Recombinant Danio rerio Leucine-rich repeat-containing protein 3 (lrrc3)

What is Leucine-rich Repeat-containing Protein 3 (lrrc3) in Danio rerio?

Leucine-rich repeat-containing protein 3 (lrrc3) is a protein-coding gene found in Danio rerio (zebrafish) that belongs to the larger family of leucine-rich repeat (LRR) proteins. The protein features characteristic leucine-rich repeat motifs with the basic pattern "LxxLxL" where "L" refers to leucine or other hydrophobic amino acids, and "x" can be any amino acid . In zebrafish, lrrc3 is one of several LRR-containing proteins that play roles in various developmental and physiological processes. The zebrafish lrrc3 protein shares sequence homology with other vertebrate LRRC3 proteins, including those found in humans, mice, and other model organisms .

How conserved is lrrc3 across different species?

The lrrc3 gene and its encoded protein demonstrate notable evolutionary conservation across various vertebrate species. Sequence analysis reveals significant homology between zebrafish lrrc3 and its orthologs in other species including Homo sapiens (human), Mus musculus (house mouse), Pan troglodytes (chimpanzee), Rattus norvegicus (Norway rat), Canis lupus familiaris (dog), Bos taurus (cattle), and Xenopus tropicalis (tropical clawed frog) . This conservation suggests functional importance across vertebrate evolution and makes zebrafish an appropriate model for studying the protein's basic functions that may translate to other species, including humans.

What are the structural characteristics of lrrc3 protein?

The lrrc3 protein in Danio rerio is characterized by its leucine-rich repeat domains, which form a solenoid structure. Each repeat unit typically consists of 20-30 amino acids with the conserved pattern containing hydrophobic residues (often leucine) at specific positions. The protein adopts a curved, horseshoe-shaped structure where the repeats stack to form the concave inner surface that often serves as a protein-protein interaction interface . Modern structure-aware annotation methods using dimensionality reduction techniques can more accurately identify these repeating structural motifs than traditional sequence-based methods, which often fail to properly annotate divergent motifs near the terminal boundaries of the LRR domain .

What expression patterns does lrrc3 show during zebrafish development?

While the search results don't provide specific expression data for lrrc3 in zebrafish, related leucine-rich repeat family proteins like lrrc8a have been shown to be ubiquitously expressed through early developmental stages (up to 12 hours post-fertilization), followed by more restricted expression around the ventricular layer of neural tubes and cardiogenic regions at 24 hours post-fertilization . Given structural and functional similarities within the leucine-rich repeat protein family, lrrc3 may exhibit comparable spatiotemporal expression patterns, though specific studies would be needed to confirm this.

How do recombinant expression systems for zebrafish lrrc3 compare in terms of yield and functionality?

The functionality assessment should include verification of proper folding through circular dichroism or limited proteolysis, as well as binding assays with known interaction partners. When expressing LRR-containing proteins, special attention should be paid to the proper formation of disulfide bonds that may be critical for structural integrity.

What are the major challenges in producing soluble recombinant lrrc3 and how can they be overcome?

Recombinant expression of leucine-rich repeat proteins like lrrc3 faces several technical challenges:

• Insolubility and inclusion body formation: LRR proteins often aggregate when overexpressed in bacterial systems due to their complex folding requirements.

• Conformational heterogeneity: The repetitive nature of LRR domains can lead to conformational heterogeneity in recombinant preparations.

• Proteolytic susceptibility: The extended solenoid structure may expose proteolytically sensitive sites.

These challenges can be addressed through several strategies:

• Fusion partners: Using solubility-enhancing fusion tags like Trigger Factor (TF), as demonstrated for similar proteins with expression at approximately 80 kDa molecular weight .

• Expression conditions: Optimizing temperature, inducer concentration, and duration can significantly improve solubility. For instance, cold-shock expression systems (pCold vectors) have shown success with LRR proteins .

• Refolding protocols: If inclusion bodies are unavoidable, specialized refolding protocols incorporating step-wise dialysis with decreasing denaturant concentrations and appropriate redox buffers can recover functional protein.

• Domain engineering: Expressing stable subdomains or introducing mutations that enhance stability without affecting function.

How can one accurately determine the repeat unit boundaries in lrrc3 for structural and functional studies?

Traditional sequence-based methods for determining LRR unit boundaries, such as those employed by LRRPredictor, often make errors when annotating divergent motifs, particularly near the C- and N-terminal boundaries . More advanced structure-aware approaches provide superior accuracy:

• AlphaFold2-based annotation: Leveraging predicted protein structures from AlphaFold2 allows for geometric information-based annotation of LRR domain features, including start/end positions, repeat unit delineation, and structural irregularities .

• Cumulative winding number analysis: This approach tracks the cumulative angular change around the solenoid axis, providing more reliable repeat unit boundaries than ML-based delineators . The winding number calculation reveals errors in traditional annotation methods and offers a more precise determination of individual repeat units.

• Four-breakpoint regression detection: For proteins containing hairpin loops or structural anomalies within the LRR domain, a four-breakpoint piecewise linear regression can identify these irregularities, which appear as short horizontal lines in the regression, representing large hairpins, insertions, or misfolding within the LRR domain .

These methods should be applied in combination with experimental validation through limited proteolysis, hydrogen-deuterium exchange mass spectrometry, or crystallographic studies when possible.

What are the functional implications of post-translational modifications on zebrafish lrrc3?

While specific data on post-translational modifications (PTMs) of zebrafish lrrc3 are not provided in the search results, leucine-rich repeat proteins commonly undergo several PTMs that can profoundly affect their function:

• Glycosylation: N-linked glycosylation sites in the concave surface of LRR domains can modulate protein-protein interactions and affect folding stability. Analysis of zebrafish lrrc3 for consensus N-glycosylation motifs (N-X-S/T) would identify potential modification sites.

• Phosphorylation: Serine, threonine, and tyrosine residues in the connecting loops between LRR units can be phosphorylated to regulate binding affinity and signaling functions.

• Disulfide bond formation: Many LRR proteins contain disulfide bonds that stabilize their tertiary structure. The presence of conserved cysteine pairs in zebrafish lrrc3 would suggest similar stabilization mechanisms.

Experimental approaches to study these modifications include mass spectrometry-based proteomics, site-directed mutagenesis of putative modification sites, and comparative functional assays of recombinant proteins expressed in systems with varying PTM capabilities.

What are the optimal protocols for recombinant zebrafish lrrc3 expression and purification?

Based on successful approaches with similar LRR-containing proteins, the following protocol is recommended:

Expression System Selection:

• Bacterial expression: Use pCold TF vector in E. coli BL21(DE3) for high-yield applications

• Eukaryotic expression: Consider HEK293F cells for functional studies requiring mammalian PTMs

Expression Protocol for E. coli System:

• Transform pCold TF-lrrc3 construct into E. coli BL21(DE3)

• Grow cultures at 37°C until OD600 reaches 0.5-0.6

• Induce with 0.5 mM IPTG and shift temperature to 15°C

• Continue expression for 16-20 hours

• Harvest cells by centrifugation (6,000 × g, 15 min, 4°C)

Purification Protocol:

• Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Perform IMAC purification using Ni-NTA resin with the His-tag on recombinant protein

• Include a gel filtration step to ensure monodispersity

• Verify protein integrity by SDS-PAGE and Western blotting with anti-His-tag antibody

This approach has been successfully employed for similar LRR-containing proteins, yielding soluble protein with apparent molecular weights of approximately 80 kDa when including the TF tag .

How can one design effective antibodies against zebrafish lrrc3?

Developing specific antibodies against LRR proteins like zebrafish lrrc3 presents unique challenges due to the highly conserved repeat structure. A successful approach demonstrated with LGR proteins (which contain LRR domains) involves:

• Antigen Selection:

  • Target the leucine-rich repeat region, which has proven successful for similar proteins

  • Express recombinant LRR domain fragments in E. coli using pET15b or pCold TF vectors

  • Verify antigen quality by SDS-PAGE and Western blotting with anti-His-tag antibody

• Immunization Strategy:

  • Consider non-mammalian hosts to overcome immune tolerance issues that arise from the high conservation of LRR domains across mammals

  • Zebrafish immunization has been successfully employed for highly conserved proteins

  • Oral immunization protocol: feed transformed E. coli expressing the target protein to 10-50 zebrafish

• Antibody Screening:

  • Perform dot blotting analysis using the recombinant protein

  • Use appropriate anti-zebrafish IgM HRP-linked secondary antibody for detection

  • Quantify fluorescence intensity to establish detection sensitivity, noting that a linear relationship typically exists at antigen concentrations below 100 ng

This approach has successfully produced antibodies against human LGR3 using zebrafish as the host, suggesting its potential applicability to zebrafish lrrc3 .

What functional assays are most informative for characterizing zebrafish lrrc3?

To comprehensively characterize zebrafish lrrc3 function, a multi-tiered approach incorporating both in vitro and in vivo assays is recommended:

In Vitro Assays:

• Protein-Protein Interaction Studies:

  • Pull-down assays with potential binding partners

  • Surface Plasmon Resonance (SPR) to determine binding kinetics

  • Yeast two-hybrid screening to identify novel interactors

• Structural Analysis:

  • Circular dichroism to assess secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to map domain boundaries

In Vivo Assays:

• Expression Analysis:

  • Whole-mount in situ hybridization to determine spatiotemporal expression patterns during development

  • RT-PCR to quantify expression levels in different tissues

• Loss-of-Function Studies:

  • Morpholino knockdown approaches, similar to those used for lrrc8a studies in zebrafish

  • CRISPR/Cas9-mediated gene editing to generate stable knockout lines

  • Phenotypic analysis focusing on developmental processes where LRR proteins typically function

• Rescue Experiments:

  • mRNA rescue experiments to confirm specificity of knockdown phenotypes

  • Structure-function analysis using mutated versions of the protein

This comprehensive approach has been successfully applied to related LRR proteins and can be adapted for zebrafish lrrc3 characterization .

How can advanced structural analysis techniques be applied to zebrafish lrrc3?

Several advanced structural analysis techniques can provide valuable insights into zebrafish lrrc3 structure and function:

This integrated computational and experimental approach provides a comprehensive structural characterization that can guide functional studies and rational protein engineering efforts .

What is the role of zebrafish lrrc3 in developmental processes?

While specific data on zebrafish lrrc3's developmental roles are not provided in the search results, we can make informed inferences based on related LRR proteins. Related family members like lrrc8a have been shown to play critical roles in brain ventricle inflation and circulation development in zebrafish . By extension, zebrafish lrrc3 may have important functions in:

• Neural development: Many LRR proteins participate in neuronal differentiation, axon guidance, and synapse formation. Investigation of lrrc3 expression in developing neural tissues can reveal potential roles in these processes.

• Organogenesis: The expression of lrrc8a around cardiogenic regions suggests that lrrc3 might similarly participate in the development of specific organs .

• Cell-cell communication: The protein-protein interaction capabilities of LRR domains often mediate cell-cell signaling events crucial for coordinated development.

Research approaches to elucidate these roles should include detailed expression analysis during development, loss-of-function studies using morpholino knockdown or CRISPR/Cas9-mediated mutagenesis, and rescue experiments to confirm specificity.

How can zebrafish lrrc3 research contribute to understanding human disease mechanisms?

Research on zebrafish lrrc3 can potentially inform our understanding of human disease mechanisms through several avenues:

• Evolutionary conservation: The high sequence conservation between zebrafish and human LRRC3 suggests functional conservation . Findings in zebrafish models may therefore translate to human biology.

• Developmental disorders: If zebrafish lrrc3 participates in developmental processes similar to those affected in human congenital disorders, the zebrafish model can provide insights into disease mechanisms.

• Functional redundancy: Understanding the interplay between lrrc3 and other LRR family proteins can reveal compensatory mechanisms relevant to disease resilience or susceptibility.

• Drug discovery: The zebrafish model offers advantages for high-throughput screening of compounds that modulate LRR protein function, potentially leading to therapeutic strategies for human diseases involving LRR proteins.

Given that other LRR-containing proteins have been implicated in human neurological disorders, immunological conditions, and cancer, zebrafish lrrc3 research may have broad translational implications for human health and disease.

What comparative insights can be gained from studying lrrc3 across different species?

Comparative analysis of lrrc3 across species offers valuable evolutionary and functional insights:

This comparative approach reveals:

What emerging technologies could advance zebrafish lrrc3 research?

Several cutting-edge technologies show promise for advancing zebrafish lrrc3 research:

• CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa): These approaches allow for temporal and spatial control of gene expression without permanent genomic alterations, enabling more nuanced functional studies than traditional knockout approaches .

• Spatial transcriptomics: This technology can reveal the precise spatial distribution of lrrc3 expression in developing zebrafish embryos, providing insights into potential tissue-specific functions.

• Cryo-electron tomography: For structural studies, this technique can visualize lrrc3 in its native cellular environment, potentially revealing physiologically relevant protein-protein interactions.

• Optogenetics: By fusing light-sensitive domains to lrrc3, researchers can achieve precise spatiotemporal control over protein function in vivo.

• AlphaFold2 and structure-aware computational methods: These approaches can guide experimental design by providing reliable structural predictions and identifying functionally important regions for targeted mutagenesis .

• Single-cell multi-omics: Integrating transcriptomic, proteomic, and epigenomic data at single-cell resolution can reveal cell-type-specific functions of lrrc3 during development and in adult tissues.

What are the most promising research questions about zebrafish lrrc3 that remain unanswered?

Several critical questions about zebrafish lrrc3 warrant investigation:

• Binding partners and signaling pathways: What molecules interact with zebrafish lrrc3, and what signaling cascades does it participate in? Protein-protein interaction studies, including co-immunoprecipitation and proximity labeling approaches, could address this question.

• Developmental functions: What is the specific role of lrrc3 in zebrafish development? Detailed phenotypic analysis of knockout and knockdown models, combined with rescue experiments, would elucidate these functions.

• Subcellular localization: Where within cells is lrrc3 localized, and does this change during development or in response to stimuli? Immunofluorescence microscopy with specific antibodies or fluorescent protein fusions could reveal this information.

• Redundancy with other LRR proteins: To what extent do other LRR proteins compensate for lrrc3 loss? Double or triple knockout studies targeting related family members could address functional redundancy.

• Evolutionary divergence of function: How has the function of lrrc3 diverged across vertebrate evolution? Comparative studies in multiple model organisms could provide insights into conserved and species-specific roles.

• Post-translational regulation: How do modifications like glycosylation or phosphorylation affect lrrc3 function? Mass spectrometry-based proteomics combined with site-directed mutagenesis would illuminate these regulatory mechanisms.