S-GlcNS6S.
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fig3: QSulf1 selectively removes 6-O sulfates from HS in vivo. [35S]HS prepared from metabolically labeled stable 293T cell lines that expressed wild-type QSulf1 and enzymatically inactive QSulf1(C-A) mutant was prepared for disaccharide analysis. The resulting disaccharide fractions were resolved by HPLC anion exchange chromatography and analyzed as described previously. The arrows correspond to the elution positions of disaccharide components. (A) The disaccharide components of 293T cells that expressed inactive QSulf1(C-A) mutant protein. (B) The disaccharide components of QSulf1-expressing stable 293T cell lines. GMS, GlcA-GlcNS6S; IMS, IdoA-GlcNS6S; ISM, IdoA2S-GlcNS; ISMS, IdoA2S-GlcNS6S.

Mentions: [35S]HS prepared from metabolically labeled 293T cells was reacted with Myc bead–purified QSulf1 or catalytic mutant QSulf1(C-A). Untreated control was [35S]HS without treatment. To test whether QSulf1 functions in vivo, [35S]HS was prepared from stable 293T cell lines expressing QSulf1 or QSulf1(C-A) by metabolic labeling with [35S]SO4. 35S-labeled disaccharides were generated by deaminative cleavage of HS, and reaction products were resolved by HPLC anion exchange chromatography. The radioactivity in each disaccharide product was quantified by scintillation counting. Results are presented as mol-percent of specific disaccharide products in three independent experiments. M in disaccharide abbreviations stands for the 2,5-anhydromannitol deamination products of GlcNS residues (see also Fig. 3).


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 selectively removes 6-O sulfates from HS in vivo. [35S]HS prepared from metabolically labeled stable 293T cell lines that expressed wild-type QSulf1 and enzymatically inactive QSulf1(C-A) mutant was prepared for disaccharide analysis. The resulting disaccharide fractions were resolved by HPLC anion exchange chromatography and analyzed as described previously. The arrows correspond to the elution positions of disaccharide components. (A) The disaccharide components of 293T cells that expressed inactive QSulf1(C-A) mutant protein. (B) The disaccharide components of QSulf1-expressing stable 293T cell lines. GMS, GlcA-GlcNS6S; IMS, IdoA-GlcNS6S; ISM, IdoA2S-GlcNS; ISMS, IdoA2S-GlcNS6S.
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Related In: Results  -  Collection

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fig3: QSulf1 selectively removes 6-O sulfates from HS in vivo. [35S]HS prepared from metabolically labeled stable 293T cell lines that expressed wild-type QSulf1 and enzymatically inactive QSulf1(C-A) mutant was prepared for disaccharide analysis. The resulting disaccharide fractions were resolved by HPLC anion exchange chromatography and analyzed as described previously. The arrows correspond to the elution positions of disaccharide components. (A) The disaccharide components of 293T cells that expressed inactive QSulf1(C-A) mutant protein. (B) The disaccharide components of QSulf1-expressing stable 293T cell lines. GMS, GlcA-GlcNS6S; IMS, IdoA-GlcNS6S; ISM, IdoA2S-GlcNS; ISMS, IdoA2S-GlcNS6S.
Mentions: [35S]HS prepared from metabolically labeled 293T cells was reacted with Myc bead–purified QSulf1 or catalytic mutant QSulf1(C-A). Untreated control was [35S]HS without treatment. To test whether QSulf1 functions in vivo, [35S]HS was prepared from stable 293T cell lines expressing QSulf1 or QSulf1(C-A) by metabolic labeling with [35S]SO4. 35S-labeled disaccharides were generated by deaminative cleavage of HS, and reaction products were resolved by HPLC anion exchange chromatography. The radioactivity in each disaccharide product was quantified by scintillation counting. Results are presented as mol-percent of specific disaccharide products in three independent experiments. M in disaccharide abbreviations stands for the 2,5-anhydromannitol deamination products of GlcNS residues (see also Fig. 3).

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