ANXA13 Human

What is ANXA13 and what is its evolutionary significance?

ANXA13 encodes a protein belonging to the annexin family of calcium-dependent phospholipid-binding proteins. It is widely considered to be the original founder gene of the 12-member vertebrate annexin A family and demonstrates the deepest-branching position in phylogenetic analyses of annexins . Evolutionary studies using comparative genetics across nine species have established ANXA13 as the probable common ancestor of all vertebrate annexins, with initial gene duplication events occurring approximately 700 million years ago, before the emergence of chordates .

The evolutionary significance of ANXA13 is particularly notable in understanding structure-function relationships in the annexin superfamily. Research indicates that the gene has acquired intestine-specific expression associated with highly differentiated intracellular transport functions . This specialized role evolved while maintaining certain core structural elements that characterize the entire annexin family.

What is the genomic structure and organization of ANXA13?

The human ANXA13 gene spans approximately 58 kb and contains 12 coding exons. It is located on chromosome 8q24.12, as determined through bacterial artificial chromosome (BAC) clone sequencing and contig analysis . The gene demonstrates a unique exon splicing pattern that is distinct from all other vertebrate annexins, a finding corroborated through comparative genomic analysis of mouse, rat, zebrafish, and pufferfish DNA .

Researchers have identified internal repetitive elements and neighboring genes through contig analysis of draft sequences from the human genome project. The putative promoter region has been characterized through phylogenetic footprinting, which identified potential binding sites for intestine-specific transcription factors . These structural features make ANXA13 an interesting subject for researchers studying gene regulation and tissue-specific expression patterns.

What unique structural features differentiate ANXA13 from other annexins?

ANXA13 possesses several protein diagnostic features that distinguish it from other members of the annexin family:

• An alternate isoform containing a KGD motif

• Elevated basic amino acid content with polyhistidine expansion in the 5′-translated region

• Conservation of 15% core tetrad residues specific to annexin A13 members

These unique structural characteristics likely contribute to the specific functional properties of ANXA13 in cellular processes. The presence of alternatively spliced transcript variants encoding different isoforms has been identified, suggesting functional versatility of this protein in different cellular contexts .

What methodologies are most effective for studying ANXA13 expression in normal and cancer tissues?

Researchers employ several complementary techniques to investigate ANXA13 expression:

Western Blotting Analysis: This remains the gold standard for quantifying ANXA13 protein levels in cell lines and tissue samples. Studies have successfully used western blotting to determine relative expression levels of annexin A13 in colorectal cancer cell lines, revealing variable expression patterns across different cell types . For example, research has demonstrated undetectable ANXA13 in SW620 cells, low expression in SW480 and Rko cells, and high expression in HCT116 and HT29 cell lines .

Genetic Manipulation Techniques:

• Overexpression: Transfection with pcDNA3.1-ANXA13 plasmids has been used to upregulate ANXA13 expression in low-expressing cells (such as SW620 and Rko cells) .

• Downregulation: siRNA treatment has proven effective for reducing ANXA13 expression in high-expressing cells (such as HCT116 and HT29) .

Functional Assays: Matrigel in vitro invasion assays are commonly employed to assess the impact of ANXA13 expression on tumor cell invasion capabilities . This methodology allows researchers to establish causal relationships between ANXA13 expression levels and cancer cell behavior.

Clinical Tissue Analysis: For translational research, immunohistochemical analysis of patient tissue samples enables correlation of ANXA13 expression with clinicopathological features and patient outcomes .

How does ANXA13 contribute to tumor cell invasion in colorectal cancer?

ANXA13 has been identified as a promoter of tumor cell invasion in colorectal cancer through several experimental approaches. Research demonstrates that ANXA13 upregulation is associated with lymph node metastasis and poor survival in CRC patients . The mechanisms underlying this association include:

While the precise molecular mechanisms remain under investigation, these findings suggest that ANXA13 modulates cellular processes involved in extracellular matrix degradation, cell motility, or other aspects of the metastatic cascade.

What signaling pathways are associated with ANXA13 in cancer cell biology?

• Calcium signaling pathways: As a member of the calcium-dependent phospholipid-binding protein family, ANXA13 likely interacts with calcium-mediated signaling pathways .

• Membrane trafficking and cellular transport: ANXA13 is associated with plasma membranes of undifferentiated, proliferating endothelial cells and differentiated villus enterocytes, suggesting a role in membrane organization and trafficking .

• Growth regulation pathways: The annexin family plays roles in the regulation of cellular growth and signal transduction pathways . While specific pathways for ANXA13 are not fully characterized, its involvement in cancer progression suggests interaction with growth and proliferation signals.

Research on other annexin family members provides clues to potential ANXA13 signaling mechanisms. For instance, ANXA6 can either activate or inactivate the Ras/MAPK signaling pathway depending on cancer type . Similar context-dependent signaling roles may exist for ANXA13.

What experimental models are optimal for studying ANXA13 function?

Based on current research, several experimental models have proven valuable for studying ANXA13:

Cell Culture Models:

• Colorectal cancer cell lines: HCT116 and HT29 (high ANXA13 expression), SW480 and Rko (low expression), and SW620 (undetectable expression) provide a spectrum of expression levels for comparative studies .

• Differentiation models: The HT-29 cell line, when grown in medium containing inosine instead of glucose, undergoes differentiation and serves as a model for studying ANXA13 in cell differentiation processes .

Animal Models:

• Mouse models: The mouse annexin A13 gene has been mapped to chromosome 15 between Sdc2 and Myc by backcross analysis, enabling genetic studies in murine systems .

• Zebrafish models: Annexin A13 has been localized to linkage group 24 in zebrafish, offering a vertebrate model for developmental studies .

Genetic Manipulation Approaches:

• Transfection and siRNA systems: These allow for controlled modulation of ANXA13 expression to study functional consequences .

• CRISPR-Cas9 gene editing: While not explicitly mentioned in the search results, this technology represents a cutting-edge approach for studying gene function.

How can researchers differentiate between the functional roles of different ANXA13 isoforms?

ANXA13 exists in multiple isoforms, including a notable alternate isoform containing a KGD motif . To differentiate between the functional roles of these isoforms, researchers can employ several strategies:

• Isoform-specific primers and antibodies: Designing primers that specifically amplify distinct isoforms enables quantification of isoform-specific expression patterns in different tissues and cell types. Similarly, isoform-specific antibodies can be developed for protein detection.

• Isoform-selective expression constructs: Transfection with plasmids containing coding sequences for specific isoforms allows for selective overexpression and functional analysis.

• Isoform-targeted siRNA or CRISPR-Cas9: Design of isoform-specific knockdown or knockout strategies enables selective reduction of individual isoforms.

• Structural and binding studies: Biochemical approaches to characterize the binding partners and structural properties of individual isoforms can reveal functional differences.

• Correlation studies: Analysis of the relative abundance of different isoforms in various tissues or disease states can provide insights into their specialized functions.

What is the prognostic significance of ANXA13 in different cancer types?

This is in contrast to other annexin family members, which have been more extensively studied across cancer types. For example:

• ANXA2 is elevated in breast cancer and stimulates fibrinolytic enzyme production, leading to angiogenesis and metastasis .

• ANXA5 is significantly expressed in non-papillary bladder cancer .

• ANXA8 is significantly enhanced in ovarian malignant tissues compared to benign tumors and normal ovarian tissues, associated with poor prognosis .

• ANXA10 has been shown to be a prognostic biomarker for papillary thyroid cancer .

What methodological approaches are used to establish ANXA13 as a potential therapeutic target?

To establish ANXA13 as a potential therapeutic target in cancer, researchers employ several methodological approaches:

• Expression modulation studies: Experimental upregulation and downregulation of ANXA13 in cancer cell lines helps establish causality between ANXA13 expression and cancer phenotypes .

• Functional assays: Invasion assays, migration experiments, and proliferation assays determine the functional consequences of modulating ANXA13 expression .

• Correlation with clinical outcomes: Analysis of ANXA13 expression in patient samples and correlation with clinical parameters (such as metastasis, survival, and treatment response) establishes clinical relevance .

• Mechanistic studies: Investigation of pathways and molecular interactions affected by ANXA13 helps identify potential intervention points.

• Comparative studies with other annexins: Understanding the roles of other annexin family members in cancer provides context for ANXA13-targeted therapeutic approaches .

While specific ANXA13 inhibitors are not discussed in the search results, researchers could potentially develop therapeutic strategies based on:

• Small molecule inhibitors that target ANXA13 directly

• siRNA or antisense oligonucleotides for ANXA13 suppression

• Antibody-based therapies targeting ANXA13 at the cell surface

• Disruption of ANXA13 interactions with binding partners

What are the challenges in isolating and purifying ANXA13 for structural and functional studies?

While the search results don't explicitly address challenges in isolating and purifying ANXA13, several technical considerations can be inferred based on the protein's properties:

• Membrane association: ANXA13's association with plasma membranes of cells suggests that isolation protocols must effectively separate the protein from membrane components.

• Calcium dependency: As a calcium-dependent phospholipid-binding protein , purification conditions must carefully control calcium concentrations to maintain proper protein conformation and function.

• Isoform complexity: The presence of alternatively spliced isoforms complicates purification efforts, potentially requiring isoform-specific approaches.

• Tissue-specific expression: ANXA13's intestine-specific expression pattern means that appropriate tissue sources must be selected for protein isolation.

Researchers typically overcome these challenges through approaches such as:

• Recombinant protein expression systems

• Affinity purification with specific tags

• Immunoprecipitation with anti-ANXA13 antibodies

• Careful optimization of buffer conditions to maintain calcium-dependent properties

How can researchers optimize detection sensitivity for ANXA13 in clinical samples?

Optimizing detection sensitivity for ANXA13 in clinical samples is crucial for both research and potential diagnostic applications. Several approaches can enhance detection sensitivity:

What are the emerging areas of investigation for ANXA13 beyond cancer research?

While current research has focused primarily on ANXA13's role in cancer, particularly colorectal cancer , several promising areas for future investigation emerge:

• Developmental biology: Given ANXA13's status as the founder gene of the annexin family and its expression in intestinal tissue, its role in embryonic development and tissue differentiation warrants further investigation.

• Intestinal physiology: The association of ANXA13 with differentiated villus enterocytes suggests potential roles in intestinal barrier function, nutrient absorption, or other aspects of intestinal physiology.

• Evolutionary biology: As the deepest-branching vertebrate annexin with an estimated gene age predating chordate emergence , ANXA13 offers unique opportunities for studying protein evolution and functional diversification.

• Inflammatory conditions: Other annexin family members have established roles in inflammation, suggesting potential functions for ANXA13 in inflammatory conditions, particularly those affecting the intestine.

• Membrane biology: ANXA13's calcium-dependent phospholipid-binding properties point to potential roles in membrane organization, repair, or trafficking that remain to be fully explored.

How might single-cell sequencing technologies advance our understanding of ANXA13 expression heterogeneity?

Single-cell sequencing technologies offer powerful approaches to understanding the heterogeneity of ANXA13 expression within tissues and cell populations:

• Identification of ANXA13-expressing cell subpopulations: Single-cell RNA sequencing (scRNA-seq) can reveal previously unrecognized subpopulations of cells with distinct ANXA13 expression patterns within tissues.

• Correlation with cellular states: Integration of ANXA13 expression data with broader transcriptomic profiles can reveal associations between ANXA13 expression and specific cellular states (proliferation, differentiation, stress responses).

• Tumor heterogeneity mapping: In cancer tissues, scRNA-seq can map ANXA13 expression across diverse tumor cell subpopulations, potentially identifying cells with enhanced metastatic potential.

• Developmental trajectories: Single-cell approaches can trace the dynamics of ANXA13 expression during development or differentiation processes.

• Spatial context: Spatial transcriptomics techniques can preserve information about the physical location of ANXA13-expressing cells within tissue architecture.

• Epigenetic regulation: Single-cell ATAC-seq can provide insights into the chromatin accessibility landscape governing ANXA13 expression in different cell types.

These advanced approaches would complement traditional bulk analyses and provide a more nuanced understanding of ANXA13's expression patterns and functional relevance across diverse biological contexts.