CCDC104 Human

What is CCDC104 and where is it primarily expressed in human tissues?

CCDC104 is a protein whose function remains largely unknown but appears biologically significant due to its high conservation across mammalian species. It is expressed in various human tissues, with particularly notable expression in the testis and spleen. CCDC104 is also expressed in the brain, though at comparatively lower levels. Analysis of CCDC104 mRNA shows increased expression in several brain regions and in testis according to the ArrayExpress Warehouse database . This expression pattern bears resemblance to CDR2, which is the antigen recognized by Yo antibodies and is primarily restricted to brain and testis tissues .

What is known about the cellular localization of CCDC104?

Immunofluorescence studies using neuroblastoma cell line SK-N-SH have demonstrated that CCDC104 is primarily localized in the nucleus (excluding the nucleolus), but is also found in specific compartments within the cytoplasm . The staining pattern is speckled, suggesting that CCDC104 is concentrated in particular subcellular structures rather than diffusely distributed. This nuclear-cytoplasmic distribution may provide clues about its biological function, potentially involving roles in both compartments .

How conserved is CCDC104 across species?

CCDC104 demonstrates remarkable evolutionary conservation, suggesting fundamental biological importance. Sequence alignment analysis using ClustalW2 has shown that CCDC104 is highly conserved among mammals, with 85% sequence identity and 93% amino acid sequence conservation. Human CCDC104 also shares substantial homology with other vertebrates, including 59% sequence identity with Gallus gallus (chicken) . This high degree of conservation implies that CCDC104 likely performs an essential biological function that has been preserved throughout evolution, similar to other paraneoplastic antigens like CDR2 .

What is the association between CCDC104 antibodies and paraneoplastic neurological syndromes?

Studies have found a significant association between antibodies against CCDC104 and Yo antibodies, which are associated with paraneoplastic cerebellar degeneration (PCD). Specifically, CCDC104 antibodies were present in 10.5% (4 of 38) of Yo-positive sera, but were not detected in patients with other onconeural antibodies such as Hu, CRMP5, amphiphysin, Ri, or Ma2 (0 of 158) . This association was statistically significant (P = 0.007, Fisher's exact test).

Despite this association with Yo antibodies, CCDC104 antibodies themselves do not appear to be directly linked to paraneoplastic neurological syndromes (PNS). Among ten patients with CCDC104 antibodies, only two had PNS . Furthermore, CCDC104 antibodies were found at similar rates in cancer patients (1.1%, 8 of 756) and healthy blood donors (0.7%, 2 of 300), indicating they are not specifically tumor-associated .

What is the structural interaction between CCDC104 and Arl3?

CCDC104 (also referred to as BARTL1 in some literature) has been shown to interact with Arl3, a small GTPase. X-ray crystallography studies have revealed detailed structural information about this interaction. Two crystal structures of the Arl3·GppNHp·CCDC104 complex have been deposited in the Protein Data Bank (PDB IDs: 4ZI2 and 4ZI3) with resolutions of 2.20Å and 2.00Å, respectively .

The crystal structure data reveals important parameters of this interaction:

The structural studies provide a foundation for understanding the molecular basis of CCDC104 function through its protein-protein interactions .

What methodologies have been employed to detect CCDC104 antibodies in patient samples?

Multiple complementary methods have been used to detect CCDC104 antibodies in patient samples:

• Immunoprecipitation assay: This technique was used as the primary screening method, with results reported as an index value. Samples with values above the established cut-off were considered positive .

• Western blot: Nine out of ten patients positive for CCDC104 antibodies by immunoprecipitation also showed reactivity with recombinant CCDC104 protein in Western blot. One patient was negative by Western blot despite being positive by immunoprecipitation, suggesting this patient had antibodies recognizing a conformational epitope rather than a linear one .

• Screening of cDNA library: For initial identification, patient serum was used to screen a rat cerebellum library, from which a positive clone was isolated and sequenced, revealing the CCDC104 gene .

These methodological approaches highlight the importance of using multiple techniques to comprehensively detect antibodies that may recognize different types of epitopes.

What is the significance of the association between Yo and CCDC104 antibodies?

The statistically significant association between Yo and CCDC104 antibodies suggests potential functional similarities between these proteins. This association is particularly interesting because Yo antibodies target the CDR2 protein, which like CCDC104, shows restricted expression in brain and testis. Furthermore, both proteins show high evolutionary conservation .

Interestingly, among the four patients positive for both Yo and CCDC104 antibodies, three were men, which is notable since Yo antibodies are typically rare in male patients . This suggests there might be gender-specific factors influencing the co-development of these antibodies.

The association between these antibodies might provide insights into shared pathogenic mechanisms or common immunological triggers, potentially helping to clarify the pathogenesis of paraneoplastic syndromes .

What immunofluorescence protocols are effective for studying CCDC104 cellular localization?

Based on published methodologies, an effective immunofluorescence protocol for CCDC104 localization includes:

• Cell preparation: Neuroblastoma cell lines such as SK-N-SH are suitable models. Cells should be seeded at approximately 7,200 cells per chamber in appropriate slides (e.g., Lab-Tek™ Chamber Permanox Slides) and grown for 3 days in Eagle's minimum essential medium with 10% fetal calf serum .

• Fixation and permeabilization: Cells should be fixed in 4% ice-cold formaldehyde for 20 minutes, rinsed in PBS, and permeabilized in 0.2% Triton X-100 for 15 minutes at room temperature .

• Blocking and antibody incubation: After washing with PBS, block cells for 1 hour in culture medium with serum. Incubate with CCDC104 antibody (diluted 1:200) for 1 hour at room temperature. After washing (3 × 10 minutes with 0.1% Triton X-100), incubate with fluorescent secondary antibody (e.g., Alexa Fluor 488) diluted 1:200 for 1 hour .

• Mounting and visualization: Mount slides with appropriate medium containing nuclear counterstain (e.g., Vectashield with DAPI). Visualization can be performed using confocal microscopy, such as Zeiss LSM 510 Meta .

For statistical reliability, at least three independent staining experiments should be conducted, with analysis of approximately 100 cells per experiment .

What tissue expression analysis methods are recommended for CCDC104 research?

For comprehensive tissue expression analysis of CCDC104, a multi-method approach is recommended:

• Western blot analysis: Using validated CCDC104 antibodies, researchers can probe protein extracts from various tissues to identify the main isoform (39 kDa) and tissue-specific variants. This method has successfully shown CCDC104 expression across human tissues, with particular prominence in testis and spleen, and lower levels in brain .

• mRNA expression analysis: Complementing protein detection, mRNA expression databases such as ArrayExpress can provide comprehensive tissue expression patterns. This approach has shown increased CCDC104 mRNA expression in several brain regions and in testis .

• Immunohistochemistry: For spatial localization within tissues, immunohistochemistry can be attempted, though some studies have noted challenges with certain antibodies. For example, CCDC104 antibody did not react with rat cerebellar tissue, even after antigen retrieval by boiling in citrate buffer .

How should researchers interpret the prevalence of CCDC104 antibodies in cancer patients versus healthy controls?

The similar prevalence of CCDC104 antibodies in cancer patients (1.1%) and healthy blood donors (0.7%) requires careful interpretation . While this suggests CCDC104 antibodies are not tumor-specific markers, several analytical considerations should be addressed:

• Statistical power: The relatively low prevalence requires large sample sizes to detect potentially meaningful differences. The studies examined 756 cancer patients and 300 blood donors, which may be insufficient to detect small but significant differences .

• Cancer subtype analysis: Aggregating all cancer types may mask associations with specific cancer subtypes. More targeted analysis of specific cancer types might reveal significant differences that are obscured in the pooled analysis.

• Temporal considerations: The presence of antibodies may fluctuate over time or correlate with disease stage. Cross-sectional sampling might miss dynamic changes in antibody status during disease progression.

• Clinical significance threshold: Even small differences in prevalence may be clinically significant if CCDC104 antibodies correlate with specific clinical features or outcomes in the cancer population.

What statistical methods are appropriate for analyzing associations between CCDC104 and other antibodies?

For analyzing associations between CCDC104 and other antibodies (such as Yo antibodies), the following statistical approaches are recommended:

• Fisher's exact test: This was appropriately used in the existing research to analyze the association between CCDC104 and Yo antibodies, yielding a significant p-value of 0.007 . This test is suitable for categorical data with small expected frequencies.

• Multiple comparison correction: When testing associations with multiple antibodies, researchers should apply corrections (e.g., Bonferroni, false discovery rate) to control the family-wise error rate.

• Stratified analysis: Stratifying by demographic factors (age, sex) and clinical parameters (cancer type, disease stage) can reveal whether associations are consistent across subgroups or dependent on specific factors.

• Logistic regression: For multivariate analysis, logistic regression can help determine whether the association between antibodies remains significant after adjusting for potential confounding variables.

What are the key considerations when interpreting structural data for CCDC104?

When interpreting structural data for CCDC104, such as the crystal structures of Arl3·GppNHp·CCDC104 complexes, researchers should consider:

• Resolution limitations: The available structures have resolutions of 2.20Å and 2.00Å , which provide good but not atomic-level detail. Some side chain conformations and water molecules may not be precisely defined.

• Crystal packing effects: Differences between the two crystal forms (space groups P 2 1 2 1 2 1 and P1 2 1 1) may reflect crystal packing influences rather than biological conformational variations .

• Biological relevance: Crystallization conditions may induce non-physiological conformations. The observed interactions should be validated through complementary biochemical and cellular approaches.

• Partial structure considerations: The reported structures cover CCDC104 fragment 133, not the full-length protein . Interactions observed with this fragment may not fully represent the behavior of the complete protein.

• Dynamic aspects: Crystal structures provide static snapshots, potentially missing important dynamic aspects of protein-protein interactions that may be functionally relevant.