DHH (C23II) His Human

What is DHH (C23II) His Human protein and its biological significance?

DHH (C23II) His Human is a recombinant protein belonging to the Hedgehog family of signaling molecules that regulate morphogenesis. The native DHH protein functions as a precursor that undergoes autocatalytic cleavage, with the N-terminal portion containing the primary signaling activity while the C-terminal portion facilitates processing. In this recombinant form, the protein features a C23II mutation (cysteine to isoleucine substitution at position 23) and includes a histidine tag for purification purposes .

DHH plays essential roles in several biological processes including:

• Male gonadal differentiation and development

• Spermatocyte survival in testes

• Perineurial development in the nervous system

• Intercellular signaling for various patterning events during development

Mutations in DHH have been associated with partial gonadal dysgenesis (PGD) and minifascicular polyneuropathy, highlighting its clinical significance in reproductive and neurological disorders .

How does DHH (C23II) His differ from native DHH protein?

The recombinant DHH (C23II) His differs from native DHH in several important aspects:

• Amino acid modification: The C23II variant contains a substitution of cysteine to isoleucine at position 23, which prevents the N-terminal fatty acid acylation that occurs in the native protein .

• His-tag addition: The recombinant protein includes a 24-amino acid histidine tag at the N-terminus to facilitate purification using metal affinity chromatography .

• N-terminal domain isolation: While native DHH undergoes autocatalytic cleavage between Gly198 and Cys199, the recombinant protein typically represents only the N-terminal domain (amino acids 23-198) that contains the signaling activity .

• Expression system: Rather than being produced by mammalian cells, DHH (C23II) His is expressed in prokaryotic systems (typically E. coli), resulting in a non-glycosylated form of the protein .

• Solubility profile: The recombinant form lacks the cholesterol modification that normally restricts the native protein to cell surfaces, potentially altering its diffusion and signaling properties .

How is DHH (C23II) His Human protein produced for research applications?

DHH (C23II) His Human recombinant protein is typically produced using Escherichia coli expression systems through the following general methodology:

• Gene construction: The human DHH gene sequence (amino acids 23-198, with C23I mutation) is fused to a histidine tag sequence and cloned into a suitable expression vector.

• Bacterial expression: The construct is transformed into E. coli, followed by induction of protein expression under optimized conditions.

• Purification process: The protein is extracted and purified using proprietary chromatographic techniques, typically including:

  • Metal affinity chromatography (utilizing the His-tag)

  • Potentially additional purification steps to achieve >90% purity

• Quality control: The final product undergoes verification of purity via SDS-PAGE and may be tested for biological activity .

The resulting product is a single, non-glycosylated polypeptide chain with a molecular mass of approximately 22.4 kDa, typically supplied as a sterile filtered clear solution .

What are the key structural and biochemical properties of DHH (C23II) His?

The protein maintains the key structural elements necessary for signaling activity, though it lacks post-translational modifications present in mammalian-expressed DHH .

What are the optimal storage and handling conditions for DHH (C23II) His protein?

To maintain the stability and activity of DHH (C23II) His protein, researchers should follow these recommended storage and handling guidelines:

Short-term storage (2-4 weeks):

• Store at 4°C if the entire vial will be used within this timeframe.

Long-term storage:

• Store frozen at -20°C.

• For extended storage periods, the addition of a carrier protein (0.1% HSA or BSA) is recommended to enhance stability.

• Use a manual defrost freezer to avoid temperature fluctuations.

Critical handling considerations:

• Avoid multiple freeze-thaw cycles which can significantly reduce protein activity.

• When reconstituting lyophilized protein, follow manufacturer recommendations (typically reconstituting at 500 μg/mL in sterile PBS).

• Allow protein to equilibrate to room temperature before opening vials to prevent condensation.

• Work under sterile conditions when preparing aliquots for long-term storage .

How can researchers validate the activity and functionality of DHH (C23II) His protein?

Validating the functionality of DHH (C23II) His protein is essential before using it in complex experiments. Several complementary approaches can be employed:

1. Binding assays:

• Surface Plasmon Resonance (SPR/Biacore) to measure binding kinetics to Patched receptor

• ELISA-based binding assays to quantify receptor interactions

• FACS analysis for cell-surface binding studies

2. Functional assays:

• Gli transcription factor activation assays (using reporter systems)

• Smoothened (SMO) activity measurements

• Cell-based hedgehog pathway activation assays

3. Validation parameters:

• Typical effective dose (ED₅₀) for hedgehog pathway activation: 15-45 μg/mL

• Binding affinity (KD) measurements should be consistent with literature values

• Association rate (kon) should be at least 10⁴ M⁻¹s⁻¹, with optimal rates above 10⁵ M⁻¹s⁻¹

4. Cross-reactivity testing:

• If relevant to the experimental design, cross-reactivity with mouse Dhh or other species variants should be assessed

• Cross-reactivity with other Hedgehog family members (Shh, Ihh) may be important to establish specificity .

How does DHH (C23II) His protein function in developmental signaling pathways?

DHH (C23II) His protein functions within the Hedgehog signaling pathway, which is critical for embryonic development and tissue homeostasis. Understanding this pathway is essential for designing experiments with DHH protein:

Canonical Hedgehog signaling mechanism:

• In the absence of DHH ligand, the twelve-pass transmembrane receptor Patched (Ptch) inhibits the activity of Smoothened (SMO), a GPCR-like protein.

• When DHH binds to Ptch, this inhibition is relieved, allowing SMO to signal through the Gli family of transcription factors.

• Activated Gli proteins translocate to the nucleus and induce transcription of target genes, including pathway components like Gli1 and Ptch.

DHH-specific signaling characteristics:

• Unlike Sonic Hedgehog (Shh), which has broad developmental roles, DHH signaling is more tissue-restricted, primarily affecting:

  • Sertoli cells in the developing testes

  • Perineurial cells in the peripheral nervous system

• In experimental systems, researchers can distinguish DHH-specific responses from other Hedgehog pathways by:

  • Using tissue-specific cellular models (e.g., testicular or Schwann cell cultures)

  • Employing DHH-specific neutralizing antibodies

  • Analyzing DHH-selective downstream targets .

What experimental models are most suitable for studying DHH signaling functions?

Selecting appropriate experimental models is crucial for investigating DHH functions. The following models offer distinct advantages for different research questions:

In vitro cellular models:

• Sertoli cell cultures - Primary or immortalized Sertoli cells respond to DHH stimulation and are ideal for studying gonadal development mechanisms.

• Perineurial cell cultures - Useful for investigating DHH's role in peripheral nerve development and maintenance.

• Cell lines with reporter constructs - Cells transfected with Gli-responsive elements linked to luciferase or other reporters provide quantifiable readouts of DHH pathway activation.

Ex vivo models:

• Testicular organ cultures - Allow for the examination of DHH effects on tissue architecture and cellular interactions in a more physiologically relevant context.

• Peripheral nerve explants - Enable the study of DHH's influence on nerve fasciculation and perineurial development.

In vivo models:

• DHH knockout mice - Display male infertility and peripheral nerve defects, serving as valuable models for loss-of-function studies.

• Conditional DHH knockout models - Allow for tissue-specific and temporally controlled deletion of DHH signaling.

• DHH overexpression models - Can reveal gain-of-function phenotypes and potential pathological consequences of excessive signaling .

How can DHH (C23II) His be applied in studies of gonadal development and disorders?

DHH plays a critical role in male gonadal development, making DHH (C23II) His protein a valuable tool for reproductive biology research. Methodological approaches include:

Fundamental developmental research:

• Sertoli cell differentiation studies - DHH (C23II) His can be used to investigate the molecular mechanisms by which Sertoli cells influence testicular development.

• Germ cell survival assays - As DHH functions as a spermatocyte survival factor, the recombinant protein can be applied in protocols assessing germ cell apoptosis and survival.

• Fetal gonad explant cultures - The protein can be added to ex vivo cultures to examine the temporal requirements of DHH signaling during critical developmental windows.

Clinical research applications:

• Disorder modeling - DHH (C23II) His can be used to rescue signaling in cells derived from patients with DHH mutations, potentially revealing mechanism-based therapeutic approaches.

• Genetic variant assessment - When novel DHH variants are identified in patients with gonadal dysgenesis, the recombinant protein (and its mutated forms) can be used to assess functional consequences.

• Biomarker development - Research using DHH (C23II) His may help identify downstream markers of DHH pathway disruption that could serve as diagnostic indicators for reproductive disorders .

What techniques can be used to study molecular interactions between DHH and its binding partners?

Understanding the molecular interactions between DHH and its partners requires sophisticated biophysical and biochemical techniques:

Quantitative binding analysis:

• Surface Plasmon Resonance (SPR) - Provides real-time measurements of association (kon) and dissociation (koff) rates between DHH and its receptors or binding partners. Typical kon rates for hedgehog family proteins are at least 10⁴ M⁻¹s⁻¹, with optimal rates above 10⁵ M⁻¹s⁻¹.

• Isothermal Titration Calorimetry (ITC) - Measures the thermodynamic parameters of DHH binding interactions, providing insights into the energetics of complex formation.

• Bio-Layer Interferometry (BLI) - Offers an alternative approach for measuring binding kinetics with the advantage of requiring smaller sample volumes.

Structural analysis methods:

• X-ray crystallography - Can resolve the atomic details of DHH-receptor complexes, revealing critical interaction interfaces.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) - Identifies regions of DHH that undergo conformational changes upon binding to partners.

• Cross-linking Mass Spectrometry - Maps the spatial proximity of specific residues between DHH and its binding partners.

Functional interaction analysis:

• Competitive binding assays - Using labeled DHH (C23II) His and potential competitors to identify molecules that interact with the same binding sites.

• Mutagenesis studies - Systematic alteration of DHH residues to identify those critical for receptor binding and pathway activation.

• Antibody blocking experiments - Using targeted binding agents with known epitopes to disrupt specific regions of the DHH-receptor interface .

How does DHH (C23II) His compare with other Hedgehog family proteins in research applications?

The Hedgehog family in mammals comprises three members: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh). Understanding their similarities and differences is essential for experimental design:

Experimental considerations when choosing between Hedgehog proteins:

• Select DHH (C23II) His for studies specifically focused on male reproductive development or peripheral nerve formation

• Consider potential cross-reactivity; some antibodies against Shh may recognize DHH due to structural similarities

• Be aware that some targeted binding agents show specificity for one Hedgehog family member over others (e.g., antibody 6D7 can bind both human and mouse Indian hedgehog but may not recognize DHH) .

What common experimental challenges occur when working with DHH (C23II) His and how can they be addressed?

Researchers working with DHH (C23II) His may encounter several technical challenges. Here are common issues and recommended solutions:

1. Protein instability and activity loss:

• Problem: DHH (C23II) His may lose activity during storage or experimental handling.

• Solution: Add carrier proteins (0.1% HSA or BSA) for long-term storage; prepare single-use aliquots; avoid multiple freeze-thaw cycles; use appropriate buffers containing DTT to maintain reducing conditions .

2. Insufficient pathway activation:

• Problem: Experiments show minimal or no activation of Hedgehog pathway components.

• Solution: Verify protein activity using positive control cells known to respond to Hedgehog signaling; ensure sufficient protein concentration (typical ED₅₀: 15-45 μg/mL); confirm expression of pathway components in target cells .

3. Non-specific binding or effects:

• Problem: Difficulty distinguishing DHH-specific effects from general Hedgehog pathway activation.

• Solution: Include appropriate controls (other Hedgehog proteins, pathway inhibitors); use DHH-specific neutralizing antibodies; validate findings in DHH knockout models .

4. Limited solubility or aggregation:

• Problem: Protein aggregation or precipitation during experiments.

• Solution: Centrifuge the protein solution before use; consider alternative formulation buffers; ensure the protein is properly thawed and mixed; use fresh preparations for critical experiments .

5. Variability in cell responsiveness:

• Problem: Inconsistent cellular responses to DHH treatment.

• Solution: Standardize cell culture conditions; control for cell passage number; verify receptor expression levels; consider serum starvation before treatment to synchronize cells .

What are the emerging applications of DHH (C23II) His in developmental biology and disease research?

DHH (C23II) His protein is finding new applications beyond its traditional research areas, opening opportunities for novel investigations:

Reproductive medicine advancements:

• Development of in vitro models for male germline development using DHH signaling modulation

• Investigation of DHH as a potential biomarker for testicular function and fertility assessment

• Exploration of DHH signaling in female reproductive tract development and disorders

Neurological research:

• Characterization of DHH's role in peripheral nerve regeneration after injury

• Investigation of DHH signaling in peripheral neuropathies beyond those directly caused by DHH mutations

• Potential therapeutic applications for peripheral nerve disorders

Cancer biology:

• Emerging evidence suggests potential roles for DHH in certain cancer types

• Investigation of DHH as a biomarker or therapeutic target in testicular and peripheral nerve sheath tumors

• Exploration of interactions between DHH and other oncogenic pathways

Stem cell and regenerative medicine:

• Application of DHH (C23II) His in differentiation protocols for generating Sertoli-like cells from stem cells

• Investigation of DHH's potential to support generation of artificial gonadal organoids

• Exploration of DHH's role in creating models of the blood-testis barrier .

How can advanced genetic and cellular techniques be combined with DHH (C23II) His to enhance research outcomes?

Integration of cutting-edge technologies with DHH (C23II) His protein research offers powerful approaches to address complex biological questions:

CRISPR/Cas9 genome editing:

• Generation of precise mutations in DHH pathway components to study structure-function relationships

• Creation of reporter cell lines with endogenous tagging of DHH receptors or downstream effectors

• Development of cellular models with inducible or tissue-specific DHH expression systems

Single-cell technologies:

• Combining DHH treatment with single-cell RNA sequencing to reveal heterogeneous cellular responses

• Single-cell proteomics to identify differential protein expression following DHH signaling

• Spatial transcriptomics to map DHH-responsive cells within complex tissues

Organoid and 3D culture systems:

• Application of DHH (C23II) His in testicular organoid development

• Investigation of DHH's role in peripheral nerve organoid formation and myelination

• Development of co-culture systems to model cellular interactions in DHH-dependent tissues

High-throughput screening platforms:

• Identification of small molecules that modulate DHH signaling

• Discovery of genetic modifiers that influence DHH pathway activity

• Characterization of combination effects between DHH and other developmental signaling pathways

Computational and systems biology approaches:

• Network analysis to map DHH-dependent signaling pathways

• Predictive modeling of DHH interaction dynamics

• Integration of multi-omics data to develop comprehensive models of DHH function in development .