Recombinant Human Uncharacterized protein C1orf101 (C1orf101), partial

What is the current molecular characterization of human C1orf101 protein?

Human C1orf101, formerly classified as an uncharacterized protein, has now been identified as CatSper epsilon (ε), a transmembrane protein subunit of the CatSper ion channel complex. The protein is encoded by the C1ORF101 gene (NCBI Accession: NM_173807) . Structurally, C1orf101 is predicted to be a single transmembrane (TM) protein with sequence homology to the C-terminal extracellular domain of CatSperδ, another CatSper subunit . Genetic mapping studies have positioned it on chromosome 1, and its expression pattern is predominantly testis-specific, suggesting a specialized role in reproductive physiology.

How does C1orf101 integrate into the CatSper ion channel complex?

C1orf101 (CatSper ε) integrates into the larger CatSper ion channel complex alongside other subunits including CatSperζ (encoded by Tex40). Biochemical analysis demonstrates that CatSper ε exhibits interdependence with other CatSper subunits, as evidenced by co-immunoprecipitation studies where it complexes with CatSper1 . Localization studies in both human and mouse sperm cells have confirmed that CatSper ε proteins are specifically localized to the principal piece of sperm tails . This precise subcellular localization is critical for the channel's function in regulating calcium influx during sperm hyperactivation.

What are the expression patterns of C1orf101 during development?

The expression of C1orf101 (CatSperε) follows a specific developmental timeline during spermatogenesis. Research indicates that both CatSperε and CatSperζ mRNAs are expressed specifically in germ cells and are detected before CatSper1 expression during postnatal development . This sequential expression pattern suggests a potential role in the assembly or trafficking of the CatSper complex during sperm cell maturation. The testis-specific expression further underscores its specialized function in male reproductive physiology.

How does the CatSper complex containing C1orf101 regulate calcium signaling in sperm?

The CatSper complex, including C1orf101 (CatSperε), forms a calcium-selective ion channel that regulates the entry of calcium ions into the sperm flagellum. This calcium influx is essential for triggering hyperactivated motility, a swimming pattern characterized by high-amplitude, asymmetrical flagellar bending that enables sperm to navigate the female reproductive tract and penetrate the egg vestments. CatSperε contributes to the structural continuity of sperm Ca²⁺ signaling domains . Without intact CatSperε, the calcium signaling is disrupted, leading to abnormal flagellar waveforms and reduced fertility.

What phenotypes are observed when C1orf101 function is disrupted?

Genetic disruption of C1orf101 (CatSperε) results in several distinct phenotypes related to sperm function:

What CRISPR-based approaches are available for studying C1orf101?

Researchers can utilize CRISPR-Cas9 gene editing technology to study C1orf101 function through targeted disruption or modification of the gene. Commercial tools such as Human C1ORF101 CRISPR gRNA + Cas9 in Lenti Particles are available with a set of 3 gRNAs covering different sequences of C1orf101 . These lentiviral particles have a titer of >1×10⁷ IU/mL and include both sgRNA and Cas9 in a single vector (pLenti-U6-sgRNA-SFFV-Cas9-2A-Puro) . For quality control of CRISPR experiments, restriction enzyme digest and sequencing using the U6 Forward Primer (5'-TACGTCCAAGGTCGGGCAGGAAGA-3') are recommended .

What imaging techniques have proven effective for studying C1orf101 localization and function?

Advanced imaging techniques have been instrumental in characterizing C1orf101 localization and function. High-speed video microscopy coupled with digital image analysis has been employed to determine swimming trajectory and flagellar waveform in detail . Additionally, immunofluorescence microscopy has been used to localize CatSperε to the principal piece of sperm tails in both human and mouse sperm cells . These imaging approaches have revealed crucial information about how CatSperε contributes to the structural organization of calcium signaling domains in the sperm flagellum.

How conserved is C1orf101 across species?

C1orf101 exhibits evolutionary conservation across mammalian species, with homologs identified in model organisms. Notably, the sea urchin Strongylocentrotus purpuratus possesses a homologous gene (LOC105438278) that encodes an "uncharacterized protein C1orf101-like" protein . In mice, the homolog C1orf101-like (currently designated Gm7068) has been characterized as CatSperε . The conservation of this protein across diverse species suggests an evolutionarily preserved role in reproductive biology, particularly in sperm function.

What functional differences exist between human C1orf101 and its homologs in model organisms?

While human C1orf101 and its homologs share structural and functional similarities, species-specific differences exist. In mice, the C1orf101 homolog (Gm7068/CatSperε) has been extensively studied and shown to be critical for sperm hyperactivated motility and fertility . The sea urchin homolog (LOC105438278) is annotated as "cation channel sperm-associated protein subunit epsilon-like" , suggesting conserved functionality in sperm calcium signaling despite evolutionary distance. Comparative functional studies between species provide valuable insights into the core conserved functions versus species-specific adaptations of this protein.

What insights can be gained from comparing different isoforms of C1orf101-like proteins?

Analysis of the sea urchin (Strongylocentrotus purpuratus) homolog reveals multiple isoforms of the C1orf101-like protein:

These different isoforms may represent functionally distinct variants or developmental stage-specific forms of the protein . Comparative analysis of these isoforms could provide insights into the structural domains essential for function versus those that may confer species-specific or context-dependent functionality.

How can recombinant C1orf101 be utilized in fertility research?

Recombinant C1orf101 (CatSperε) provides a valuable tool for studying male fertility mechanisms. Researchers can use purified recombinant protein for generating specific antibodies, performing binding assays to identify interaction partners, or developing high-throughput screens for compounds that modulate CatSper function. Given its critical role in sperm hyperactivation and fertility, recombinant C1orf101 could also be employed in the development of novel male contraceptive approaches or diagnostic tools for unexplained male infertility.

What are the challenges in expressing functional recombinant C1orf101?

Expressing functional recombinant C1orf101 presents several challenges. As a membrane protein that functions as part of a multi-subunit complex, C1orf101 likely requires co-expression with other CatSper subunits for proper folding and function. Previous attempts to reconstitute functional CatSper channels, even with simultaneous expression of all known subunits, have been unsuccessful . Researchers may need to consider specialized expression systems, optimization of detergents for membrane protein solubilization, and potentially co-expression with chaperones or other CatSper subunits to obtain correctly folded, functional protein.

What research questions remain unexplored regarding C1orf101 function?

Despite recent advances, several critical questions about C1orf101 function remain unanswered:

• The precise stoichiometry of C1orf101 within the CatSper complex

• The specific protein-protein interactions that mediate C1orf101 incorporation into the complex

• Post-translational modifications that regulate C1orf101 function

• Potential roles beyond sperm calcium signaling, particularly in tissues where low-level expression might occur

• Genetic variations in C1orf101 that might contribute to unexplained male infertility cases

Addressing these questions will require interdisciplinary approaches combining structural biology, electrophysiology, advanced imaging, and clinical genetics.

How might C1orf101 dysfunction contribute to male infertility?

C1orf101 (CatSperε) dysfunction could significantly contribute to male infertility through disruption of sperm calcium signaling. Research has demonstrated that genetic disruption of CatSperε reduces CatSper currents and hyperactivated motility, resulting in severe subfertility . The specific swimming defects—restricted flagellar envelope and inefficient rheotaxis—impair sperm navigation through the female reproductive tract. These findings suggest that mutations or altered expression of C1orf101 could underlie some cases of unexplained male infertility, particularly those characterized by normal sperm counts but poor fertilization outcomes.

What are the most promising methodological approaches for clinical studies of C1orf101?

For clinical studies investigating the relationship between C1orf101 and male fertility, several methodological approaches show promise:

• Targeted sequencing of C1orf101 in cohorts of men with unexplained infertility

• Development of specific antibodies for immunodetection of C1orf101 in human sperm samples

• Patch-clamp electrophysiology to measure CatSper currents in sperm from patients with suspected channel dysfunction

• High-resolution imaging of calcium dynamics and flagellar movement in patient sperm samples

• CRISPR-based correction of identified mutations in patient-derived cells for causality confirmation