Recombinant Bovine Transmembrane and coiled-coil domain-containing protein 5B (TMCO5B)

What is TMCO5B and how does it relate to the TMCO gene family?

TMCO5B is a member of the Transmembrane and Coiled-coil domains (TMCO) gene family, which includes several proteins characterized by the presence of coiled-coil domains in the N-terminal region and transmembrane domains in the C-terminal region . The TMCO family has been implicated in various cellular functions, particularly related to membrane organization and protein trafficking. TMCO5B shares significant homology with TMCO5 (sometimes designated as TMCO5A), which has been characterized in mouse models and shown to be expressed exclusively in elongating spermatids .

While mouse TMCO5 has been studied more extensively, bovine TMCO5B shows conservation of key structural domains, suggesting potential functional similarities despite species-specific variations. Researchers should note that comprehensive characterization of the bovine ortholog remains an emerging area of study compared to the more extensively documented mouse variant.

What are the key structural features of TMCO5B that influence its functionality?

TMCO5B is characterized by distinct structural domains that contribute to its cellular functions:

• N-terminal coiled-coil domain: This region facilitates protein-protein interactions and may be involved in oligomerization or interactions with cytoskeletal elements .

• C-terminal transmembrane domain: This hydrophobic region anchors the protein to cellular membranes and likely determines its subcellular localization .

• Intervening sequences: The regions between these domains may contain regulatory elements or binding sites for interaction partners.

How conserved is TMCO5B across mammalian species, and what evolutionary insights does this provide?

While the search results don't provide direct comparative data across species for TMCO5B specifically, studies on the related TMCO5 protein suggest conservation of function in mammalian spermatogenesis. Research comparing expression patterns between mouse and rat TMCO5 has noted some differences in timing and localization, which may reflect species-specific adaptations in spermatogenesis cycles .

Mouse and rat spermatogenesis cycles differ in both duration (233.6 hours versus 310.8 hours) and staging (12 versus 13 stages respectively), which may account for observed variations in TMCO5 expression patterns between species . These differences highlight the importance of species-specific characterization when studying TMCO5B orthologs.

What is the tissue-specific expression pattern of TMCO5B in bovine systems?

Based on studies of mouse TMCO5, we can infer that bovine TMCO5B likely exhibits highly tissue-specific expression. In mice, TMCO5 is expressed exclusively in the testis and cannot be detected in other tissues including ovary, skeletal muscle, brain, skin, stomach, intestine, colon, and spleen, even using highly-sensitive chemiluminescent detection methods .

Within the testis, expression appears to be developmental stage-specific, with protein detection beginning at approximately 4 weeks of age in mice, coinciding with the appearance of elongating spermatids . Notably, TMCO5 is not detectable in epididymal tissue, indicating it is not a component of mature sperm .

What is the subcellular localization of TMCO5B and how does this relate to its function?

Studies in mouse models have shown that TMCO5 localizes specifically to the manchette in elongating spermatids . The manchette is a transient cytoskeletal structure consisting predominantly of microtubules and actin filaments that forms during spermatid elongation . This structure serves as a transport pathway for Golgi-derived non-acrosomal vesicles.

When experimentally expressed in CHO cells, TMCO5 co-localizes with β-tubulin, supporting its association with microtubule structures . Interestingly, induced expression of TMCO5 in CHO cells resulted in reorganization of the Golgi apparatus, with Golgi elements concentrating at the center of TMCO5 distribution . This suggests a potential role for TMCO5B in organizing cellular structures through interaction with the cytoskeleton.

At what stages of spermatogenesis is TMCO5B expressed, and how is this determined?

Based on immunohistochemical analysis of mouse tissues, TMCO5 is expressed specifically in step 9-12 elongating spermatids . This has been confirmed through careful staging of seminiferous tubules using Hematoxylin staining to identify developmental stages based on cellular arrangement and nuclear morphology .

These findings align with immunoblotting results showing TMCO5 detection in mouse testes beginning at 4 weeks of age, which corresponds to the appearance of step 9-12 spermatids in the first round of spermatogenesis . This timing reflects the developmental specificity of TMCO5 expression and suggests a specialized role in the elongation phase of spermiogenesis.

It's worth noting that some discrepancies exist in the literature regarding the expression window, with some researchers reporting TMCO5 expression in round and almost developed spermatids as well . These differences may reflect species-specific variations (mouse versus rat) or methodological differences in antibody specificity .

What expression systems are most effective for recombinant bovine TMCO5B production?

Based on successful recombinant production of mouse TMCO5, bacterial expression systems using E. coli BL21(DE3) pLysS with pRSET vectors have proven effective for producing partial TMCO5 protein fragments . The documented approach involves:

• PCR amplification of the target region (nucleotide positions 162-536 in mouse TMCO5)

• Cloning into pRSET A vector using NheI and HindIII restriction sites

• Transformation into BL21(DE3) pLysS competent cells

• Protein purification using TALON Metal Affinity chromatography

For full-length protein expression, especially for proteins with transmembrane domains like TMCO5B, researchers may need to explore eukaryotic expression systems. The search results describe successful expression of full-length TMCO5 in CHO cells using a tetracycline-inducible system (Tet-on) , which may be preferable for maintaining proper folding and post-translational modifications.

What are the optimal purification strategies for recombinant TMCO5B?

For bacterially-expressed TMCO5 fragments, metal affinity chromatography has been successfully employed . The documented protocol used TALON Metal Affinity chromatography, which is based on cobalt-based resin with high specificity for His-tagged proteins.

For full-length TMCO5B containing transmembrane domains, more complex purification strategies may be required:

• Detergent solubilization to extract membrane-associated proteins

• Affinity chromatography as an initial capture step

• Size exclusion or ion exchange chromatography for further purification

The choice of detergents is critical when working with transmembrane proteins to maintain native conformation while effectively solubilizing the protein from membranes.

What challenges are commonly encountered when expressing recombinant TMCO5B, and how can they be addressed?

While the search results don't explicitly detail challenges specific to TMCO5B expression, several common issues with transmembrane proteins can be anticipated:

• Protein misfolding and aggregation: The presence of hydrophobic transmembrane domains can lead to aggregation in bacterial systems. This can be addressed by:

  • Using lower induction temperatures (16-20°C)

  • Employing specialized E. coli strains designed for membrane protein expression

  • Adding solubilizing agents or fusion partners

• Low expression yields: Transmembrane proteins often express poorly in heterologous systems. Consider:

  • Optimizing codon usage for the expression host

  • Testing different promoter strengths

  • Using eukaryotic expression systems for complex proteins

• Protein instability: The search results indicate successful production of a partial mouse TMCO5 fragment (amino acids corresponding to nucleotide positions 162-536) , suggesting this region may be more amenable to recombinant expression than the full-length protein.

How can specific antibodies against bovine TMCO5B be developed and validated?

The search results detail a successful approach for developing monoclonal antibodies against mouse TMCO5 that can serve as a template for bovine TMCO5B antibody development:

• Immunogen preparation: Express and purify recombinant protein fragments (partial coding region of TMCO5) .

• Immunization protocol: Immunize female rats with 300 μg of purified protein with Freund's adjuvant three times at 2-week intervals, followed by a final immunization with 300 μg of purified protein alone .

• Hybridoma generation: Harvest spleen cells 3 days after final immunization, fuse with P3U1 myeloma cells, and perform HAT selection in RPMI-1640 medium with 10% FBS .

• Screening and selection: Screen hybridomas using ELISA with recombinant protein and colorimetric immunohistochemistry on tissue sections. Clone positive hybridomas by limited dilution .

• Validation: Validate antibody specificity through immunoblotting against multiple tissue samples and developmental stages, confirming absence of cross-reactivity with non-target tissues .

What experimental protocols yield optimal results for immunohistochemical detection of TMCO5B?

Based on the successful detection of mouse TMCO5, the following immunohistochemistry approaches can be adapted for bovine TMCO5B:

For tissue sections:

• Fix tissues appropriately (4% paraformaldehyde)

• Embed in paraffin or optimal cutting temperature compound

• Section tissues at appropriate thickness

• For colorimetric detection:

  • Block endogenous peroxidase activity

  • Incubate with primary antibody (e.g., culture supernatant of anti-TMCO5B)

  • Use appropriate secondary antibody system

  • Counterstain with Hematoxylin for developmental staging

For cultured cells:

• Grow cells on collagen-coated coverslips

• Fix with 4% paraformaldehyde-PBS for 10 min at room temperature

• Block with appropriate blocking solution

• Incubate with primary antibodies (can be multiplexed with other markers like β-tubulin)

• Detect with fluorescently-labeled secondary antibodies

• Counterstain nuclei with DAPI

• Observe using confocal microscopy

How should Western blotting be optimized for reliable detection of TMCO5B?

The search results provide a detailed protocol for Western blotting of mouse TMCO5 that can be adapted for bovine TMCO5B:

• Sample preparation: Extract tissues with 5 times volume (v/w) of SDS sample buffer (5% 2-mercaptoethanol, 10% glycerol, 2% SDS, 0.005% Bromophenol Blue, and 63 mM Tris-HCl pH 6.8) and boil for 5 min. Centrifuge at 17,400 x g for 10 min and collect supernatants .

• Gel electrophoresis: Load 15 μl of samples into 10% SDS-PAGE gel .

• Transfer: Transfer proteins to nitrocellulose membrane .

• Blocking: Briefly wash with TBST (150 mM NaCl, 0.1% Tween 20, and 50 mM Tris–HCl pH7.5), then block with 0.5% casein in TBST for 30 min .

• Antibody incubation:

  • Primary antibody: Incubate for 1 hour with culture supernatant containing anti-TMCO5B antibody

  • Wash 3 times with TBST

  • Secondary antibody: Incubate for 1 hour with appropriate conjugated secondary antibody

• Detection options:

  • Colorimetric: Use alkaline phosphatase-conjugated secondary antibody (1:5,000 dilution) and BCIP-NBT Solution Kit

  • Chemiluminescent: Use horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution) and ECL kit with appropriate imaging system (e.g., LAS4000min)

How can the interaction between TMCO5B and microtubules be characterized?

Based on the reported co-localization of mouse TMCO5 with β-tubulin, several approaches can be employed to characterize TMCO5B-microtubule interactions:

• Co-localization studies: Use confocal microscopy with antibodies against TMCO5B and β-tubulin to examine spatial relationships in cellular contexts . The search results indicate TMCO5 and β-tubulin share distribution patterns, though TMCO5 regions appear slightly thinner than β-tubulin regions .

• Co-immunoprecipitation: Develop protocols to isolate TMCO5B-containing complexes and identify associated proteins, particularly tubulins and microtubule-associated proteins.

• In vitro binding assays: Express and purify recombinant TMCO5B domains to test direct binding to purified microtubules or tubulin subunits.

• Cellular perturbation: Use microtubule-disrupting agents (e.g., nocodazole) to examine effects on TMCO5B localization and distribution.

• Heterologous expression systems: The search results demonstrate that expression of TMCO5 in CHO cells results in co-localization with β-tubulin , providing a model system for structure-function studies.

What approaches are most effective for studying TMCO5B's role in Golgi organization?

Induced expression of mouse TMCO5 in CHO cells caused reorganization of the Golgi apparatus, with Golgi elements concentrating at one point at the center of TMCO5 distribution . This finding suggests TMCO5B may influence Golgi organization, which can be studied through:

• Inducible expression systems: Utilize tetracycline-inducible expression systems (Tet-on) in appropriate cell lines, possibly with Golgi markers (like the EGFP-tagged Golgi system used in the search results) .

• Time-course analysis: Monitor changes in Golgi morphology and distribution at various time points after TMCO5B induction to establish temporal relationships.

• Structure-function analysis: Generate TMCO5B variants with mutations or deletions in specific domains to identify regions required for Golgi effects.

• Depletion studies: Develop knockdown or knockout models of endogenous TMCO5B in relevant cell types to assess Golgi organization in the absence of the protein.

• Pharmacological approaches: Use drugs that affect microtubule dynamics or vesicular trafficking to probe the mechanisms of TMCO5B's effects on Golgi organization.

How might TMCO5B contribute to vesicular transport during spermatogenesis?

Based on the manchette localization of mouse TMCO5 and its potential role in Golgi organization, TMCO5B may function in vesicular transport during spermatogenesis. This can be investigated through:

• Ultrastructural analysis: Employ electron microscopy to visualize the relationship between TMCO5B, the manchette, and vesicular structures during spermatid elongation.

• Vesicle tracking: Utilize live-cell imaging with fluorescently tagged vesicular markers in cell models expressing TMCO5B to monitor vesicle movement along microtubules.

• Identification of cargo proteins: Develop methods to isolate TMCO5B-associated vesicles and characterize their contents to identify transported proteins.

• Functional perturbation: Create knockout or knockdown models of TMCO5B to assess effects on vesicular transport and spermatid morphogenesis.

How do post-translational modifications regulate TMCO5B function in spermatogenesis?

While the search results don't specifically address post-translational modifications of TMCO5B, this represents an important research area given the complex regulation of proteins during spermatogenesis. Researchers should consider:

• Identification of modifications: Use mass spectrometry to identify phosphorylation, glycosylation, ubiquitination, or other modifications on TMCO5B during different stages of spermatogenesis.

• Functional significance: Generate mutant forms of TMCO5B lacking specific modification sites to assess impacts on localization, interactions, and function.

• Regulatory enzymes: Identify kinases, phosphatases, or other enzymes that modify TMCO5B and investigate their expression patterns during spermatogenesis.

• Temporal dynamics: Characterize changes in modification patterns throughout spermatid development to correlate with functional transitions.

What methodological approaches can resolve discrepancies in TMCO5 expression patterns reported in the literature?

The search results note discrepancies in reported expression patterns of TMCO5, with some studies suggesting expression in round and almost developed spermatids in addition to elongating spermatids . To resolve such discrepancies, researchers should consider:

• Antibody validation: Rigorously characterize antibody specificity using multiple approaches, including:

  • Western blot analysis across developmental stages

  • Immunohistochemistry with detailed staging analysis

  • Controls using tissues from knockout models

  • Comparison of multiple antibodies targeting different epitopes

• Species considerations: Account for species-specific differences in spermatogenesis cycles (e.g., 233.6h vs. 310.8h in mice vs. rats) and staging (12 vs. 13 stages) .

• Transcript analysis: Combine protein detection with in situ hybridization or RT-PCR to correlate mRNA and protein expression patterns.

• Single-cell approaches: Utilize single-cell RNA sequencing to precisely map expression patterns across the continuum of spermatogenesis.

How do the functions of TMCO5A and TMCO5B differ, and what experimental approaches can distinguish their roles?

To investigate potential functional differences between TMCO5A and TMCO5B:

• Comparative expression analysis: Determine whether TMCO5A and TMCO5B exhibit distinct tissue or developmental expression patterns through parallel analysis with isoform-specific probes and antibodies.

• Domain structure comparison: Analyze differences in protein domains that might confer distinct functions or interactions.

• Interaction partner identification: Perform comparative interactome analysis to identify shared and unique binding partners.

• Isoform-specific perturbation: Develop genetic models with selective knockout or knockdown of each isoform to assess distinct phenotypic consequences.

• Complementation studies: Test whether one isoform can functionally replace the other in appropriate model systems.

What are common pitfalls in recombinant TMCO5B expression, and how can they be resolved?

How can non-specific binding be minimized in immunohistochemical detection of TMCO5B?

Based on the successful immunohistochemistry protocols described for mouse TMCO5, researchers should implement the following strategies to minimize non-specific binding:

• Optimization of blocking conditions: Use appropriate blocking agents (0.5% casein in TBST was effective in the reported protocols) .

• Antibody dilution optimization: Systematically test different dilutions of primary antibody to determine optimal signal-to-noise ratio.

• Extended washing steps: Perform multiple washes with TBST (three times for 5-10 minutes each) to remove unbound antibody .

• Controls: Include negative controls (omitting primary antibody) and tissue-specific controls (tissues known not to express TMCO5B) in each experiment.

• Antibody pre-absorption: Consider pre-absorbing antibodies with unrelated tissues to reduce non-specific binding.

• Monoclonal antibodies: Use well-characterized monoclonal antibodies (like RTm01 described in the search results) rather than polyclonal antibodies to increase specificity .

What strategies can resolve difficulties in detecting TMCO5B in specific developmental contexts?

For researchers encountering challenges in detecting TMCO5B during specific developmental stages:

• Sample preparation optimization: Ensure appropriate fixation protocols that preserve epitope accessibility while maintaining tissue morphology.

• Antigen retrieval: Test various antigen retrieval methods (heat-induced, enzymatic, pH variations) to expose potentially masked epitopes.

• Signal amplification: Consider employing tyramide signal amplification or other enhancement methods for low-abundance targets.

• Alternative detection systems: Compare colorimetric versus fluorescent detection methods for optimal sensitivity.

• Combination with staging markers: Use co-staining with stage-specific markers to precisely identify developmental windows of interest.

• High-sensitivity Western blotting: The search results mention using highly-sensitive chemiluminescent detection methods for challenging samples , which can be particularly helpful for developmental studies.