Recombinant Human Transmembrane and coiled-coil domain-containing protein 5A (TMCO5A)

What is the structural composition of TMCO5A?

TMCO5A belongs to the Transmembrane and coiled-coil domains (TMCO) gene family. The protein has a distinctive domain organization with a coiled-coil domain in the N-terminal region and a transmembrane domain in the C-terminal region . This structural arrangement is critical to its cellular localization and function. Experimental evidence using truncated versions lacking the transmembrane region (TMCO5AΔC) has demonstrated that the transmembrane domain is essential for proper localization to the endoplasmic reticulum-nuclear membrane (ER-NM) .

What experimental approaches can identify TMCO5A expression in tissues?

Multiple complementary techniques have proven effective in characterizing TMCO5A expression:

• In situ hybridization: Useful for detecting mRNA expression patterns in tissue sections

• Immunoblotting: Can detect protein expression in tissue extracts using specific antibodies

• Immunohistochemistry: Allows visualization of protein localization within cells and tissues

High-throughput in situ hybridization screening has successfully identified TMCO5A expression in round and elongated spermatids in mouse seminiferous tubules . For protein detection, monoclonal antibodies against recombinant TMCO5A have demonstrated high specificity in immunoblotting and immunohistochemistry applications .

How is TMCO5A isolated and identified?

The TMCO5A gene was originally isolated using polymerase chain reaction (PCR)-based subtraction techniques . For protein studies, researchers have successfully amplified partial coding regions (nucleotide positions 162-536, NM_026104) using adult testis cDNA libraries with specifically designed primers containing restriction enzyme sites for subsequent cloning . The amplified fragments can be cloned into expression vectors like pRSET A and transformed into bacterial expression systems such as BL21 (DE3) pLysS for recombinant protein production .

What is the tissue-specific expression pattern of TMCO5A?

TMCO5A exhibits a highly restricted expression pattern. It is predominantly expressed in the testes, with expression beginning at approximately 4 weeks of postnatal development in rats . Comprehensive immunoblotting analysis across multiple tissues (testis, ovary, skeletal muscle, brain, skin, stomach, intestine, colon, and spleen) has confirmed that TMCO5A is expressed exclusively in the testis of adult male mice . This tissue-specific expression suggests a specialized role in male reproductive biology.

What is the subcellular localization of TMCO5A?

TMCO5A displays distinctive subcellular localization patterns that provide insights into its function:

• In heterologous expression systems: When expressed in COS7 cells, TMCO5A is distributed in the endoplasmic reticulum-nuclear membrane (ER-NM) as a membrane-associated protein .

• In spermatids: TMCO5A is localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappears from epididymal spermatozoa . In mouse elongating spermatids, it is specifically localized to the manchette, a transient cytoskeletal structure consisting predominantly of microtubules and actin filaments .

• Co-localization patterns: TMCO5A is closely associated with developing manchette microtubules but does not completely colocalize with them . Importantly, almost all TMCO5A colocalizes with SUN4, a linker of nucleoskeleton and cytoskeleton (LINC) complex protein present at the posterior part of spermatid nuclei .

How does TMCO5A expression change during spermatogenesis?

TMCO5A expression follows a precise temporal pattern during spermatogenesis:

• Developmental timeline: Expression begins around 4 weeks of postnatal development in mice, corresponding to the appearance of elongating spermatids in the first wave of spermatogenesis .

• Stage-specific expression: In mice, enzymatic immunohistochemistry has demonstrated that TMCO5A protein is expressed exclusively in the elongating spermatids of step 9 to 12, specifically in the head region . This restricted expression window coincides with critical nuclear shaping events during spermiogenesis.

• Species differences: There may be differences in expression patterns between mice and rats. While mouse studies show expression restricted to step 9-12 spermatids, some rat studies have reported expression in round and almost developed spermatids as well, possibly due to differences in species-specific spermatogenesis timing or antibody specificity .

What is the proposed function of TMCO5A in spermiogenesis?

Based on its localization and expression patterns, TMCO5A is likely involved in the process of spermiogenesis . Specifically, it may participate in:

• Manchette organization: The close association with manchette microtubules suggests a role in organizing or regulating this transient structure .

• Nuclear shaping: Given its localization closer to the nuclei than the manchette microtubules and co-localization with the nuclear envelope protein SUN4, TMCO5A may contribute to nuclear shaping during spermatid elongation .

• Vesicular transport: The manchette serves in the transport of Golgi-derived non-acrosomal vesicles, and TMCO5A's association with this structure suggests it may participate in vesicle transport along the manchette .

How does TMCO5A interact with cellular structures?

TMCO5A demonstrates specific interactions with multiple cellular components:

• Microtubule association: Double immunolabeling with anti-TMCO5A and anti-β-tubulin antibodies shows that TMCO5A is closely associated with developing manchette microtubules . In CHO cells with induced TMCO5A expression, the protein colocalizes with β-tubulin, although TMCO5A distribution appears slightly thinner than β-tubulin in these cells .

• Golgi apparatus reorganization: Induced expression of TMCO5A in CHO cells results in the reorganization of the Golgi apparatus, causing it to concentrate at one point in the center of the region where TMCO5A is distributed . This suggests a role in Golgi organization, similar to other TMCO family members like Tango6 (TMCO7) .

• Nuclear membrane proximity: TMCO5A colocalizes with SUN4, a LINC complex protein at the posterior part of spermatid nuclei, indicating it is positioned closer to the nuclei than to manchette microtubules .

How can researchers produce recombinant TMCO5A for experimental studies?

The following methodology has proven effective for recombinant TMCO5A production:

• Gene amplification: Amplify the partial coding region of TMCO5A (e.g., nucleotide position 162-536, NM_026104) using PCR with specifically designed primers containing appropriate restriction enzyme sites .

• Cloning strategy:

  • Digest the amplified fragment with appropriate restriction enzymes (e.g., NheI and HindIII)

  • Clone into an expression vector such as pRSET A

  • Transform into competent cells like BL21 (DE3) pLysS

• Protein purification: Purify the recombinant protein using metal affinity chromatography (e.g., TALON Metal Affinity chromatography) following the manufacturer's protocol .

This approach has successfully generated recombinant TMCO5A suitable for antibody production and other experimental applications.

What is the recommended procedure for developing antibodies against TMCO5A?

The following protocol has been validated for generating specific monoclonal antibodies against TMCO5A:

• Immunization:

  • Immunize rats or other suitable animals with purified recombinant protein (e.g., 300 μg) with Freund's adjuvant three times at 2-week intervals

  • Administer a final immunization with purified protein alone

• Hybridoma generation:

  • Harvest spleen cells 3 days after final immunization

  • Fuse with myeloma cells (e.g., P3U1)

  • Perform HAT selection in appropriate medium with 10% FBS

• Antibody screening:

  • Screen using ELISA with recombinant protein

  • Confirm specificity with immunohistochemistry on relevant tissue sections (e.g., adult mouse testes)

  • Clone positive hybridomas by limited dilution method

This approach has generated highly specific monoclonal antibodies (e.g., RTm01) suitable for immunoblotting, immunohistochemistry, and other applications .

What cellular models are appropriate for studying TMCO5A function?

Several cellular systems have proven useful for investigating TMCO5A:

• COS7 cells: Effective for studying TMCO5A subcellular localization and the role of specific domains. Expression in these cells demonstrates distribution in the ER-NM as a membrane-associated protein .

• CHO cells with Tet-on system: Valuable for inducible expression studies to examine effects on cellular organization:

  • Use a Tet-on system comprising two vectors (pcDNA 4/TO for target protein expression and pcDNA 6/TR for repressor production)

  • Co-transfect these plasmids into CHO cells

  • Select for integration using appropriate antibiotics (e.g., Zeocin, Blasticidin)

  • Induce expression with tetracycline (1 μg/ml)

• CHO-GolEGF cells: Particularly useful for studying effects on Golgi organization, as these cells have EGFP-tagged Golgi apparatus .

These models allow examination of TMCO5A's effects on subcellular structures and protein-protein interactions.

What methodological factors might explain differences between mouse and rat TMCO5A studies?

Several factors could contribute to discrepancies in TMCO5A findings between species:

• Species-specific spermatogenesis timing:

  • Mice and rats have different spermatogenesis cycle periods (233.6 hours versus 310.8 hours)

  • They have different numbers of stages (12 versus 13 stages respectively)

  • These differences could account for variations in gene expression timing

• Antibody characteristics:

  • Different antibody types (monoclonal versus polyclonal)

  • Different target antigens (recombinant proteins versus synthetic oligopeptides)

  • Monoclonal antibodies may recognize specific three-dimensional structures, potentially missing some forms of the protein

• Detection methods: Variations in sensitivity and specificity of detection methods can influence results. Highly sensitive chemiluminescent methods may detect expression not observable with less sensitive techniques .

Understanding these methodological differences is crucial when comparing studies across species or laboratories.

What are the potential interactions between TMCO5A and LINC complex proteins?

The co-localization of TMCO5A with SUN4, a component of the LINC complex, suggests functional interactions between these proteins. The LINC complex (Linker of Nucleoskeleton and Cytoskeleton) plays crucial roles in nuclear positioning and shaping during spermatid development . The observation that almost all TMCO5A colocalizes with SUN4 at the posterior part of spermatid nuclei indicates a potential role in the nucleoskeleton-cytoskeleton connection .

Further investigation of these interactions could involve:

• Co-immunoprecipitation studies to confirm physical interactions

• Proximity ligation assays to visualize protein-protein interactions in situ

• Knockout or knockdown studies to assess functional relationships

These approaches would help elucidate the role of TMCO5A in nuclear shaping during spermiogenesis.

What are the critical unanswered questions about TMCO5A function?

Several important aspects of TMCO5A biology remain to be fully elucidated:

• Molecular mechanisms: How does TMCO5A contribute to manchette organization and function? What molecular partners mediate its effects on microtubules and the Golgi apparatus?

• Human relevance: While studies have focused on rodent models, the role of TMCO5A in human spermatogenesis and potential implications for male fertility remain largely unexplored.

• Regulation of expression: The precise mechanisms controlling the stage-specific expression of TMCO5A during spermatogenesis are not fully understood. Investigation of transcriptional and post-transcriptional regulatory mechanisms would provide valuable insights.

What emerging technologies could advance TMCO5A research?

Several cutting-edge approaches could significantly enhance our understanding of TMCO5A:

• CRISPR/Cas9 genome editing: Generation of knockout or knockin models to study the consequences of TMCO5A deletion or modification in vivo.

• Super-resolution microscopy: Advanced imaging techniques could provide more detailed visualization of TMCO5A's subcellular localization and interactions with other proteins and cellular structures.

• Proteomics approaches: Identification of TMCO5A-interacting proteins through techniques such as BioID, proximity labeling, or immunoprecipitation coupled with mass spectrometry.

These approaches would contribute to a more comprehensive understanding of TMCO5A's role in spermatogenesis and potentially identify new therapeutic targets for male infertility.