Recombinant Rat Echinoderm microtubule-associated protein-like 5 (Eml5), partial

What is the molecular structure of rat EML5 and how does it compare to other species?

Rat EML5 belongs to the Echinoderm Microtubule-Associated Protein-Like (EML) family, which has five members in mammals (EML1 through EML5) but only one homolog in both Drosophila (ELP-1) and C. elegans (ELP-1) . The protein contains 11 WD40 domain repeats and 3 HELP domains that form three TAPE domains. A distinctive structural feature of EML5 is the presence of an α-helical coiled-coil between the N-terminus and central regions, suggesting potential for homodimerization or heterodimerization with other EML family members, particularly EML3 or EML4 .

Unlike other EML family members, EML5 uniquely lacks a calcium-binding EF-hand domain . The WD40 domains form β-propeller structures that have been modeled using multiple approaches including HHpred/MODELLER, TrRosetta/YASARA, and AlphaFold2 . These models reveal that key residues like R1654 (in bovine EML5) are located at the end of the penultimate blade of the N-terminal β-propeller and protrude into the solvent, potentially affecting protein-protein interactions .

For researchers investigating structural conservation, it's worth noting that C. elegans ELP-1 contains a unique exon (exon 5) that is highly conserved between C. elegans and C. briggsae (88% identical) but not detected in other EMAP-like proteins, suggesting nematode-specific functions .

Where is EML5 predominantly expressed in rats and what does this suggest about its function?

In rats, EML5 is primarily expressed within the nervous system. It was first detected in rat brain, with the highest expression levels in the cerebellum, hippocampus, and olfactory bulb . This neuronal expression pattern suggests potential roles in neuronal development, synaptic function, or microtubule organization within specific brain regions.

Interestingly, expression patterns differ between species. In cattle, the Bgee database indicates that EML5 expression is highest in the reproductive system, adrenal gland, retina, and cerebellum . This cross-species variation suggests potentially diverse tissue-specific functions.

For researchers studying EML5 in specific contexts, these expression patterns provide valuable guidance for selecting appropriate experimental systems and designing tissue-specific investigations.

What are the optimal approaches for cloning and expressing recombinant rat EML5?

Based on published methodologies for related EML proteins, researchers can design species-specific primers using NCBI Primer-BLAST with the appropriate reference genome. For PCR amplification, a protocol similar to that used for bovine EML5 can be applied:

• Design forward and reverse primers that specifically amplify the target EML5 sequence

• Perform PCR using an optimized reaction mixture (e.g., 1 μl of gDNA (20 ng/μl), 1 μl of each primer (10 μM), 3 μl of master mix, 4 μl of nuclease-free water)

• Run approximately 30 cycles of denaturation (94°C for 30s), annealing (60°C for 60s), and extension (72°C for 30s)

• Confirm PCR products by electrophoresis on 1% agarose gel

• Clean up PCR products using a purification kit

• Verify DNA concentration using spectrophotometry

• Clone the purified product into an expression vector with appropriate tags

For recombinant protein expression, bacterial systems like E. coli or eukaryotic systems like insect cells may be used depending on whether post-translational modifications are required for functional studies.

What techniques are most effective for studying EML5 localization and interactions?

Multiple complementary approaches can be used to study EML5 localization and interactions:

• Fluorescent protein tagging: Creating EML5::GFP fusion constructs allows visualization of localization patterns in live cells. Both full-length and domain-specific constructs should be considered, as truncated constructs may show different localization patterns compared to full-length proteins .

• Immunofluorescence microscopy: Developing specific antibodies against EML5 allows detection of endogenous protein. This can be combined with markers for subcellular structures (e.g., microtubules, dense bodies) to assess co-localization .

• Super-resolution microscopy: For detailed analysis of EML5 association with cytoskeletal elements or other subcellular structures.

• Protein-protein interaction studies: Co-immunoprecipitation, yeast two-hybrid, or proximity labeling techniques can identify EML5 binding partners.

• Microtubule co-sedimentation assays: These can determine direct interaction with microtubules, as observed with other EML family members and their homologs .

When interpreting localization data, researchers should be aware that membrane components and associated structures (like dense bodies in muscle cells) can sometimes be separated from internal cytoskeletal elements during sample preparation, affecting apparent protein localization .

How can structural modeling help predict the effects of EML5 mutations?

Structural modeling provides valuable insights into how mutations might affect EML5 function:

• Multiple modeling approaches: Using complementary methods like HHpred/MODELLER, TrRosetta/YASARA, and AlphaFold2 can provide more robust structural predictions. These approaches have been successfully applied to model the C-terminal region of EML5 (residues E1369-C1942), with the three methods producing similar models of the EML5 TAPE domain .

• Visualizing mutation effects: Tools like PyMOL's mutagenesis wizard can model the effects of specific mutations, as demonstrated for the R1654W mutation in bovine EML5 . This allows visualization of how mutations might alter surface properties, protein folding, or interaction interfaces.

• Validating predictions experimentally: After generating structural predictions, researchers should validate them through experimental approaches such as circular dichroism spectroscopy, thermal stability assays, or direct binding assays comparing wild-type and mutant proteins.

Figure 1B from the literature shows a top view of models of the N-terminal β-propeller of the EML5 TAPE domain (residues G1378-S1698), generated by different modeling approaches and superimposed onto the crystal structure of the corresponding region of EML1 . This demonstrates how structural modeling can reveal the position of specific residues (like R1654) within the protein's three-dimensional structure.

What are the known functional roles of EML5 in neuronal systems?

While specific functions of rat EML5 in neurons require further investigation, its high expression in the cerebellum, hippocampus, and olfactory bulb suggests important roles in these regions . Based on studies of related proteins and homologs, several potential functions can be proposed:

• Microtubule organization: Other EML family members and the C. elegans homolog ELP-1 interact with microtubules , suggesting EML5 may regulate neuronal microtubule dynamics or organization.

• Synaptic function: The enrichment in regions like the hippocampus suggests potential roles in synaptic development or plasticity.

• Neuronal development: EML proteins may influence neuronal migration, axon guidance, or dendrite formation through cytoskeletal regulation.

To investigate these functions, researchers could employ techniques such as:

• Neuronal-specific knockdown or knockout of EML5

• Live imaging of EML5-GFP in developing neurons

• Electrophysiological recordings to assess effects on synaptic transmission

• Axon regeneration assays to test roles in neuronal repair

What is the evidence for EML5's role in reproductive biology?

Several lines of evidence suggest EML5 involvement in reproductive functions:

• Expression patterns: In cattle, EML5 expression is highest in the reproductive system, suggesting tissue-specific roles .

• Genetic associations: Polymorphisms within the EML5 gene have been associated with decreased sperm motility , indicating potential roles in male fertility.

• EML family precedent: Other EML family members have established reproductive functions - EML4 undergoes tyrosine phosphorylation during sperm capacitation, and EML6 participates in oocyte meiotic division regulation .

For researchers investigating EML5's reproductive functions, approaches might include:

• Detailed expression analysis throughout gametogenesis

• Generation of tissue-specific knockout models

• Assessment of sperm parameters in models with EML5 mutations

• Protein interaction studies in reproductive tissues

Could EML5 have potential roles in disease pathogenesis?

While the search results don't directly implicate rat EML5 in disease, evidence from other EML family members suggests potential pathological relevance:

• Cancer associations: EML1-ABL1 fusion proteins constitutively activate oncogenic signaling pathways (ERK, Stat5, and Src) . Similarly, EML4 fusions with anaplastic lymphoma kinase (ALK) occur in non-small cell lung cancers . These precedents suggest EML5 might also form oncogenic fusion proteins in certain contexts.

• Neurological disorders: Given its high neuronal expression, mutations or dysregulation of EML5 could potentially contribute to neurological disorders.

• Fertility issues: The association between EML5 polymorphisms and sperm motility suggests potential involvement in fertility disorders .

Researchers investigating disease associations could:

• Screen patient samples for EML5 mutations or expression changes

• Analyze public genome-wide association studies for EML5 variants linked to specific conditions

• Generate disease-relevant cellular or animal models with altered EML5 function

How does the WD40 domain architecture of EML5 influence its functions?

The 11 WD40 domain repeats in EML5 form β-propeller structures that likely mediate protein-protein interactions . Research approaches to understand their functional significance include:

• Domain deletion studies: Creating constructs lacking specific WD40 repeats to determine their contribution to localization and function.

• Point mutation analysis: Introducing mutations in conserved residues (like the R1654 studied in bovine EML5) to assess effects on protein structure and interactions .

• Protein interaction screening: Using techniques like BioID or yeast two-hybrid with isolated WD40 domains to identify domain-specific binding partners.

The literature shows that R1654 in bovine EML5 is located at the end of the penultimate blade of the β-propeller and protrudes into the solvent, potentially forming part of a protein interaction surface . Similar structurally important residues could be identified in rat EML5 through comparative analysis.

What challenges exist in creating specific antibodies against rat EML5?

Developing specific antibodies against rat EML5 presents several challenges:

• Sequence conservation: High homology between EML family members may lead to cross-reactivity. Researchers should carefully select unique epitopes specific to EML5.

• Conformational epitopes: The complex tertiary structure of EML5, particularly the β-propeller domains, may mean that linear epitopes fail to recognize the native protein.

• Validation strategies: Researchers should validate antibody specificity using multiple approaches:

  • Testing on recombinant EML5 protein

  • Western blotting of tissues with known EML5 expression

  • Comparing with GFP-tagged EML5 localization patterns

  • Testing in EML5 knockout/knockdown samples as negative controls

How can contradictory findings in EML5 research be reconciled?

When facing seemingly contradictory results in EML5 research, several methodological approaches can help reconcile disparate findings:

• Context-dependent effects: As seen with the different expression patterns between rat and cattle EML5 , function may be species or tissue-specific. Systematic comparison across experimental systems can clarify these differences.

• Isoform-specific effects: Checking for alternative splicing or post-translational modifications that might generate functionally distinct EML5 variants.

• Methodological differences: Different techniques (e.g., antibody staining vs. GFP fusion) might reveal different aspects of EML5 biology, as observed with C. elegans ELP-1 where truncated and full-length constructs showed different localization patterns .

• Technical artifacts: Some contradictions may result from experimental artifacts, such as the separation of membrane components and associated structures during sample preparation .

How might EML5 research contribute to understanding neurological disorders?

Given EML5's high expression in the cerebellum, hippocampus, and olfactory bulb , research in this area may:

• Illuminate cytoskeletal regulation in neurons: Microtubule organization is crucial for neuronal function, and EML5's potential role in this process could provide insights into disorders involving neuronal cytoskeletal abnormalities.

• Identify novel disease mechanisms: Screening for EML5 mutations or expression changes in patients with unexplained neurological disorders, particularly those affecting regions with high EML5 expression.

• Suggest therapeutic targets: If EML5 dysfunction contributes to specific disorders, it could represent a novel therapeutic target, particularly if its enzymatic activities or protein interactions can be modulated pharmacologically.

• Improve understanding of neuronal connectivity: The potential role of EML5 in microtubule organization might influence axonal pathfinding or synapse formation, processes disrupted in many neurodevelopmental disorders.

What are the most promising directions for EML5 research in reproductive biology?

Based on the association between EML5 polymorphisms and sperm motility , several research directions appear promising:

• Detailed characterization of EML5 in gametes: Investigating the precise localization and dynamics of EML5 in sperm and potentially oocytes.

• Mechanistic studies of sperm motility regulation: Determining whether EML5 directly influences flagellar microtubule organization or interacts with other motility-related proteins.

• Clinical correlation studies: Analyzing EML5 variants in humans with unexplained infertility to determine clinical relevance.

• Therapeutic implications: Exploring whether modulating EML5 function could address certain forms of infertility, particularly those involving reduced sperm motility.

The finding that polymorphisms in EML5 are associated with decreased sperm motility provides a foundation for these investigations.

How can emerging technologies advance EML5 research?

Several cutting-edge technologies offer new approaches to understanding EML5 biology:

• Cryo-electron microscopy: Could provide high-resolution structural information about EML5 alone or in complex with binding partners or microtubules.

• Single-cell transcriptomics: May reveal cell-type specific expression patterns of EML5 and co-expressed genes in complex tissues like brain or testis.

• CRISPR-Cas9 genome editing: Enables precise manipulation of EML5 in cellular and animal models, including introduction of specific mutations identified in human disorders.

• Proximity labeling proteomics: Techniques like BioID or APEX can identify proteins in close proximity to EML5 in living cells, helping map its interaction network.

• Live super-resolution microscopy: Can reveal the dynamics of EML5-microtubule interactions with unprecedented spatial and temporal resolution.

These technologies, combined with the foundational knowledge summarized in these FAQs, provide a roadmap for researchers investigating the complex biology of rat EML5.