EDAR Human, Sf9

What is EDAR Human, Sf9 and how is it structurally characterized?

EDAR Human produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 413 amino acids (residues 27-187) with a molecular mass of 45.6kDa. When analyzed via SDS-PAGE, it typically appears at approximately 40-57kDa. The commercially available form is expressed with a 249 amino acid hIgG-His-tag at the C-Terminus and is purified using proprietary chromatographic techniques .

Structurally, the extracellular region of EDAR consists of three cysteine-rich domains (CRDs) and adopts an extended conformation spanning approximately 65 Å in length. Each EDAR CRD binds across the convex surface of a single EDA·A1 THD (TNF homology domain) in the trimer, burying about 1340 Ų of total solvent-accessible surface area at the intermolecular interface .

What is the functional significance of EDAR in developmental biology?

EDAR plays a conserved role in the development of ectodermally derived organs across species. In both humans and mice, loss of function mutations in the genes coding for the ligand EDA-A1, EDAR, and EDARADD lead to strikingly similar phenotypes characterized by defective hair development, absence of eccrine glands, and missing or misshapen teeth .

The receptor is part of the TNF receptor superfamily and has a unique 1:1 binding mode with its ligand EDA·A1, which is distinctly different from other TNF-receptor complex structures where each receptor typically binds into the concaved groove formed by two adjacent ligand protomers .

What are the optimal expression conditions for producing functional EDAR in Sf9 cells?

When expressing EDAR in Sf9 baculovirus cells, researchers should consider the following methodological approach:

• Vector selection: The protein coding sequence should be optimized for expression in insect cells and cloned into a suitable vector containing a strong promoter (such as polyhedrin or p10) for high-level expression.

• Protein tags: Expression with a C-terminal tag (such as the 249 amino acid hIgG-His-tag used in commercial preparations) facilitates purification while preserving the native N-terminal structure critical for ligand binding .

• Post-translational modifications: Monitor glycosylation status, as EDAR typically exhibits glycosylation when expressed in Sf9 cells. PNGase F treatment can be used to assess glycosylation patterns, as demonstrated in EDA·A1 studies where both glycosylated and non-glycosylated forms were observed .

• Culture conditions: Maintain Sf9 cells at 27°C in serum-free medium optimized for insect cell expression to maximize protein yield while preserving proper folding.

• Purification strategy: Implement a multi-step chromatography approach, typically involving affinity chromatography (utilizing the His-tag), followed by size exclusion and/or ion exchange chromatography to achieve high purity.

How can researchers validate the functional activity of purified EDAR Human, Sf9?

Functional validation of purified EDAR should include:

• Binding assays: Surface plasmon resonance (SPR) or pull-down assays to measure the interaction between EDAR and its ligand EDA·A1. The binding affinity and kinetics should be consistent with published values .

• Structural integrity assessment: Circular dichroism (CD) spectroscopy to confirm proper folding of the protein's secondary structure.

• Signaling pathway activation: Cell-based assays measuring NF-κB activation following ligand binding, as EDAR is known to signal through this pathway .

• Thermal stability testing: Differential scanning fluorimetry (DSF) to assess protein stability and batch-to-batch consistency.

• Glycosylation analysis: Western blot analysis with and without PNGase F treatment to characterize glycosylation patterns, which may affect function .

How can EDAR Human, Sf9 be utilized in evolutionary genetics studies?

EDAR variants, particularly the EDARV370A allele, represent one of the strongest candidates for recent positive selection in human evolution. Researchers can use purified EDAR Human, Sf9 in several advanced applications:

• Comparative binding studies: Using purified wildtype and variant (e.g., V370A) EDAR proteins to quantitatively compare binding affinities with EDA·A1 via surface plasmon resonance or isothermal titration calorimetry.

• Structural analysis: Crystallography studies comparing wildtype and variant EDAR structures, particularly focusing on the Death Domain (DD) required for interaction with the downstream signal transducer EDARADD .

• Signaling pathway analysis: In vitro assessment of NFκB signaling activation differences between variants, which has been reported to be up-regulated with the 370A variant compared to 370V .

• Protein-protein interaction screens: Using purified EDAR to identify novel interaction partners that might differ between populations expressing different EDAR variants.

• Evolutionary timeline reconstruction: Molecular dating analysis suggests the EDARV370A allele arose in Central China approximately 30,000 years ago, providing a framework for population genetics studies using modern EDAR variants .

What methodological approaches are recommended for studying EDAR's role in eccrine gland development?

Research into EDAR's role in eccrine gland development should consider:

• Quantitative phenotyping: Development of precise methods for counting eccrine glands, similar to the approaches used in the EDARV370A mouse model studies where gland numbers were quantitatively assessed .

• Rescue experiments: Testing the ability of purified EDAR proteins (wildtype vs. variants) to rescue eccrine gland phenotypes in models with EDAR loss-of-function mutations, such as the downless (dlj, E379K) mouse model .

• Signaling pathway manipulation: Modulating the EDAR-NF-κB pathway at different developmental timepoints to determine critical windows for eccrine gland specification and development.

• Human association studies: Correlating EDAR genotypes with eccrine gland density in diverse human populations, expanding on findings that EDARV370A is associated with increased active eccrine gland numbers in Han Chinese populations .

• Single-cell transcriptomics: Analyzing gene expression changes in developing eccrine gland progenitors following EDAR pathway activation to identify downstream effectors specific to this tissue.

What are the critical interface residues mediating EDAR-EDA·A1 binding and how do they impact function?

The EDAR-EDA·A1 interface contains several critical residues essential for binding:

• Key EDA·A1 residues:

  • Ala259 forms a saddle point at the interface

  • Ser258 and Gln261 create the "pommel and cantle" of this saddle-shaped depression

  • Asp265 contributes to the acidic surface area critical for binding

  • Tyr310, Tyr311, Lys340, Thr341, and Tyr343 form a panel with aromatic and basic side chains

• Key EDAR residues:

  • Glu94 interacts with Lys340 and Thr341 of EDA·A1 via direct electrostatic interactions

  • Gly95 is positioned to avoid steric hindrance (replacement with valine in XEDAR prevents binding)

  • Asp92 interacts with Tyr310 of EDA·A1

Functional studies have demonstrated that mutations at the interface completely disrupt binding. For example, the A259E and D265G mutations in EDA·A1 THD abolished interaction with EDAR CRDs in pull-down and surface plasmon resonance assays. These mutations are clinically associated with non-syndromic tooth agenesis (NSTA) and X-linked hypohidrotic ectodermal dysplasia (XL-HED), respectively .

How does the binding specificity between EDAR and EDA·A1 differ from related receptor-ligand pairs?

The binding specificity between EDAR and EDA·A1 is defined by several unique features:

• 1:1 binding mode: Unlike other TNF-receptor complexes (such as TNF-TNFR2 and RANKL-RANK) where each receptor binds into the concaved groove formed by two adjacent ligand protomers, EDAR binds in a 1:1 fashion across a single EDA·A1 THD in the trimer .

• Structural determinants of specificity: Comparing EDA·A1 with the related ligand EDA·A2 reveals that two extra amino acids (Val307 and Glu308) in EDA·A1 are critical for reshaping local geometry to enable EDAR binding. These residues influence the positioning of key aromatic residues like Tyr310, Tyr311, and Phe314 .

• Receptor discrimination: EDAR specifically binds EDA·A1 but not EDA·A2, while the related receptor XEDAR binds EDA·A2 but not EDA·A1. This specificity is mediated by key differences, including:

  • XEDAR Val66 (equivalent to EDAR Gly95) creates steric hindrance with EDA·A1 Tyr310

  • XEDAR Thr63 (replacing EDAR Asp92) abolishes interaction with EDA·A1 Tyr310

  • The hydrophobic nature of helix α1 in XEDAR CRD2 eliminates hydrogen-bonding interactions seen in the EDA·A1-EDAR complex

These structural differences establish the molecular basis for ligand-receptor specificity in the EDA signaling system and provide insight into the evolution of this developmentally critical pathway.

How do disease-causing mutations in EDAR affect protein structure and function?

Disease-causing mutations in EDAR can impact protein structure and function through several mechanisms:

• Interface disruption: Mutations at the EDA-EDAR interface disrupt ligand binding. For example:

  • A259E mutation severely deforms the saddle-shaped depression of EDA·A1 necessary for EDAR binding

  • D265G mutation reduces the acidic surface area at the interface

  • R276C mutation impacts the interaction surface

• Signaling impairment: Mutations in the EDAR Death Domain (DD) required for interaction with EDARADD, such as the V370A variant, can alter downstream NFκB signaling efficiency .

• Functional consequences: The severity of phenotypic effects correlates with the degree of binding and signaling disruption:

  • Complete disruption of EDA-EDAR interaction leads to severe X-linked hypohidrotic ectodermal dysplasia (XL-HED)

  • Partial weakening of interaction results in milder non-syndromic manifestations

  • Some variants like V370A may actually enhance signaling, leading to phenotypes associated with thicker hair and increased eccrine gland number

• Structure-function relationships: Crystal structures of the EDA·A1 THD-EDAR CRDS complex reveal that disease-causing mutations cluster at functionally significant regions of the protein-protein interface, providing mechanistic explanations for the observed phenotypes .

What experimental approaches can be used to model EDAR variants and assess their functional consequences?

Several experimental approaches have proven valuable for modeling EDAR variants:

• Knock-in mouse models: Generation of precise genetic modifications to create mouse models expressing human EDAR variants, such as the EDARV370A knock-in mouse. This approach offers the advantage of evaluating phenotypes on a genetically homogeneous background, isolating the effects of a single variant .

• In vitro binding assays: Pull-down assays and surface plasmon resonance (SPR) using purified ectodomains of EDA·A1 and EDAR to quantitatively measure how variants affect binding affinity and kinetics .

• Signaling pathway assessment: NF-κB reporter assays to measure downstream signaling activation in response to variant EDAR proteins .

• Protein glycosylation analysis: Deglycosylation studies using PNGase F to determine if variants affect post-translational modifications .

• Cross-species validation: Testing conserved function of variants across species (e.g., human and mouse) to validate evolutionary conservation of critical residues .

• Human genotype-phenotype correlation: Association studies in human populations to correlate EDAR variants with quantitative traits (hair thickness, tooth morphology, eccrine gland number) .

• Rescue experiments: Testing the ability of variant EDAR proteins to rescue phenotypes in loss-of-function models, such as the experiment demonstrating that 370A/379K heterozygous animals had more eccrine glands than 370V/379K animals .

What quality control measures should be implemented when working with EDAR Human, Sf9?

Rigorous quality control measures for EDAR Human, Sf9 research should include:

• Purity assessment: SDS-PAGE analysis to confirm protein size (expected 40-57kDa range) and purity .

• Glycosylation characterization: Western blot analysis with and without PNGase F treatment to identify glycosylated versus non-glycosylated forms, as observed in related studies where EDA·A1 ectodomain showed two close bands, with the higher molecular-weight band corresponding to a glycosylated form .

• Functional validation: Binding assays with known ligand (EDA·A1) to confirm biological activity, using techniques such as pull-down assays or surface plasmon resonance .

• Structural integrity: Circular dichroism or thermal shift assays to confirm proper folding and stability.

• Batch consistency: Lot-to-lot comparison of critical quality attributes including purity, activity, and glycosylation patterns.

• Endotoxin testing: Limulus amebocyte lysate (LAL) assay to ensure preparations are endotoxin-free for cell-based applications.

• Storage stability: Accelerated and real-time stability testing to establish appropriate storage conditions and shelf-life.

What are the most effective experimental protocols for studying EDAR's role in hair follicle development?

Effective protocols for studying EDAR's role in hair follicle development should include:

• Histological analysis: Standardized sectioning and staining protocols to quantitatively assess hair shaft thickness, follicle density, and morphology in model systems .

• Ex vivo hair follicle culture: Organ culture systems allowing manipulation of EDAR signaling in isolated follicles to study direct effects on growth and development.

• Genotype-phenotype correlation studies: Methods for precise phenotyping of hair traits (thickness, density, shape) in human populations with different EDAR variants, building on findings that EDARV370A has been associated with increased scalp hair thickness .

• Developmental timing studies: Stage-specific manipulation of EDAR signaling to determine critical windows for hair follicle induction, morphogenesis, and cycling.

• Single-cell transcriptomics: Analysis of gene expression changes in different hair follicle cell populations following EDAR pathway activation.

• 3D skin organoid models: Development of three-dimensional skin models incorporating different EDAR variants to study hair follicle morphogenesis in a controlled environment.

• Comparative models: Cross-species analysis leveraging the conserved role of the Ectodysplasin pathway in the development of ectodermally derived organs across vertebrates .