Limits...
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.

Show MeSH

Related in: MedlinePlus

QSulf1 is an HS sulfatase with substrate specificities distinct from lysosomal GlcNR6Sase. (A) QSulf1 is enzymatically active in [35S]SO4 release from metabolically labeled [35S]GAGs. [35S]GAGs were reacted overnight (16 h) with QSulf1 in cell lysate. Myc bead–purified QSulf1 is similarly active. Control lysates were prepared from 293 cells transfected with either pAG empty vector (Ctrl) or a catalytic mutant (QSulf1[C-A]–MycHis). [35S]SO4 release was assayed by spin column fractionation of reaction products and quantitation by scintillation counting. QSulf1 released ∼5% of total radioactivity from the [35S]GAGs, whereas control extracts had no activity. (B) QSulf1 desulfates HS-enriched GAGs. CS was depleted from [35S]GAG preparations by enzymatic digestion with chondroitinase ABC, which eliminated ∼22% of the labeled GAGs. QSulf1 released ∼7% of the [35S]SO4 from HS-enriched GAGs, equivalent to the loss from total [35S]GAG substrates. (C) QSulf1 does not desulfate CS-enriched GAGs. HS was depleted from [35S]GAG preparations by enzymatic digestion with heparinases I and II. QSulf1 is inactive in [35S]SO4 release from CS-enriched GAG substrate. (D) Glypican1 is a substrate for QSulf1. [35S]Glypican1–Myc was prepared by metabolically labeling Glypican1–Myc-transfected cells with [35S]SO4, followed by Myc bead purification, and subsequently used as the substrate in the enzymatic assay. QSulf1 released [35S]SO4 from [35S]Glypican1, whereas control extracts from 293 cells transfected with pAG empty vector or catalytic mutant QSulf1(C-A)–MycHis plasmids had no [35S]SO4 release activity. (E) QSulf1 is inactive in 6-O sulfate release on monosaccharide GlcNAc6S substrates. GlcNAc6S was reacted with control, QSulf1, or lysosomal GlcNR6Sase at pH 5.5 and 7.5, in the presence of PbCl2. QSulf1 did not desulfate GlcNAc6S, whereas lysosomal GlcNR6Sase was highly active at both pHs. (F) QSulf1 showed pH optima at pH 7.5. Metabolically labeled [35S]GAGs were reacted overnight (16 h) with Ctrl, QSulf1, or lysosomal GlcNR6Sase at pH 5.5, 7.5, and 9.5. QSulf1 was most active in [35S]SO4 release at pH 7.5, whereas GlcNR6Sase showed low or little activity toward [35S]GAGs under these pHs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172803&req=5

fig1: QSulf1 is an HS sulfatase with substrate specificities distinct from lysosomal GlcNR6Sase. (A) QSulf1 is enzymatically active in [35S]SO4 release from metabolically labeled [35S]GAGs. [35S]GAGs were reacted overnight (16 h) with QSulf1 in cell lysate. Myc bead–purified QSulf1 is similarly active. Control lysates were prepared from 293 cells transfected with either pAG empty vector (Ctrl) or a catalytic mutant (QSulf1[C-A]–MycHis). [35S]SO4 release was assayed by spin column fractionation of reaction products and quantitation by scintillation counting. QSulf1 released ∼5% of total radioactivity from the [35S]GAGs, whereas control extracts had no activity. (B) QSulf1 desulfates HS-enriched GAGs. CS was depleted from [35S]GAG preparations by enzymatic digestion with chondroitinase ABC, which eliminated ∼22% of the labeled GAGs. QSulf1 released ∼7% of the [35S]SO4 from HS-enriched GAGs, equivalent to the loss from total [35S]GAG substrates. (C) QSulf1 does not desulfate CS-enriched GAGs. HS was depleted from [35S]GAG preparations by enzymatic digestion with heparinases I and II. QSulf1 is inactive in [35S]SO4 release from CS-enriched GAG substrate. (D) Glypican1 is a substrate for QSulf1. [35S]Glypican1–Myc was prepared by metabolically labeling Glypican1–Myc-transfected cells with [35S]SO4, followed by Myc bead purification, and subsequently used as the substrate in the enzymatic assay. QSulf1 released [35S]SO4 from [35S]Glypican1, whereas control extracts from 293 cells transfected with pAG empty vector or catalytic mutant QSulf1(C-A)–MycHis plasmids had no [35S]SO4 release activity. (E) QSulf1 is inactive in 6-O sulfate release on monosaccharide GlcNAc6S substrates. GlcNAc6S was reacted with control, QSulf1, or lysosomal GlcNR6Sase at pH 5.5 and 7.5, in the presence of PbCl2. QSulf1 did not desulfate GlcNAc6S, whereas lysosomal GlcNR6Sase was highly active at both pHs. (F) QSulf1 showed pH optima at pH 7.5. Metabolically labeled [35S]GAGs were reacted overnight (16 h) with Ctrl, QSulf1, or lysosomal GlcNR6Sase at pH 5.5, 7.5, and 9.5. QSulf1 was most active in [35S]SO4 release at pH 7.5, whereas GlcNR6Sase showed low or little activity toward [35S]GAGs under these pHs.

Mentions: QSulf1 has sequence homology with the catalytic domain of GlcNR6Sase, a 6-O exosulfatase involved in the lysosomal catabolism of HS. Mutation of a critical Cys89 in the QSulf1 catalytic domain blocks its Wnt signaling activity, suggesting that QSulf1 is an enzymatically active sulfatase (Dhoot et al., 2001). To investigate the enzymatic activity of QSulf1 on sulfated glycosaminoglycan (GAG) substrates, 293 cells were metabolically labeled with [35S]SO4, and high molecular mass, 35S-labeled GAGs were isolated for enzymatic analysis. Myc-tagged QSulf1 expressed by transient transfection of 293 cells was isolated and purified 15-fold by affinity purification on Myc beads, as determined by Western blot analysis (unpublished data). QSulf1 in cell lysates and Myc bead–purified preparations was incubated with [35S]GAGs and then assayed for [35S]SO4 release using a spin column method to monitor enzyme activity. As controls, cell extracts were prepared from 293 cells transfected with either empty expression vector or a vector expressing mutant QSulf1(C-A), which has Ala substitutions at Cys89,90 to block N-formylglycine modification, which is required for catalytic activity of all sulfatases (Schmidt et al., 1995). Wild-type QSulf1 was enzymatically active and released ∼5% of [35S]SO4 from the [35S]GAG substrate (Fig. 1 A). QSulf1 activity is optimal at pH 7.5 (Fig. 1 F), requires Mg2+, is enhanced by Pb2+, and is inhibited by 25 mM phosphate or sulfate. Control cell extracts prepared from cultures transfected with pAG empty vector or mutant QSulf1(C-A) were inactive in [35S]SO4 release, confirming the requirement of that Cys89,90 and N-formylglycine modification for enzyme activity and showing that cell extracts do not have measurable endogenous sulfatase activities. As GAGs are a mixture of HS and chondroitin sulfate (CS), [35S]GAGs were differentially treated with heparinases I and II and chondroitinase ABC to selectively deplete HS or CS, which accounted for 78 and 22% of radioactivity in [35S]GAG preparations, respectively. QSulf1 was fully active in [35S]SO4 release from HS-enriched [35S]GAGs (Fig. 1 B) and was inactive on CS-enriched [35S]GAGs (Fig. 1 C), establishing that QSulf1 is an HS sulfatase. QSulf1 is also active in HS desulfation on metabolically labeled [35S]Glypican1 (Fig. 1 D), a cell surface HSPG that mediates Wnt signaling (Lin and Perrimon, 1999; Tsuda et al., 1999; Baeg et al., 2001), and, therefore, is the likely cellular substrate for QSulf1.


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 is an HS sulfatase with substrate specificities distinct from lysosomal GlcNR6Sase. (A) QSulf1 is enzymatically active in [35S]SO4 release from metabolically labeled [35S]GAGs. [35S]GAGs were reacted overnight (16 h) with QSulf1 in cell lysate. Myc bead–purified QSulf1 is similarly active. Control lysates were prepared from 293 cells transfected with either pAG empty vector (Ctrl) or a catalytic mutant (QSulf1[C-A]–MycHis). [35S]SO4 release was assayed by spin column fractionation of reaction products and quantitation by scintillation counting. QSulf1 released ∼5% of total radioactivity from the [35S]GAGs, whereas control extracts had no activity. (B) QSulf1 desulfates HS-enriched GAGs. CS was depleted from [35S]GAG preparations by enzymatic digestion with chondroitinase ABC, which eliminated ∼22% of the labeled GAGs. QSulf1 released ∼7% of the [35S]SO4 from HS-enriched GAGs, equivalent to the loss from total [35S]GAG substrates. (C) QSulf1 does not desulfate CS-enriched GAGs. HS was depleted from [35S]GAG preparations by enzymatic digestion with heparinases I and II. QSulf1 is inactive in [35S]SO4 release from CS-enriched GAG substrate. (D) Glypican1 is a substrate for QSulf1. [35S]Glypican1–Myc was prepared by metabolically labeling Glypican1–Myc-transfected cells with [35S]SO4, followed by Myc bead purification, and subsequently used as the substrate in the enzymatic assay. QSulf1 released [35S]SO4 from [35S]Glypican1, whereas control extracts from 293 cells transfected with pAG empty vector or catalytic mutant QSulf1(C-A)–MycHis plasmids had no [35S]SO4 release activity. (E) QSulf1 is inactive in 6-O sulfate release on monosaccharide GlcNAc6S substrates. GlcNAc6S was reacted with control, QSulf1, or lysosomal GlcNR6Sase at pH 5.5 and 7.5, in the presence of PbCl2. QSulf1 did not desulfate GlcNAc6S, whereas lysosomal GlcNR6Sase was highly active at both pHs. (F) QSulf1 showed pH optima at pH 7.5. Metabolically labeled [35S]GAGs were reacted overnight (16 h) with Ctrl, QSulf1, or lysosomal GlcNR6Sase at pH 5.5, 7.5, and 9.5. QSulf1 was most active in [35S]SO4 release at pH 7.5, whereas GlcNR6Sase showed low or little activity toward [35S]GAGs under these pHs.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: QSulf1 is an HS sulfatase with substrate specificities distinct from lysosomal GlcNR6Sase. (A) QSulf1 is enzymatically active in [35S]SO4 release from metabolically labeled [35S]GAGs. [35S]GAGs were reacted overnight (16 h) with QSulf1 in cell lysate. Myc bead–purified QSulf1 is similarly active. Control lysates were prepared from 293 cells transfected with either pAG empty vector (Ctrl) or a catalytic mutant (QSulf1[C-A]–MycHis). [35S]SO4 release was assayed by spin column fractionation of reaction products and quantitation by scintillation counting. QSulf1 released ∼5% of total radioactivity from the [35S]GAGs, whereas control extracts had no activity. (B) QSulf1 desulfates HS-enriched GAGs. CS was depleted from [35S]GAG preparations by enzymatic digestion with chondroitinase ABC, which eliminated ∼22% of the labeled GAGs. QSulf1 released ∼7% of the [35S]SO4 from HS-enriched GAGs, equivalent to the loss from total [35S]GAG substrates. (C) QSulf1 does not desulfate CS-enriched GAGs. HS was depleted from [35S]GAG preparations by enzymatic digestion with heparinases I and II. QSulf1 is inactive in [35S]SO4 release from CS-enriched GAG substrate. (D) Glypican1 is a substrate for QSulf1. [35S]Glypican1–Myc was prepared by metabolically labeling Glypican1–Myc-transfected cells with [35S]SO4, followed by Myc bead purification, and subsequently used as the substrate in the enzymatic assay. QSulf1 released [35S]SO4 from [35S]Glypican1, whereas control extracts from 293 cells transfected with pAG empty vector or catalytic mutant QSulf1(C-A)–MycHis plasmids had no [35S]SO4 release activity. (E) QSulf1 is inactive in 6-O sulfate release on monosaccharide GlcNAc6S substrates. GlcNAc6S was reacted with control, QSulf1, or lysosomal GlcNR6Sase at pH 5.5 and 7.5, in the presence of PbCl2. QSulf1 did not desulfate GlcNAc6S, whereas lysosomal GlcNR6Sase was highly active at both pHs. (F) QSulf1 showed pH optima at pH 7.5. Metabolically labeled [35S]GAGs were reacted overnight (16 h) with Ctrl, QSulf1, or lysosomal GlcNR6Sase at pH 5.5, 7.5, and 9.5. QSulf1 was most active in [35S]SO4 release at pH 7.5, whereas GlcNR6Sase showed low or little activity toward [35S]GAGs under these pHs.
Mentions: QSulf1 has sequence homology with the catalytic domain of GlcNR6Sase, a 6-O exosulfatase involved in the lysosomal catabolism of HS. Mutation of a critical Cys89 in the QSulf1 catalytic domain blocks its Wnt signaling activity, suggesting that QSulf1 is an enzymatically active sulfatase (Dhoot et al., 2001). To investigate the enzymatic activity of QSulf1 on sulfated glycosaminoglycan (GAG) substrates, 293 cells were metabolically labeled with [35S]SO4, and high molecular mass, 35S-labeled GAGs were isolated for enzymatic analysis. Myc-tagged QSulf1 expressed by transient transfection of 293 cells was isolated and purified 15-fold by affinity purification on Myc beads, as determined by Western blot analysis (unpublished data). QSulf1 in cell lysates and Myc bead–purified preparations was incubated with [35S]GAGs and then assayed for [35S]SO4 release using a spin column method to monitor enzyme activity. As controls, cell extracts were prepared from 293 cells transfected with either empty expression vector or a vector expressing mutant QSulf1(C-A), which has Ala substitutions at Cys89,90 to block N-formylglycine modification, which is required for catalytic activity of all sulfatases (Schmidt et al., 1995). Wild-type QSulf1 was enzymatically active and released ∼5% of [35S]SO4 from the [35S]GAG substrate (Fig. 1 A). QSulf1 activity is optimal at pH 7.5 (Fig. 1 F), requires Mg2+, is enhanced by Pb2+, and is inhibited by 25 mM phosphate or sulfate. Control cell extracts prepared from cultures transfected with pAG empty vector or mutant QSulf1(C-A) were inactive in [35S]SO4 release, confirming the requirement of that Cys89,90 and N-formylglycine modification for enzyme activity and showing that cell extracts do not have measurable endogenous sulfatase activities. As GAGs are a mixture of HS and chondroitin sulfate (CS), [35S]GAGs were differentially treated with heparinases I and II and chondroitinase ABC to selectively deplete HS or CS, which accounted for 78 and 22% of radioactivity in [35S]GAG preparations, respectively. QSulf1 was fully active in [35S]SO4 release from HS-enriched [35S]GAGs (Fig. 1 B) and was inactive on CS-enriched [35S]GAGs (Fig. 1 C), establishing that QSulf1 is an HS sulfatase. QSulf1 is also active in HS desulfation on metabolically labeled [35S]Glypican1 (Fig. 1 D), a cell surface HSPG that mediates Wnt signaling (Lin and Perrimon, 1999; Tsuda et al., 1999; Baeg et al., 2001), and, therefore, is the likely cellular substrate for QSulf1.

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