Recombinant Danio rerio Selenoprotein N (sepn1)

How is recombinant Danio rerio Selenoprotein N expressed and purified for research purposes?

Expression of recombinant sepn1 typically employs one of two systems:

E. coli expression system:

• Usually tagged with N-terminal 10xHis-tag for purification

• Stored in Tris-based buffer with 50% glycerol

• Challenging due to the need to incorporate selenocysteine

Mammalian expression systems:

• Can be expressed in HEK 293 or NIH 3T3 cells with C-terminal tags

• Requires co-expression of selenocysteine incorporation machinery

• Expression constructs must include the native SECIS element in the 3' UTR to direct selenocysteine incorporation

Purification methodology:

• Affinity chromatography using the incorporated tag (His-tag)

• Size exclusion chromatography to separate monomeric and oligomeric forms

• Ion exchange chromatography for further purification

• Concentration determination via spectrophotometry

• Storage at -20°C or -80°C for extended periods, with working aliquots at 4°C

When working with recombinant sepn1, it's critical to avoid repeated freeze-thaw cycles as this can compromise protein activity .

What are the functional domains and post-translational modifications of Selenoprotein N in zebrafish?

Selenoprotein N contains several functional domains and undergoes important post-translational modifications:

Key functional domains:

• EF-hand domain: Binds calcium with an affinity in the range of ER calcium concentration; mutations in this domain affect calcium affinity and conformational changes

• Thioredoxin-like domain: Contains the selenocysteine residue critical for redox activity

• Transmembrane domain: Anchors the protein to the ER membrane as a type II transmembrane protein

Post-translational modifications:

• N-glycosylation: Occurs at specific asparagine residues, verified by endoglycosidase H treatment causing a mobility shift in SDS-PAGE

• Disulfide bond formation: Particularly involving C108, which mediates protein oligomerization

• Selenium incorporation: As selenocysteine, the 21st amino acid, incorporated via a specialized translation mechanism involving a SECIS element

Functional studies show that SelN oligomeric state changes in response to calcium levels, with oligomers prevalent under basal calcium conditions dissociating into monomers upon calcium depletion, enhancing its reductase activity toward targets like SERCA2 .

What expression patterns does Selenoprotein N show during zebrafish development?

Selenoprotein N shows a dynamic expression pattern during zebrafish development:

• Highest expression: In early development, particularly in the tailbud presomitic mesoderm (PSM) at 6, 8, 10, 14, 18, and 24 hours post-fertilization (hpf)

• Developmental trajectory: Expression levels peak in wild-type embryos at 1 day post-fertilization (dpf) and decrease by 6 dpf

• Tissue specificity: Primarily expressed in developing muscle tissues including the myotome

• Cell types: Single-cell RNA sequencing analysis reveals expression in tailbud PSM cell clusters, with incremental expression increases through development (6-24 hpf)

Interestingly, in sepn1 mutant zebrafish, transcript levels at 1 dpf are approximately half of wild-type levels, suggesting potential autoregulation of the gene .

This temporal expression pattern aligns with the proposed role of SelN in early muscle development, potentially explaining why deficiencies lead to congenital myopathies.

What methods are effective for studying the redox properties of recombinant Selenoprotein N?

Investigating the redox properties of sepn1 requires specialized techniques due to its reactive selenocysteine residue:

Redox state analysis:

• AMS modification assay: Treating reduced samples with 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) causes a mobility shift of 0.9 kDa per reduced disulfide bond on SDS-PAGE

• DTT/TCEP reduction: Samples are reduced with 10 mM DTT (redox potential -332 mV at pH 7.0) or 10 mM TCEP followed by alkylation

• Non-reducing vs. reducing SDS-PAGE: Comparing migration patterns to determine disulfide-mediated oligomerization

Functional redox assays:

• Redox trapping experiments: To identify redox-dependent interacting partners (e.g., SERCA2)

• [³H]Ryanodine binding assays: Measuring the effect of sepn1 on RyR channel activity under different redox conditions

• Glutathione (GSH) redox state measurement: Quantifying the ratio between oxidized (GSSG) and reduced (GSH) forms in sepn1-deficient models versus controls

For example, studies have shown that sepn1 oligomers are stabilized by disulfide bonds involving C108, but not all oligomers are covalently linked, as demonstrated by sucrose gradient analysis under denaturing conditions . Additionally, experimental data shows that wild-type sepn1 can restore redox sensitivity to RyR channels from SEPN1-diseased human muscle tissue .

How can researchers effectively model SEPN1-related myopathies using zebrafish?

Zebrafish offer several advantages for modeling SEPN1-related myopathies:

Generation of sepn1-deficient models:

• Knockout strategies: Using CRISPR/Cas9 or homologous recombination techniques

• Example knockout model: Homologous recombination-based technique replacing a 222-bp region of the sepn1 gene with a neomycin cassette

• Gene-specific mutations: Creating in-frame deletions or substitutions (e.g., p.G66VdelL67) that model human disease mutations

Phenotypic analysis methods:

• Motor function assessment: Swimming behavior analysis using automated tracking systems

• Muscle histology: Examining fiber type, size, and ultrastructure at different developmental stages

• Calcium imaging: Measuring calcium transients in muscle cells during contraction

• Electron microscopy: Evaluating mitochondrial and sarcoplasmic reticulum integrity

Molecular and functional analyses:

• Transcriptional profiling: qRT-PCR or RNA-seq to measure expression of selenoproteome genes

• Calcium dynamics: Using calcium-sensitive dyes or genetically encoded calcium indicators

• Redox homeostasis: Measuring glutathione redox states in sepn1-deficient models

• Muscle regeneration: Evaluating satellite cell maintenance and muscle repair after injury

Recent studies have demonstrated that sepn1-deficient zebrafish show altered glutathione redox homeostasis, providing insight into potential pathomechanisms of SEPN1-related myopathies .

What strategies are used to assess calcium sensing by Selenoprotein N in zebrafish models?

Investigating calcium sensing by sepn1 requires specialized approaches:

Structural analysis:

• EF-hand domain characterization: Mutagenesis of the calcium-binding EF-hand domain (e.g., D80A mutation) to assess its role in calcium sensing

• Protein conformational analysis: Using non-reducing SDS-PAGE and sucrose gradient fractionation to detect calcium-dependent oligomerization changes

Functional assays:

• Calcium depletion experiments: Treating cells/tissues with thapsigargin (Tg) to deplete ER calcium stores and monitor sepn1 conformational changes

• Calcium binding measurement: In vitro assessment of calcium binding affinity using purified recombinant protein and calcium sensors

• SERCA activity assays: Measuring calcium uptake into microsomes in the presence of wild-type or mutant sepn1

Interaction studies:

• Co-immunoprecipitation: Under varying calcium conditions to identify calcium-dependent binding partners

• Proximity labeling: Using BioID or APEX approaches to identify proteins in close proximity to sepn1 in different calcium states

• FRET-based sensors: To monitor real-time changes in protein interactions or conformations in response to calcium fluctuations

For example, research has shown that under basal ER calcium conditions, sepn1 exists predominantly in an oligomeric state (96.2% of total), but upon calcium depletion with thapsigargin, it shifts to a monomeric form (61.8% of total), indicating its role as a calcium sensor .

How does Selenoprotein N interact with ryanodine receptors in zebrafish muscle?

The interaction between sepn1 and ryanodine receptors (RyRs) is critical for muscle function:

Biochemical interaction analysis:

• Co-immunoprecipitation: To detect physical association between sepn1 and RyRs

• Surface plasmon resonance: For measuring binding kinetics and affinity

• Crosslinking studies: To identify specific interaction regions

Functional assessment:

• [³H]Ryanodine binding assays: Demonstrates that sepn1 affects RyR sensitivity to redox conditions

• Single-channel recordings: Measuring RyR channel activity in the presence of purified sepn1

• Calcium flux measurements: Using calcium-sensitive dyes to monitor RyR-mediated calcium release

Disease-relevant findings:

• Zebrafish sepn1 can partially restore binding capacity of human RyRs from SEPN1-diseased tissue (Bmax= 1.43 ± 0.19 pmol/mg)

• Most significantly, addition of zebrafish sepn1 restores the ability of human RyRs from diseased tissue to respond to changes in redox potential

• RyRs from SEPN1-diseased muscle lose their normal sensitivity to redox conditions, which can be rescued by recombinant sepn1

These findings indicate that sepn1 functions as a redox-dependent modifier of RyR channels, explaining why mutations in either gene lead to similar myopathies .

What techniques are effective for analyzing transcriptional changes in selenoproteins in zebrafish models?

Analyzing selenoprotein transcription in zebrafish requires specialized approaches:

Quantitative expression analysis:

• Quantitative real-time PCR (qRT-PCR): Using TaqMan-based assays with fluorogenic-labeled probes for specific amplification products

• Microarray analysis: For genome-wide expression profiling, though this may have limitations for detecting subtle changes in selenoproteome genes

• RNA sequencing: Particularly useful for developmental time course studies, with statistical models accounting for time and treatment effects

Experimental design considerations:

• Sex-specific effects: Include both sexes in analysis as selenoprotein expression shows sex-dependent responses to selenium supplementation

• Time-course design: Assess expression at multiple time points (e.g., 1, 7, and 14 days) to capture dynamic responses

• Sample size: Larger sample sizes (>10 per group) are necessary to detect subtle expression changes

Statistical analysis approaches:

• Mixed model ANCOVA: Using reference gene expression (e.g., 18S) as a covariate for normalization

• Model selection: Testing full models including all main effects, interactions, and covariates, then reducing to the model with the smallest Akaike information criterion

A comprehensive qRT-PCR panel for selenoproteome genes should include:

• Selenoprotein genes (sepp1a, sepp1b, gpx3, gpx4a, txnrd1, dio2)

• Selenoprotein synthesis machinery (secp43, staf, secisbp2, sla, eefsec, sps1, sps2)

• Reference genes (18S rRNA)

Example primers and probes for key selenoprotein genes:

This methodological approach can detect subtle expression changes that might be missed by other techniques .

What are the key considerations for designing rescue experiments with recombinant Selenoprotein N?

Rescue experiments with recombinant sepn1 require careful design:

Protein preparation considerations:

• Selenocysteine incorporation: Ensure proper incorporation of selenocysteine, as cysteine mutants have different redox properties

• EF-hand domain integrity: Verify calcium binding capacity, as this affects protein function

• Oligomeric state: Characterize monomeric/oligomeric distribution and maintain appropriate storage conditions to preserve native state

• Protein quality control: Verify activity before use through reductase activity assays

Experimental design for rescue studies:

• Dosage determination: Titrate recombinant protein to determine optimal concentrations for rescue

• Delivery methods:

  • For cell culture: Direct addition to medium or transfection of expression constructs

  • For zebrafish: Microinjection of protein or mRNA into embryos

• Timing: Apply treatment at developmental stages where endogenous sepn1 would normally be active

• Controls: Include inactive protein controls (e.g., selenocysteine-to-cysteine mutants)

Assessment of rescue efficacy:

• Functional readouts: RyR activity, calcium homeostasis, muscle contractility

• Molecular markers: Restoration of normal redox state, gene expression profiles

• Morphological assessment: Muscle histology and ultrastructure

• Behavioral analysis: Swimming performance in zebrafish models

A successful example is the rescue of RyR function in SEPN1-disease patient muscle homogenates by addition of purified recombinant zebrafish sepn1, which partially restored binding capacity (Bmax= 1.43 ± 0.19 pmol/mg) and, more importantly, restored redox sensitivity of the RyR channels .

How can researchers assess the role of Selenoprotein N in early development using zebrafish models?

Zebrafish offer unique advantages for studying sepn1's developmental roles:

Embryonic development analysis:

• Temporal expression profiling: Using in situ hybridization or reporters to track sepn1 expression throughout development

• Single-cell RNA sequencing: To identify cell populations expressing sepn1 during development and co-expressed genes

• Time-lapse imaging: Of transgenic lines with fluorescently tagged sepn1 or muscle markers

Developmental perturbation approaches:

• Morpholino knockdown: For transient reduction of sepn1 during specific developmental windows

• Temperature-sensitive mutants: For temporal control of protein function

• Inducible expression systems: To rescue sepn1 function at specific developmental stages

Analytical methods:

• Somite formation assessment: Examining timing and patterning of muscle segment formation

• Myogenesis markers: Evaluating expression of myogenic regulatory factors and muscle-specific genes

• Muscle precursor analysis: Assessing proliferation and differentiation of muscle progenitor cells

• Cell fate mapping: Tracking the developmental trajectory of sepn1-expressing cells

Recent single-cell RNA sequencing analyses have revealed incremental expression increases of sepn1 in tailbud presomitic mesoderm cells at 6, 8, 10, 14, 18, and 24 hpf, suggesting a role in the somite segmentation process . Furthermore, co-expression analysis identified correlations between sepn1 and components of the glutathione redox pathway, indicating potential functional relationships in developmental contexts .

How does Selenoprotein N contribute to redox homeostasis in zebrafish models?

Selenoprotein N plays a crucial role in cellular redox balance:

Redox homeostasis assessment methods:

• Glutathione redox state: Quantifying the ratio between oxidized (GSSG) and reduced (GSH) forms

• ROS detection: Using fluorescent probes (e.g., DCF-DA, MitoSOX) to measure reactive oxygen species levels

• Protein oxidation: Measuring carbonyl content or using redox proteomics approaches

• Antioxidant enzyme activity: Assessing catalase, superoxide dismutase, and glutathione peroxidase activities

Connections to calcium signaling:

• Sepn1 functions as a reductase that is activated when calcium levels in the ER are low

• This establishes a feedback mechanism where sepn1 senses calcium levels and modulates downstream signaling

• Redox-dependent regulation of RyR activity provides a mechanistic link between sepn1's redox function and calcium handling in muscle

These findings support the concept that sepn1 is one of the long-sought reductases of the ER, playing a critical role in maintaining cellular redox balance, particularly in the context of calcium signaling pathways .