Recombinant Chicken Methyltransferase-like protein 9 (METTL9)

What is METTL9 and what is its primary function?

METTL9 (Methyltransferase Like 9) is a protein coding gene that functions as a histidine methyltransferase. Its primary enzymatic activity involves the N1-specific methylation of histidine residues, particularly targeting the second histidine within "His-x-His" (HxH) motifs, where 'x' typically represents an amino acid with a small side chain. This methylation activity is dependent on S-adenosylmethionine (SAM) as a methyl donor .

The enzyme recognizes the first histidine in the HxH motif as a signature element while methylating the second histidine. This specificity is achieved through an intimate engagement between METTL9 and a pentapeptide motif, where the small "x" residue becomes embedded and confined within the substrate pocket. During complex formation, the N3 atom of the histidine imidazole ring is stabilized by an aspartate residue, which positions the N1 atom for methylation by S-adenosylmethionine .

How conserved is METTL9 between chicken and other species?

While the search results don't provide specific sequence identity percentages between chicken METTL9 and other species, there is evidence of evolutionary conservation. The fact that recombinant proteins have been produced for multiple species including human, chicken, zebrafish, and mouse suggests functional conservation .

The SAM/SAH binding residues in METTL9 are highly conserved across species, with only Leu175 showing variations. This conservation indicates a preserved mode of cofactor binding that is essential to the core methyltransferase activity . Researchers working with chicken METTL9 can likely draw some functional parallels from studies of the human ortholog, though species-specific differences in substrate specificity and regulatory mechanisms should be considered.

What is the typical protein structure of METTL9?

METTL9 adopts a canonical seven-β-strand (7BS) methyltransferase architecture. This structure is characterized by a seven-strand β-sheet arranged in the order β4-β3-β2-β5-β6-β10-β9. The cofactor S-adenosylhomocysteine (SAH) or S-adenosylmethionine (SAM) binds in a deep groove formed by β2, β3, β5, α7, and α8 structural elements .

In crystal structures obtained at resolutions as high as 1.69 Å, the SAH molecule is observed to interact with METTL9 primarily through hydrogen bonding. The adenosine ring is sandwiched between Leu175 and Leu211. Specific hydrogen bonds form between the ribose O3' and O4' atoms and Glu174, between the SAH amide and the main chain of Leu209, and between the carboxylate groups of SAH and the side chains of Asn210 and Tyr295 .

What expression systems are commonly used for producing recombinant chicken METTL9?

Based on the available information, recombinant chicken METTL9 has been successfully produced, though specific expression system details for chicken METTL9 are not provided in the search results . For human METTL9, HEK293 cells have been utilized as an expression system, which suggests that mammalian expression systems might be appropriate for chicken METTL9 as well, particularly when post-translational modifications and proper folding are concerns .

When designing an expression system for chicken METTL9, researchers should consider:

• Codon optimization for the chosen expression host

• The addition of purification tags (such as Myc/DDK tags used for human METTL9)

• Expression conditions that maximize soluble protein yield

• Purification strategies that preserve enzymatic activity

How can the enzymatic activity of recombinant chicken METTL9 be assessed in vitro?

To assess chicken METTL9 enzymatic activity in vitro, researchers can adapt methods used for human METTL9, which include:

• Mass Spectrometry (MS) Analysis: Incubate purified METTL9 with synthetic peptide substrates containing the "HxH" motif in the presence of S-adenosylmethionine (SAM) as a methyl donor. After the reaction, use MS to detect mass shifts corresponding to methyl group addition (+14 Da per methylation) .

• Enzyme Kinetics Assay: Determine the kinetic parameters (kcat, Km) using varying concentrations of peptide substrates and monitoring product formation over time. For human METTL9 variants, kcat/Km values around 0.37 μM⁻¹ h⁻¹ have been reported, providing a benchmark for comparison .

• Isothermal Titration Calorimetry (ITC): Assess binding affinities between chicken METTL9 and potential peptide substrates. For human METTL9, binding affinities (Kd values) typically range from 2.3–9.3 μM .

• Mutational Analysis: Create point mutations in key residues (based on human METTL9 studies, such as E174A or Y306A/L308A) to verify the importance of specific amino acids for substrate binding and catalytic activity .

When comparing chicken METTL9 activity to human METTL9, researchers should pay attention to potential differences in substrate specificity, particularly regarding the amino acid preferences surrounding the "HxH" motif.

What are the recommended approaches for studying METTL9 substrate specificity in chicken tissues?

To study chicken METTL9 substrate specificity in tissue contexts, researchers can employ several complementary approaches:

• Peptide Array Screening: Design a peptide array containing various HxH motifs with different flanking sequences to determine the sequence preferences of chicken METTL9. This approach can reveal whether chicken METTL9 shares the preference for small side chain residues at the "x" position as observed in human METTL9 .

• Proteomics Identification of Methylated Substrates:

  • Perform immunoprecipitation using anti-METTL9 antibodies from chicken tissue lysates

  • Analyze co-precipitated proteins by mass spectrometry

  • Look for proteins containing HxH motifs and verify methylation status

  • Compare methylation patterns between wild-type and METTL9-knockdown samples

• Targeted Analysis of Potential Substrates: Based on known human METTL9 substrates, examine the conservation of these proteins in chicken and assess their methylation status. For example, if SLC39A5 is a substrate in humans, its chicken ortholog could be investigated .

• Tissue-Specific Expression Analysis: Determine the expression profile of METTL9 across different chicken tissues using qPCR, similar to the approach used for METTL21C which showed enrichment in chicken soleus and gastrocnemius muscles . This can provide clues about tissue-specific functions and potential substrate availability.

How can structural studies of chicken METTL9 inform substrate design and inhibitor development?

Structural studies of chicken METTL9 can significantly advance our understanding of its function and facilitate rational substrate and inhibitor design. Based on human METTL9 structural insights, researchers should consider:

• X-ray Crystallography Approach:

  • Express and purify chicken METTL9 (potentially using mutations similar to the M6 variant used for human METTL9 if crystallization is challenging)

  • Co-crystallize with SAH/SAM and various peptide substrates

  • Determine structures at high resolution (aim for <2.0 Å)

  • Compare with human METTL9 structures (PDB: 8GZF, 7YF2, 7Y9C, 7YF3, 7YF4)

• Key Structural Features to Analyze:

  • The substrate binding pocket that accommodates the "HxH" motif

  • The position of the aspartate residue that stabilizes the N3 atom of histidine

  • The SAM-binding groove formed by conserved residues

  • Potential species-specific differences in the substrate recognition region

• Substrate Design Considerations:

  • Optimize the amino acid at the "x" position in the "HxH" motif

  • Explore flanking sequences that enhance binding affinity

  • Design peptides with multiple "HxH" motifs to study the "C-to-N" directional methylation preference

• Inhibitor Development Strategy:

  • Design SAM analogs that compete for the cofactor binding site

  • Develop peptide-based inhibitors that mimic the "HxH" motif but resist methylation

  • Target species-specific features if chicken METTL9 shows structural differences from human METTL9

A detailed structure-function analysis will help researchers understand if chicken METTL9 shares the same mechanistic features as human METTL9, particularly the N1-specific methylation without requiring substantial conformational changes during catalysis .

What are the potential biological implications of METTL9-mediated methylation in chicken development and disease?

Based on what is known about METTL9 in other species, several important biological implications could be investigated in chickens:

• Developmental Roles:

  • Analyze METTL9 expression during chicken embryonic development

  • Investigate potential roles in tissue-specific development, particularly in muscles where methyltransferases like METTL21C have shown enrichment

  • Examine the temporal regulation of METTL9 expression and activity during different developmental stages

• Pathological Implications:

  • Explore potential roles in diseases analogous to those associated with human METTL9, such as hearing impairments (related to Deafness, Autosomal Recessive 22) and inflammatory conditions

  • Investigate whether METTL9 has oncogenic properties in chicken cell models, similar to its reported role in human hepatocellular carcinoma

  • Study the relationship between METTL9 and ferroptosis regulation through potential substrates like SLC7A11

• Cellular Pathway Analysis:

  • Determine if chicken METTL9 affects specific cellular pathways through methylation of key proteins

  • Investigate whether METTL9-mediated methylation affects protein-protein interactions, protein stability, or subcellular localization

  • Examine crosstalk between METTL9 and other post-translational modification systems

• Evolutionary Considerations:

  • Compare substrate specificity between chicken and mammalian METTL9 to identify conserved and divergent targets

  • Analyze whether METTL9 function has been evolutionarily adapted for avian-specific biological processes

How can CRISPR/Cas9 gene editing be optimized for studying METTL9 function in chicken cell lines?

CRISPR/Cas9 gene editing offers powerful approaches for studying chicken METTL9 function:

• Guide RNA Design Strategy:

  • Target conserved regions in chicken METTL9 exons, particularly those encoding catalytic residues

  • Design multiple gRNAs targeting different regions to increase editing efficiency

  • Perform in silico off-target analysis using chicken genome databases

  • Consider targeting regulatory regions to study expression control mechanisms

• Editing Approaches:

  • Complete knockout: Design gRNAs to create frameshift mutations or large deletions

  • Point mutations: Use homology-directed repair to introduce specific mutations in catalytic residues (e.g., equivalent to E174A or Y306A in human METTL9)

  • Domain deletion: Target specific functional domains while preserving others

  • Endogenous tagging: Add reporter tags to study localization and interactions

• Validation Methods:

  • Genomic PCR and sequencing to confirm edits

  • Western blotting to verify protein expression changes

  • Enzymatic activity assays to assess functional consequences

  • RNA-seq to identify genes affected by METTL9 modification

• Experimental Applications:

  • Generate METTL9 knockout chicken cell lines to identify cellular processes dependent on its activity

  • Create catalytically inactive METTL9 mutants to distinguish between enzymatic and potential scaffolding functions

  • Perform rescue experiments with wild-type or mutant METTL9 to confirm specificity of observed phenotypes

  • Use inducible CRISPR systems for temporal control of METTL9 disruption

What is the recommended protocol for purifying recombinant chicken METTL9?

While specific purification protocols for chicken METTL9 are not detailed in the search results, the following protocol can be adapted based on approaches used for human METTL9 and standard protein purification techniques:

• Expression System Selection:

  • Consider using HEK293 cells for mammalian expression as used for human METTL9

  • Alternative systems include E. coli or insect cells if proper folding can be achieved

• Construct Design:

  • Include a C-terminal or N-terminal affinity tag (Myc/DDK tags have been used for human METTL9)

  • Consider adding a cleavable linker between the protein and tag

  • Optimize chicken METTL9 coding sequence for the expression host

• Purification Steps:

  a. Cell Lysis:

  • Harvest cells and resuspend in lysis buffer (typically 25 mM Tris-HCl pH 7.3, 150 mM NaCl, 1% Triton X-100, protease inhibitors)

  • Sonicate or use mechanical disruption for thorough lysis

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

  b. Affinity Chromatography:

  • For His-tagged constructs: Use Ni-NTA resin

  • For FLAG/DDK-tagged constructs: Use anti-FLAG affinity resin

  • Wash extensively to remove non-specific binding

  c. Additional Purification:

  • Ion exchange chromatography (based on theoretical pI of chicken METTL9)

  • Size exclusion chromatography to ensure monodispersity and remove aggregates

• Quality Control:

  • SDS-PAGE to assess purity (aim for >80% as used for human METTL9)

  • Western blot to confirm identity

  • Mass spectrometry to verify protein integrity

  • Dynamic light scattering to check for aggregation

• Storage:

  • Store in buffer containing 25 mM Tris-HCl pH 7.3, 100 mM glycine

  • Flash-freeze aliquots in liquid nitrogen

  • Store at -80°C to maintain stability

  • Avoid repeated freeze-thaw cycles

What analytical techniques are most effective for characterizing chicken METTL9 methylation activity?

To comprehensively characterize chicken METTL9 methylation activity, researchers should consider a multi-technique approach:

• Mass Spectrometry-Based Analysis:

  • Targeted MS/MS to identify methylated histidine residues and distinguish between N1 and N3 methylation

  • Quantitative approaches (MRM/PRM) to measure methylation stoichiometry

  • Top-down proteomics to analyze intact methylated proteins

  • MALDI-TOF for rapid screening of methylation on synthetic peptides

• Biochemical Assays:

  • Radiometric assays using [³H]-SAM to measure methyltransferase activity

  • Coupled enzymatic assays monitoring SAH production

  • Fluorescence-based approaches using methylation-sensitive fluorescent probes

  • Enzyme kinetics determination for various substrates (Km, kcat, kcat/Km)

• Structural Analysis:

  • X-ray crystallography of chicken METTL9 with substrates and product analogs

  • NMR studies to examine dynamic aspects of substrate binding

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational changes

  • Molecular dynamics simulations to predict substrate binding modes

• Comparative Analysis:

  • Side-by-side assessment of chicken and human METTL9 activity on the same substrates

  • Analysis of species-specific substrate preferences

  • Evaluation of the effect of "HxH" motif variations on methylation efficiency

When designing these experiments, researchers should take into account the catalytic parameters observed for human METTL9 variants (kcat/Km value of approximately 0.37 μM⁻¹ h⁻¹) and substrate binding affinities (Kd values in the range of 2.3–9.3 μM) as reference points .

What are the current limitations in studying chicken METTL9 function and how might they be overcome?

Several challenges exist in studying chicken METTL9, along with potential strategies to address them:

• Limited Specific Research:

  • Challenge: Relatively few studies have focused specifically on chicken METTL9 compared to human METTL9

  • Solution: Leverage the evolutionary conservation between species to apply findings from human studies while validating chicken-specific aspects

• Substrate Identification:

  • Challenge: Comprehensive identification of physiological substrates remains difficult

  • Solution: Combine proteomics approaches with bioinformatics prediction of proteins containing "HxH" motifs in the chicken proteome

• Functional Redundancy:

  • Challenge: Potential overlapping functions with other methyltransferases may complicate phenotypic analysis

  • Solution: Generate combinatorial knockouts or use specific inhibitors to distinguish unique METTL9 functions

• Tissue-Specific Roles:

  • Challenge: Understanding tissue-specific functions in the context of chicken development

  • Solution: Develop conditional knockout models or use tissue-specific promoters for expression studies

• Technical Limitations:

  • Challenge: Obtaining high-quality recombinant protein with proper folding and activity

  • Solution: Optimize expression conditions, consider using chicken cell lines for expression, and explore different tags and purification strategies

How might METTL9 be involved in chicken muscle development and what experimental approaches could test this?

Given that methyltransferases like METTL21C have shown enrichment in chicken muscles , METTL9 might also play a role in muscle development. To investigate this potential function:

• Expression Analysis:

  • Perform qPCR and Western blot analysis of METTL9 expression across different muscle types and developmental stages

  • Use in situ hybridization to visualize spatial expression patterns in embryonic and adult muscles

  • Compare expression between fast-twitch and slow-twitch fibers

• Loss-of-Function Studies:

  • Generate METTL9 knockdown or knockout in chicken myoblast cell lines

  • Analyze effects on myoblast proliferation, differentiation, and fusion

  • Examine changes in muscle-specific gene expression programs

  • Investigate potential alterations in myofiber type specification

• Substrate Identification in Muscle Context:

  • Perform proteomics analysis of methylated proteins in chicken muscle tissues

  • Focus on muscle-specific proteins containing "HxH" motifs

  • Investigate whether key myofibrillar proteins or regulatory factors are METTL9 substrates

• Functional Analysis:

  • Examine the impact of METTL9 modulation on muscle contractile properties

  • Assess effects on muscle regeneration following injury

  • Investigate potential roles in muscle metabolism and energy utilization

• Comparative Studies:

  • Compare METTL9 functions between different chicken breeds with varying muscle characteristics

  • Analyze differences between birds raised in free-range versus caged conditions

  • Examine potential interactions between METTL9 and other muscle-enriched methyltransferases