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Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY - Elife (2015)

Bottom Line: These results explain numerous disease-associated mutations.Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition.The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

No MeSH data available.


Related in: MedlinePlus

Expression and purification of biologically active recombinant Norrin.(A) Schematic diagrams of the expression constructs including Norrin (a signal peptide, SP, followed by Norrin and Rho-1D4 tag at C-terminus) and SUMO-Norrin (a SP followed by a Strep-tag II, an octahistidine, SUMO, HRV 3C protease cleavage site, Norrin, and Rho-1D4 tag at C-terminus). (B and C) Conditioned media from transfected HEK293T cells were immunoblotted (IB) with the anti-Rho-1D4 antibody. (B) SUMO fusion improves Norrin secreted expression. (C) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid. (D) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines. (E) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).DOI:http://dx.doi.org/10.7554/eLife.06554.003
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fig1: Expression and purification of biologically active recombinant Norrin.(A) Schematic diagrams of the expression constructs including Norrin (a signal peptide, SP, followed by Norrin and Rho-1D4 tag at C-terminus) and SUMO-Norrin (a SP followed by a Strep-tag II, an octahistidine, SUMO, HRV 3C protease cleavage site, Norrin, and Rho-1D4 tag at C-terminus). (B and C) Conditioned media from transfected HEK293T cells were immunoblotted (IB) with the anti-Rho-1D4 antibody. (B) SUMO fusion improves Norrin secreted expression. (C) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid. (D) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines. (E) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).DOI:http://dx.doi.org/10.7554/eLife.06554.003

Mentions: To address the challenge of producing Norrin in large quantities, we screened conditions and constructs for Norrin expression (Figure 1A). We found that fusion of Norrin to the C-terminus of small ubiquitin-like modifier (SUMO) (Peroutka et al., 2008), in combination with addition of valproic acid (Backliwal et al., 2008), a putative histone deacetylase inhibitor, substantially boosted expression of the secreted protein in human embryonic kidney (HEK) 293T cells (Figure 1B,C). After removal of the SUMO fusion tag, the recombinant Norrin shows a monodispersed state in size-exclusion chromatography (SEC; Figure 1D) and is biologically active in a cell-based luciferase reporter assay (Figure 1E).10.7554/eLife.06554.003Figure 1.Expression and purification of biologically active recombinant Norrin.


Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY - Elife (2015)

Expression and purification of biologically active recombinant Norrin.(A) Schematic diagrams of the expression constructs including Norrin (a signal peptide, SP, followed by Norrin and Rho-1D4 tag at C-terminus) and SUMO-Norrin (a SP followed by a Strep-tag II, an octahistidine, SUMO, HRV 3C protease cleavage site, Norrin, and Rho-1D4 tag at C-terminus). (B and C) Conditioned media from transfected HEK293T cells were immunoblotted (IB) with the anti-Rho-1D4 antibody. (B) SUMO fusion improves Norrin secreted expression. (C) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid. (D) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines. (E) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).DOI:http://dx.doi.org/10.7554/eLife.06554.003
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4497409&req=5

fig1: Expression and purification of biologically active recombinant Norrin.(A) Schematic diagrams of the expression constructs including Norrin (a signal peptide, SP, followed by Norrin and Rho-1D4 tag at C-terminus) and SUMO-Norrin (a SP followed by a Strep-tag II, an octahistidine, SUMO, HRV 3C protease cleavage site, Norrin, and Rho-1D4 tag at C-terminus). (B and C) Conditioned media from transfected HEK293T cells were immunoblotted (IB) with the anti-Rho-1D4 antibody. (B) SUMO fusion improves Norrin secreted expression. (C) The expression level of SUMO tagged Norrin was further boosted for HEK-293T cells treated with valproic acid. (D) SEC elution profile and SDS-PAGE under reducing conditions with fractions analysed marked by red lines. (E) Purified recombinant untagged Norrin actives the canonical Wnt/β-catenin pathway in the luciferase reporter assay. RLU: relative light unit. Error bars indicate standard deviations (n = 3).DOI:http://dx.doi.org/10.7554/eLife.06554.003
Mentions: To address the challenge of producing Norrin in large quantities, we screened conditions and constructs for Norrin expression (Figure 1A). We found that fusion of Norrin to the C-terminus of small ubiquitin-like modifier (SUMO) (Peroutka et al., 2008), in combination with addition of valproic acid (Backliwal et al., 2008), a putative histone deacetylase inhibitor, substantially boosted expression of the secreted protein in human embryonic kidney (HEK) 293T cells (Figure 1B,C). After removal of the SUMO fusion tag, the recombinant Norrin shows a monodispersed state in size-exclusion chromatography (SEC; Figure 1D) and is biologically active in a cell-based luciferase reporter assay (Figure 1E).10.7554/eLife.06554.003Figure 1.Expression and purification of biologically active recombinant Norrin.

Bottom Line: These results explain numerous disease-associated mutations.Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition.The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

View Article: PubMed Central - PubMed

Affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

No MeSH data available.


Related in: MedlinePlus