ANXA4 Human

What is the basic structure of human ANXA4?

Human Annexin A4 (ANXA4) belongs to the annexin family of calcium-dependent phospholipid-binding proteins. Its structure consists of a unique N-terminal domain followed by a homologous C-terminal core domain containing the phospholipid-binding sites. The C-terminal domain comprises four 60-70 amino acid annexin repeats forming a tightly packed disc structure known as the annexin domain. This structural arrangement enables ANXA4 to function as a membrane-binding protein, predominantly localized to the cytosolic face of the plasma membrane. The molecular weight of ANXA4 is approximately 36 kDa, as detected through Western blot analysis in various cell lines including human epithelial carcinoma cells (A431) .

How conserved is ANXA4 across species?

ANXA4 shows remarkable evolutionary conservation across vertebrate species, indicating its fundamental biological importance. Human ANXA4 shares 92% amino acid sequence identity with mouse and rat ANXA4 . Evolutionary conservation analysis across 13 species including human, chimpanzee, pongo abelii, green monkey, mouse, rat, cattle, pig, dog, horse, rabbit, and goat demonstrates that certain regions, particularly those containing functional domains, are highly conserved. This conservation is especially evident in regions containing specific mutations such as the G8 residue, which when mutated to aspartic acid (G8D) has been shown to have pathological implications . The high degree of conservation suggests that ANXA4 serves essential biological functions that have been maintained throughout vertebrate evolution.

What are the primary cellular functions of ANXA4?

ANXA4 performs several critical cellular functions as a calcium-dependent membrane-binding protein. It modulates membrane permeability and membrane trafficking, which are essential for maintaining cellular homeostasis. Additionally, ANXA4 participates in cellular growth regulation and apoptotic pathways. It plays roles in cytoskeletal interactions, phospholipase inhibition, and intracellular signal transduction pathways . In the context of cancer biology, ANXA4 has been shown to enhance tumor invasion capabilities, which explains its upregulation in various epithelial tumors. Within the reproductive system, ANXA4 is localized to the glandular and luminal epithelium of the human endometrium and is present at high levels throughout the menstrual cycle, except during the early secretory phase, suggesting its involvement in reproductive processes .

What methods are most effective for detecting ANXA4 in different biological samples?

Multiple validated techniques exist for detecting and quantifying ANXA4 in biological samples. Western blotting is effective for detecting ANXA4 in cell lysates, with specific bands appearing at approximately 36 kDa under reducing conditions. This technique has been validated in various cell lines including A431 human epithelial carcinoma cells, NIH-3T3 mouse embryonic fibroblasts, and Rat-2 rat embryonic fibroblasts . Immunohistochemistry (IHC) can effectively detect ANXA4 in tissue sections, as demonstrated in human kidney samples where specific staining was localized to cell membranes and nuclear envelopes of epithelial cells in convoluted tubules . For quantitative analysis, enzyme-linked immunosorbent assay (ELISA) provides reliable measurements of ANXA4 in serum and plasma samples, with recovery rates averaging 94% across different sample types .

How reliable are ELISA methods for quantifying ANXA4 in human samples?

ELISA methods demonstrate high reliability for quantifying ANXA4 in human biological samples. Recovery tests conducted on multiple sample types show consistent and reproducible results. The average recovery rates for serum samples range from 88% to 101% (average 94%), for EDTA plasma from 87% to 102% (average 94%), and for heparin plasma from 90% to 103% (average 94%) . Linearity tests for sample dilutions also demonstrate reliable results:

These data indicate that ELISA methods provide consistent quantification across different dilution ranges, making them suitable for various experimental designs and sample limitations .

What sample preparation protocols are recommended for optimal ANXA4 detection?

For optimal ANXA4 detection, sample preparation protocols should be tailored to the detection method and sample type. For Western blotting, cell lysates should be prepared under reducing conditions using appropriate immunoblot buffer systems (such as Immunoblot Buffer Group 2). PVDF membranes are recommended for protein transfer, with detection using specific anti-ANXA4 antibodies (such as Mouse Anti-Human Annexin A4 Monoclonal Antibody) at concentrations around 0.1 μg/mL, followed by appropriate HRP-conjugated secondary antibodies . For immunohistochemistry of tissue sections, heat-induced epitope retrieval using basic antigen retrieval reagents is recommended before primary antibody incubation (typically at 15 μg/mL overnight at 4°C) . For ELISA applications, samples should be properly stored at -20°C or -80°C until analysis, avoiding repeated freeze-thaw cycles. Appropriate dilution buffers should be used according to the kit specifications, with attention to temperature conditions specified in the protocol .

What is the role of ANXA4 in cancer progression and metastasis?

ANXA4 has been implicated in cancer progression and metastasis across multiple cancer types. Research indicates that ANXA4 is significantly upregulated in various clinical epithelial tumors, including colorectal, pancreatic, ovarian, breast, and prostate cancers . This upregulation correlates with enhanced tumor invasion capabilities, suggesting that ANXA4 plays a role in promoting metastatic potential. The mechanisms through which ANXA4 contributes to cancer progression involve its influence on cell migration, invasion, and adhesion properties. Functional studies have shown that specific mutations in ANXA4 can inhibit these cellular processes, indicating that normal ANXA4 function may promote these behaviors in cancer cells . Additionally, ANXA4's roles in membrane organization, signal transduction, and calcium-dependent processes may contribute to altered cellular behaviors characteristic of cancer progression. Researchers investigating ANXA4 in cancer contexts should consider its potential as both a biomarker and therapeutic target, with particular attention to cancer-specific expression patterns.

How does ANXA4 contribute to reproductive disorders such as recurrent spontaneous abortion?

Recent research has identified a novel role for ANXA4 in reproductive disorders, particularly recurrent spontaneous abortion (RSA). A study examining 325 RSA patients identified a novel mutation in the ANXA4 gene (c.23G > A, p.G8D) in one patient, which was not found in 941 control subjects or public genetic databases . This mutation was confirmed through Sanger sequencing and is located in the first exon of the ANXA4 gene on chromosome 2p13.3. Evolutionary conservation analysis revealed that the affected amino acid residue (G8) is highly conserved across 13 vertebrate species, suggesting its functional importance. Functional assays demonstrated that this mutation inhibits cell migration, invasion, and adhesion, which are critical processes in placentation and embryo implantation . Additionally, ANXA4 is known to be localized to the glandular and luminal epithelium of the human endometrium and is present at high levels throughout the menstrual cycle (except during the early secretory phase), further supporting its role in reproductive processes .

What genetic variations in ANXA4 have clinical significance?

The most clinically significant genetic variation in ANXA4 identified to date is the missense mutation c.23G > A (p.G8D). This mutation was discovered through whole exome sequencing (WES) in a patient with recurrent spontaneous abortion and was subsequently validated by Sanger sequencing . The pathogenicity of this mutation was assessed through multiple lines of evidence: First, it was absent in 941 control subjects and in public databases (dbSNP, 1,000 Genomes Project, ExAC, and BGI in-house databases), indicating its rarity. Second, evolutionary conservation analysis demonstrated that the G8 residue is highly conserved across 13 vertebrate species, suggesting functional importance. Third, the SIFT program prediction and structural modeling analysis indicated that this mutation is likely harmful. Fourth, functional assays revealed that the mutation inhibits cell migration, invasion, and adhesion in human endometrial stromal cells . The clinical significance of this mutation is further supported by follow-up observations showing that the affected patient experienced another recurrent miscarriage despite appropriate fertility preservation treatment, suggesting a causal relationship between the ANXA4 mutation and reproductive disorders .

What cell models are appropriate for studying ANXA4 function?

Several established cell models have proven effective for studying ANXA4 function across different research contexts. For cancer-related research, A431 human epithelial carcinoma cells demonstrate measurable ANXA4 expression and are suitable for investigating its role in epithelial cancers . For studying basic cellular functions, NIH-3T3 mouse embryonic fibroblasts and Rat-2 rat embryonic fibroblast cell lines have documented ANXA4 expression and can be used for comparative studies across species . For reproductive biology research, THESCs (human endometrial stromal cell line) have been successfully employed to investigate ANXA4 function in the context of reproductive processes and disorders . These cells respond well to transfection with ANXA4 expression constructs using reagents such as FuGENE HD, allowing for wild-type and mutant ANXA4 functional studies. When selecting a cell model, researchers should consider the tissue-specific expression patterns of ANXA4 and choose models that naturally express the protein or that represent tissues where ANXA4 plays significant physiological roles.

How can researchers effectively generate and validate ANXA4 mutations for functional studies?

To generate and validate ANXA4 mutations for functional studies, researchers should follow a structured approach that ensures accurate mutation creation and comprehensive validation. First, obtain wild-type ANXA4 cDNA (such as NM_001153.5) and insert it into an appropriate expression vector (e.g., pcDNA3.1). Second, use site-directed mutagenesis techniques, such as those provided in KOD-Plus-Mutagenesis Kits, to generate specific mutations in the wild-type construct . All generated constructs must be confirmed by Sanger sequencing to verify the presence of the desired mutation and absence of unintended alterations. For functional validation, transfect both wild-type and mutant constructs into appropriate cell lines (such as THESCs) using optimized transfection protocols. Specifically, seed approximately 1 × 10^5 cells in 6-well plates, culture for 24 hours, and then transfect using an appropriate ratio of DNA to transfection reagent (e.g., 2 μg of plasmid and 5 μL of FuGENE HD transfection reagent per well) . After transfection, confirm expression of the wild-type and mutant proteins using Western blot and then proceed to functional assays that assess processes relevant to ANXA4 function, such as calcium binding, membrane association, cell migration, invasion, and adhesion.

What functional assays best characterize ANXA4 activity in different experimental contexts?

Several functional assays effectively characterize ANXA4 activity across different experimental contexts. Cell migration assays, such as wound healing or Transwell migration assays, are valuable for assessing ANXA4's influence on cellular motility, which is particularly relevant in both cancer research and reproductive biology . Cell invasion assays using Matrigel-coated Transwell inserts can evaluate ANXA4's impact on invasive capabilities, crucial for understanding its role in tumor progression and placentation. Cell adhesion assays, measuring attachment to extracellular matrix components, help elucidate ANXA4's function in cell-matrix interactions, relevant to both cancer metastasis and embryo implantation . Calcium binding assays, such as calcium overlay or fluorescence-based calcium flux measurements, can assess ANXA4's calcium-dependent functions. Membrane association assays, including subcellular fractionation and immunofluorescence microscopy, can evaluate ANXA4's translocation to membranes upon calcium stimulation. Phospholipid binding assays using liposomes or lipid strips can characterize ANXA4's interaction with specific phospholipids. For studies involving mutations, researchers should conduct comparative functional analyses between wild-type and mutant ANXA4 to determine how specific alterations affect protein function and downstream cellular processes.

How can ANXA4 expression be effectively manipulated in experimental systems?

ANXA4 expression can be manipulated through several complementary approaches depending on research objectives. For overexpression studies, transfection of expression constructs containing wild-type or mutated ANXA4 cDNA into appropriate cell lines is effective. Optimized transfection protocols using reagents such as FuGENE HD with precisely calibrated DNA:reagent ratios (e.g., 2 μg plasmid: 5 μL reagent) yield reliable results . For stable expression, lentiviral or retroviral transduction systems provide longer-term expression with integration into the host genome. For knockdown/knockout studies, RNA interference using siRNA or shRNA targeting ANXA4 mRNA can achieve transient or stable knockdown, respectively. CRISPR-Cas9 gene editing enables precise genomic alterations for knockout studies or for introducing specific mutations. When designing manipulation strategies, researchers should include appropriate controls: empty vector controls for overexpression studies, non-targeting siRNA/shRNA for knockdown studies, and wild-type cells for CRISPR-edited lines. Validation of manipulation efficacy through quantitative RT-PCR, Western blotting, or immunofluorescence is essential before proceeding to functional analyses to ensure the desired expression changes have been achieved.

What are the emerging roles of ANXA4 in cellular stress responses?

ANXA4, as a calcium-binding protein involved in membrane organization, plays emerging roles in cellular stress responses that warrant further investigation. Its calcium-dependent membrane association positions ANXA4 as a potential responder to calcium flux disturbances during cellular stress. Research should focus on how ANXA4 responds to specific stressors such as oxidative stress, endoplasmic reticulum stress, hypoxia, and DNA damage. Particular attention should be given to ANXA4's potential roles in membrane repair following stress-induced damage, as other annexin family members have demonstrated functions in this process. The relationship between ANXA4 and stress-activated signaling pathways, such as the unfolded protein response (UPR), mitogen-activated protein kinase (MAPK) cascades, and NF-κB signaling, represents an important research direction. Additionally, ANXA4's interactions with other stress-responsive proteins should be characterized through co-immunoprecipitation, proximity ligation assays, or yeast two-hybrid screening. Time-course analyses of ANXA4 expression, localization, and post-translational modifications following stress induction would provide valuable insights into its dynamic roles in stress responses. These investigations could reveal novel therapeutic targets for conditions involving dysregulated stress responses, including cancer, neurodegenerative diseases, and inflammatory disorders.

How do post-translational modifications affect ANXA4 function?

Post-translational modifications (PTMs) likely serve as critical regulators of ANXA4 function, though this area remains incompletely characterized and presents rich opportunities for investigation. Researchers should focus on identifying PTMs including phosphorylation, acetylation, ubiquitination, SUMOylation, and glycosylation of ANXA4 using mass spectrometry-based proteomics approaches. The calcium-binding properties and membrane association of ANXA4 may be modulated by phosphorylation events, particularly at serine, threonine, or tyrosine residues within or adjacent to the calcium-binding regions. To study these modifications, researchers should employ site-directed mutagenesis to create phosphomimetic (e.g., S→D or T→E) or phospho-deficient (e.g., S→A or T→A) mutants, followed by functional assays comparing their behavior to wild-type ANXA4. The enzymatic regulators of ANXA4 modification—including specific kinases, phosphatases, acetyltransferases, deacetylases, and E3 ligases—should be identified through inhibitor studies, co-immunoprecipitation, and siRNA-mediated knockdown approaches. Additionally, the stimulus-dependent dynamics of ANXA4 modifications should be characterized in response to calcium flux, growth factor stimulation, and various cellular stressors. Understanding the PTM landscape of ANXA4 will provide insights into its functional regulation and potential as a therapeutic target.

What are common challenges in ANXA4 antibody selection and validation?

Selecting and validating antibodies for ANXA4 research presents several challenges that researchers should address systematically. First, cross-reactivity with other annexin family members is a significant concern due to the high sequence homology, particularly in the C-terminal core domain. Researchers should select antibodies specifically validated for non-cross-reactivity with related annexins (A1, A2, A3, A5, A6, A8) through direct ELISA and Western blot testing . Second, epitope accessibility may vary depending on ANXA4's conformational state and calcium-binding status. Using antibodies targeting different epitopes can help ensure detection across various conditions. Third, application-specific performance varies; an antibody performing well in Western blots may not be optimal for immunohistochemistry or immunoprecipitation. Validation should be performed for each specific application. For Western blotting, validation should include positive controls (cell lines with known ANXA4 expression, such as A431 cells) and negative controls (knockdown or knockout samples) . For immunohistochemistry, researchers should validate through peptide competition assays and comparison with in situ hybridization data. For co-immunoprecipitation studies, validation should include reciprocal pull-downs and mass spectrometry confirmation. Researchers should maintain detailed records of antibody performance across different lots and experimental conditions to ensure reproducibility.

How can researchers optimize ELISA protocols for ANXA4 detection in diverse sample types?

Optimizing ELISA protocols for ANXA4 detection across diverse sample types requires systematic adjustment of several parameters. First, sample preparation should be standardized with consistent collection, processing, and storage protocols. For serum and plasma samples, processing time should be minimized and samples stored at -80°C until analysis, with avoidance of repeated freeze-thaw cycles . Sample dilution optimization is critical; preliminary experiments should establish optimal dilution factors for different sample types (serum, EDTA plasma, heparin plasma) to ensure measurements fall within the standard curve's linear range . Standard curve preparation should be performed with high-quality recombinant ANXA4 protein, using sufficient calibration points (at least 7-8 points) and appropriate curve-fitting methods. Researchers should validate detection antibody specificity and optimal concentration through titration experiments. Incubation times and temperatures significantly impact assay performance; systematic comparison of different conditions (e.g., overnight at 4°C versus shorter incubations at room temperature) should be performed. Washing steps are critical for reducing background; optimizing wash buffer composition and washing protocol can improve signal-to-noise ratio. For challenging samples with potential matrix effects, the addition of blocking agents or optimization of the sample dilution buffer composition may improve results. Finally, researchers should include appropriate controls in each assay, including blank wells, negative controls, positive controls, and spike-in recovery samples to validate assay performance .

What strategies help resolve data inconsistencies in ANXA4 expression studies?

When facing data inconsistencies in ANXA4 expression studies, researchers should implement a structured troubleshooting approach. First, perform methodological standardization by establishing detailed standard operating procedures (SOPs) for all aspects of sample collection, processing, storage, and analysis to minimize technical variability. Second, employ multiple detection methods; when discrepancies arise between techniques (e.g., qPCR, Western blot, ELISA, immunohistochemistry), additional orthogonal methods should be used to resolve inconsistencies . Third, carefully validate all reagents, particularly antibodies, across different lots and through specificity testing against related annexin proteins . Fourth, account for biological variables including cell confluence, passage number, tissue heterogeneity, and patient demographics that may influence ANXA4 expression. Fifth, consider post-translational modifications and protein degradation that might affect detection but not transcript levels, potentially explaining discrepancies between protein and mRNA measurements. Sixth, examine subcellular localization shifts that might affect extraction efficiency in different fractionation protocols. Seventh, implement appropriate normalization strategies using stable reference genes or proteins validated specifically for the experimental conditions and tissue types under study. Finally, perform statistical power analysis to ensure sufficient biological replicates are included to detect meaningful differences despite biological variability. When reporting results, researchers should transparently document all methodological details and acknowledge any unresolved inconsistencies.

What emerging technologies will advance ANXA4 functional characterization?

Several emerging technologies show promise for advancing ANXA4 functional characterization in the coming years. CRISPR-based gene editing advances, including base editing and prime editing, will enable more precise genetic manipulation of ANXA4 with minimal off-target effects, allowing researchers to introduce specific mutations or regulatory elements to study their functional consequences. Live-cell imaging techniques with improved spatial and temporal resolution, combined with genetically encoded calcium indicators, will provide insights into ANXA4's dynamic behavior during calcium signaling events and membrane processes. Proximity labeling approaches such as BioID or APEX2 can identify the proximal interactome of ANXA4 in different cellular compartments and under various conditions, revealing novel interaction partners and potential functions. Advanced proteomics approaches including targeted mass spectrometry will facilitate more precise quantification of ANXA4 post-translational modifications and their dynamics during cellular processes. Single-cell technologies including single-cell RNA-seq, proteomics, and spatial transcriptomics will help characterize cell-type-specific expression patterns and functions of ANXA4 within heterogeneous tissues. Organoid and microphysiological systems that better recapitulate tissue architecture will provide more physiologically relevant contexts for studying ANXA4 functions in complex cellular environments. These technologies, especially when applied in combination, will substantially enhance our understanding of ANXA4's multifaceted roles in normal physiology and disease states.

How might ANXA4-targeted therapeutics be developed for reproductive disorders?

The development of ANXA4-targeted therapeutics for reproductive disorders represents an emerging frontier following the discovery of ANXA4's role in recurrent spontaneous abortion (RSA) . The therapeutic development pathway should begin with detailed characterization of ANXA4 expression, localization, and function in normal endometrial tissue compared to patients with reproductive disorders. This should include transcriptomic, proteomic, and spatial analyses across the menstrual cycle and during early pregnancy. Drug discovery efforts should focus on multiple modalities: small molecule modulators that could enhance or inhibit ANXA4 function depending on the specific mechanism involved; biologics such as antibodies or recombinant proteins that could supplement ANXA4 function or block pathological interactions; and genetic approaches including antisense oligonucleotides or siRNA delivery systems for transient modulation of ANXA4 expression. High-throughput screening platforms using cell lines expressing wild-type or mutant ANXA4 (such as the p.G8D variant) could identify compounds that rescue functional deficits in migration, invasion, or adhesion . Lead compounds should be validated in progressively more complex models, including organoids, ex vivo endometrial tissue cultures, and eventually animal models of reproductive disorders. Biomarker development should proceed in parallel to identify patients most likely to benefit from ANXA4-targeted approaches and to monitor treatment efficacy. Given the complexity of reproductive disorders, combination approaches targeting ANXA4 alongside other pathways may ultimately prove most effective.

What are the most promising interdisciplinary approaches for understanding ANXA4 in human disease?

Understanding ANXA4's roles in human disease will benefit significantly from interdisciplinary approaches that integrate diverse methodologies and perspectives. Systems biology approaches combining multi-omics data (genomics, transcriptomics, proteomics, metabolomics) with computational modeling can reveal how ANXA4 functions within broader cellular networks and how its dysregulation contributes to disease pathogenesis. Translational research bridging basic ANXA4 biology with clinical observations will accelerate the identification of therapeutic opportunities, particularly through biobanking initiatives that collect samples from patients with conditions potentially involving ANXA4 dysregulation, such as recurrent pregnancy loss and epithelial cancers. Structural biology and biophysics methods, including cryo-electron microscopy and advanced spectroscopic techniques, can provide insights into ANXA4's molecular mechanisms, particularly its calcium-dependent conformational changes and membrane interactions. Engineering approaches, including biomaterials and microfluidic devices, can create controlled environments for studying ANXA4 function at tissue interfaces relevant to implantation biology or tumor invasion. Population genetics and epidemiology can identify additional ANXA4 variants associated with disease risk across diverse populations, expanding upon the initial finding of the p.G8D variant . Patient-derived models, including induced pluripotent stem cells differentiated into relevant cell types, organoids, and xenografts, can provide personalized platforms for studying how specific ANXA4 variants affect cellular functions in genetic backgrounds matching affected individuals. These interdisciplinary approaches, supported by collaborative research networks, will accelerate understanding of ANXA4's roles in human health and disease.