Recombinant Macaca mulatta Transmembrane O-methyltransferase (LRTOMT)

What is the genomic organization of LRTOMT in primates versus rodents?

LRTOMT in primates is a fusion gene with 10 exons that produces five different alternatively spliced transcripts. Interestingly, exons 5, 7, and 8 are translated in two alternative reading frames, encoding either the C-terminus of LRTOMT1 or the N-terminus of LRTOMT2. In contrast, rodents have two separate genes, Lrrc51 and Tomt, which together are orthologous to primate LRTOMT. Despite extensive attempts using various primer combinations and RACE analysis, fusion transcripts between Lrrc51 and Tomt have not been detected in rodent tissue (brain, liver, heart), although they are readily amplifiable from human tissues . This evolutionary divergence has important implications for researchers using rodent models to study LRTOMT function.

How does the protein structure of Macaca mulatta LRTOMT compare to human LRTOMT?

Macaca mulatta, along with four other primate species, expresses transcripts that include nearly all exons of LRTOMT as well as a separate transcript equivalent to rodent Lrrc51 . The protein expressed in Macaca mulatta is structurally similar to human LRTOMT, reflecting their close evolutionary relationship. Western blot analyses have demonstrated that antisera directed against mouse TOMT recognized a 38 kDa LRTOMT2 protein in human tissues, which is consistent with the predicted size of human isoform D' (32.2 kDa deduced). This suggests structural conservation of the functional domains between human and Macaca mulatta LRTOMT proteins, particularly in the catalytic catechol-O-methyltransferase domain .

What are the specific domains and functional regions of LRTOMT in Macaca mulatta?

LRTOMT in Macaca mulatta, similar to humans, encodes two distinct proteins. LRTOMT1 contains two leucine-rich repeats, while LRTOMT2 features a catechol-O-methyltransferase domain. The catechol-O-methyltransferase domain is particularly important for enzymatic function. Molecular modeling based on rat COMT crystal structure (pdb-code 1h1d) demonstrates that key functional residues in LRTOMT2 are located in helix 1 (p.R81), helix 2 (p.W105), and the loop following helix 2 (p.E110) . These structural elements form critical salt bridges, hydrogen bonds, and hydrophobic interactions that maintain protein stability and influence substrate binding. The catalytic domain is essential for the methyltransferase activity that may be crucial for auditory function.

What are the recommended methods for recombinant expression of Macaca mulatta LRTOMT?

For recombinant expression of Macaca mulatta LRTOMT, researchers should consider several methodological approaches:

• Gene Synthesis: Custom synthesis of the LRTOMT coding sequence optimized for the expression system of choice, based on Macaca mulatta sequence data.

• Expression System Selection: Mammalian expression systems (HEK293, CHO cells) are preferred over bacterial systems to ensure proper post-translational modifications. For structural studies requiring higher protein yields, insect cell systems (Sf9, High Five) may be considered.

• Vector Design: Incorporate a fusion tag (His, GST, or FLAG) for purification, with a protease cleavage site for tag removal. Include species-appropriate Kozak sequence for optimal translation initiation.

• Transcript Verification: Use RT-PCR and sequencing to confirm correct splicing of all 10 exons, particularly focusing on the alternative reading frames in exons 5, 7, and 8 that are critical for producing the correct protein isoforms .

• Protein Expression Validation: Western blotting with specific antibodies against either LRTOMT1 or LRTOMT2 domains to verify expression of the correct protein variants (approximately 28-30 kDa for TOMT isoform and 38 kDa for LRTOMT2) .

What are the most effective methods for analyzing LRTOMT mutations and their functional impacts?

Several complementary approaches are recommended for comprehensive analysis of LRTOMT mutations:

• Genomic Screening: Initial screening can be performed using PCR-Single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) to detect potential mutations, followed by Sanger sequencing for confirmation .

• Bioinformatic Analysis: Utilize prediction tools such as SIFT (deleterious threshold score: ≤0.05) and Mutation Taster (disease-causing probability near 1.0) to assess potential pathogenicity of identified variants .

• Structural Analysis: Apply molecular modeling using established crystal structures (e.g., rat COMT structure) to predict the impact of mutations on protein folding and function. Key effects to examine include disruption of secondary structures, such as conversion from organized α-helix to random conformation .

• Transcript Analysis: RT-PCR using lymphoblastoid cell lines or recombinant expression systems to evaluate effects on splicing, as demonstrated with mutations affecting exon 8 inclusion .

• Functional Assays: Develop methyltransferase activity assays to quantitatively measure the impact of mutations on enzymatic function, particularly for variants affecting the catechol-O-methyltransferase domain.

What experimental controls are critical when working with recombinant LRTOMT?

When conducting experiments with recombinant Macaca mulatta LRTOMT, the following controls are essential:

• Species Controls: Include both human and rodent LRTOMT/Tomt as comparative controls to account for species-specific differences in structure and function .

• Isoform Controls: Generate constructs expressing each specific isoform (LRTOMT1, LRTOMT2) separately to distinguish isoform-specific effects, as the fusion gene encodes proteins with distinct functions .

• Mutation Controls: Include known pathogenic variants (e.g., p.R81Q, p.W105R, p.E110K) as positive controls for functional disruption .

• Tissue-specific Expression Controls: When studying expression patterns, compare multiple tissues (cochlea, brain, liver, kidney) as LRTOMT shows differential expression across tissues .

• Site-directed Mutagenesis Controls: For mutation studies, create positive control samples via site-directed mutagenesis to validate detection methods .

• Western Blot Controls: Use specific antibodies that can distinguish between LRTOMT1 and LRTOMT2 proteins, with expected band sizes of approximately 28-30 kDa for TOMT and 38 kDa for LRTOMT2 .

How do mutations in the catechol-O-methyltransferase domain affect LRTOMT2 protein function?

Mutations in the catechol-O-methyltransferase domain of LRTOMT2 can significantly disrupt protein function through several mechanisms:

What are the key differences between LRTOMT isoforms and their specific functions?

LRTOMT produces multiple protein isoforms with distinct structural features and likely different functional roles:

The functional distinction between these isoforms is highlighted by the fact that pathogenic mutations causing hearing loss primarily affect the LRTOMT2 protein, particularly its catechol-O-methyltransferase domain . The dual reading frame feature of exons 5, 7, and 8 allows a single genetic locus to encode proteins with entirely different functions, representing an unusual example of genomic economy .

What is the evolutionary significance of the fusion gene structure in primates versus separate genes in rodents?

The evolutionary divergence between primate LRTOMT fusion gene and the separate rodent Lrrc51 and Tomt genes represents a fascinating case study in molecular evolution:

• Genomic Economy: The fusion gene in primates allows for production of two functionally distinct proteins from overlapping genomic sequences, utilizing alternative reading frames in exons 5, 7, and 8. This represents an efficient use of genomic material .

• Regulatory Coordination: The fusion may allow for coordinated regulation of both proteins in primates, whereas rodents would require separate regulatory mechanisms for each gene.

• Evolutionary Constraint: In rodents, a hypothetical fusion transcript would be non-functional because if the first translation start codon (ATG in exon 5) were used, an in-frame translation stop codon would occur just four codons downstream. Additionally, the first exon of Tomt in rodents lacks an in-frame consensus splice acceptor site .

• Primate-Specific Innovation: This fusion appears to be primate-specific, as it is observed in five different primate species, suggesting it occurred after the divergence of rodents and primates but before the radiation of modern primates .

• Functional Conservation: Despite the structural difference, functional conservation is evident as mutations in human LRTOMT and mouse Tomt both result in hearing impairment, suggesting the methyltransferase function is the critical component for auditory function .

How can LRTOMT research in Macaca mulatta inform human hearing loss mechanisms?

Research on LRTOMT in Macaca mulatta provides valuable insights into human hearing loss mechanisms through several avenues:

• Evolutionary Proximity: Macaca mulatta is genetically closer to humans than rodent models, particularly those from Nepal which show greater similarity to Indian-origin than Chinese-origin rhesus macaques . This makes them excellent translational models for human LRTOMT function.

• Structural Conservation: The catechol-O-methyltransferase domain shows significant conservation between species, with 39% sequence identity between human LRTOMT2 and rat COMT in the modeled region (residues 79-290) . This conservation enables structural insights from macaque studies to be applied to human hearing mechanisms.

• Mutation Analysis: Studies of naturally occurring or engineered LRTOMT mutations in Macaca mulatta can provide insights into how similar mutations affect auditory function in humans, potentially revealing the precise biochemical and cellular mechanisms of pathogenesis.

• Therapeutic Development: Macaque models can serve as a platform for testing gene therapy or other interventions before human clinical trials, particularly important since early intervention (such as cochlear implants) appears to improve auditory outcomes in humans with LRTOMT mutations .

• Developmental Insights: Studying LRTOMT expression and function during cochlear development in macaques may reveal critical windows for intervention in human hearing loss.

What are the challenges in developing recombinant LRTOMT for research applications?

Researchers face several significant challenges when developing recombinant Macaca mulatta LRTOMT:

• Alternative Splicing Complexity: The gene produces five different alternatively spliced transcripts, making it challenging to determine which specific isoform(s) to express for particular research applications .

• Dual Reading Frames: The unusual feature of exons 5, 7, and 8 being translated in two alternative reading frames complicates expression system design, as special care must be taken to ensure correct translation of the intended protein (LRTOMT1 or LRTOMT2) .

• Post-translational Modifications: Ensuring proper folding and post-translational modifications is critical, particularly for the catechol-O-methyltransferase domain, which may require specific cofactors or processing steps for activity.

• Expression System Selection: Different expression systems (bacterial, insect, mammalian) may produce proteins with varying levels of activity and structure, necessitating careful system selection and validation.

• Protein Solubility: Transmembrane proteins often present challenges for recombinant expression and purification due to hydrophobic domains and complex folding requirements.

• Functional Assays: Developing reliable assays to measure methyltransferase activity requires identification of the natural substrate, which remains poorly defined for LRTOMT.

What genetic screening approaches are most effective for identifying novel LRTOMT mutations in research populations?

Based on successful identification of pathogenic LRTOMT variants in previous studies, the following approaches are recommended for comprehensive genetic screening:

• Initial Assessment: Begin with known deafness-associated genes like GJB2 to exclude common causes before proceeding to LRTOMT analysis, as mutations in GJB2 are the most common genetic etiology of sensorineural hearing impairment (SNHI) .

• Targeted Sequencing: For focused studies, Sanger sequencing of specific LRTOMT exons (particularly exons 5, 7, and 8 that encode the dual reading frames) can be effective. This approach identified a novel variant (c.179 T>C; p.Leu60Pro) in Mauritanian children with congenital deafness .

• PCR-SSCP and Heteroduplex Analysis: These methods provide a cost-effective initial screening approach, with site-directed mutagenesis used to create positive controls for validation .

• Whole Exome Sequencing (WES): For comprehensive analysis, particularly in cases with consanguinity or multiple affected family members, WES has proven effective for identifying novel LRTOMT variants after excluding common causes .

• Bioinformatic Analysis Pipeline: Apply prediction tools (SIFT, Mutation Taster) followed by structural modeling to assess the functional impact of identified variants, with interpretation according to American College of Medical Genetics and Genomics (ACMG) guidelines .

• Co-segregation Analysis: Verify that candidate variants segregate with the hearing loss phenotype in families to confirm pathogenicity .

What protein purification strategies yield the highest activity for recombinant LRTOMT?

For optimal purification of functional recombinant Macaca mulatta LRTOMT, researchers should consider the following strategies:

How can researchers effectively model the effects of LRTOMT mutations on protein structure?

To effectively model the structural impact of LRTOMT mutations, researchers should employ a multi-tiered approach:

• Template Selection: Use the crystal structure of rat COMT (pdb-code 1h1d) as a template for modeling the catechol-O-methyltransferase domain of human LRTOMT2, which shares 39% sequence identity in the modeled region (residues 79-290) .

• Modeling Software: Utilize tools like WHAT IF-server (http://swift.cmbi.ru.nl) for initial model building, followed by energy minimization and analysis with Yasara (http://www.yasara.org) or similar programs .

• Mutation Analysis: Introduce specific mutations (like p.R81Q, p.W105R, p.E110K) to assess their impact on:

  • Salt bridges and hydrogen bonds (particularly relevant for p.R81Q and p.E110K)

  • Hydrophobic interactions (critical for p.W105R)

  • Secondary structure elements (helices and loops)

  • The groove that binds the putative methyl acceptor

• Molecular Dynamics Simulations: Perform MD simulations to observe dynamic structural changes over time, providing insights beyond static models.

• Visualization: Generate 3D structural comparisons between wild-type and mutant forms using tools like UCSF Chimera to visualize disruptions in protein architecture, as demonstrated with the comparison of wild-type and mutant LRTOMT .

• Validation: Compare computational predictions with experimental data from CD spectroscopy or thermal stability assays to confirm structural changes predicted by models.

What considerations are important when designing primers for LRTOMT amplification across species?

When designing primers for LRTOMT amplification, particularly for cross-species studies, researchers should consider:

• Evolutionary Divergence: Account for the fundamental difference between primate fusion gene (LRTOMT) and separate rodent genes (Lrrc51 and Tomt). For rodent studies, design separate primer sets for each gene .

• Conserved Regions: Target highly conserved exonic regions, particularly within the catechol-O-methyltransferase domain, which shows 39% sequence identity between human and rat .

• Splice Variant Coverage: Design primers to detect all five known alternatively spliced transcripts, including the crucial exons 5, 7, and 8 that contain dual reading frames .

• Species-Specific Optimization:

  • For primates: Design primers that can amplify fusion transcripts spanning the junction between what would be separate genes in rodents

  • For Macaca mulatta: Consider subspecies differences (Nepal/Indian vs. Chinese origin) that may affect primer binding sites

• Technical Approach:

  • Use nested PCR strategies for low-abundance transcripts

  • Consider including RACE primers for complete transcript characterization

  • Verify amplicon specificity through sequencing, particularly for novel species or variants

• Controls: When assessing evolutionary conservation, include positive controls from species with known sequence information (human, mouse) alongside experimental samples .

• Validation: Test designed primers on cDNA from multiple tissues, as expression patterns vary across tissues. Previous studies successfully used brain, liver, heart, and cochlear cDNA for validation .

What are the most promising therapeutic approaches targeting LRTOMT for hearing loss?

Based on current understanding of LRTOMT's role in hearing loss, several therapeutic approaches show promise:

• Gene Therapy: Delivering functional copies of LRTOMT using viral vectors (AAV) to cochlear hair cells represents a potential approach for treating DFNB63-related hearing loss. Macaca mulatta models would provide valuable pre-clinical validation.

• Early Intervention: Clinical data suggests that early fitting of cochlear implants can improve auditory outcomes in patients with LRTOMT mutations, highlighting the importance of early genetic screening and intervention .

• Small Molecule Stabilizers: Based on structural modeling of LRTOMT2 mutations, developing small molecules that could stabilize mutant proteins (particularly those with destabilizing mutations like p.R81Q, p.W105R, and p.E110K) might restore partial function .

• Methyltransferase Bypass: Identifying the downstream targets of LRTOMT2's methyltransferase activity could potentially allow for direct modification of these targets, bypassing the need for functional LRTOMT.

• Combination Approaches: Integrating genetic diagnosis, cochlear implantation, and molecular therapies may provide comprehensive management strategies for patients with LRTOMT-associated hearing loss.

Future research should focus on elucidating the precise molecular functions of both LRTOMT1 and LRTOMT2 in the auditory system, as well as developing Macaca mulatta models that accurately recapitulate human DFNB63 phenotypes for therapeutic testing.

How can cross-species LRTOMT research improve our understanding of non-syndromic hearing loss?

Cross-species analysis of LRTOMT offers unique insights into non-syndromic hearing loss mechanisms:

• Evolutionary Conservation Analysis: Comparing the fusion gene structure in primates to separate genes in rodents helps identify functionally critical domains that have been conserved despite structural reorganization .

• Variant Interpretation: Mutations discovered in human populations (Middle Eastern, North African) can be modeled in Macaca mulatta to determine their functional consequences in a genetically similar system .

• Tissue-Specific Expression: Comparative analysis of LRTOMT expression across species and tissues (particularly cochlear tissues) can reveal regulatory mechanisms important for auditory function .

• Subspecies Differences: Genetic characterization of rhesus macaques from different origins (Nepal, India, China) provides insight into population-specific variants that may influence susceptibility to hearing impairment .

• Translational Models: Macaca mulatta represents an ideal intermediate model between rodents and humans for studying LRTOMT function, allowing for more accurate prediction of therapeutic outcomes in humans.