CA13 Human

What is the structural characterization of human CA13?

Human CA13 exhibits a globular molecular structure with high structural similarity to other cytosolic carbonic anhydrase isozymes (CA I, II, and III). Structural modeling studies have confirmed that CA13 maintains the characteristic fold of the α-carbonic anhydrase family . Like other active carbonic anhydrases, CA13 contains a zinc ion in its active site, qualifying it as a metalloenzyme . The protein consists of 262 amino acids in its mature form, with a molecular mass of approximately 31.8 kDa . Recombinant forms typically include additional amino acids such as a histidine tag for purification purposes, resulting in a 285-amino acid polypeptide with the complete sequence starting with MGSSHHHHHH SSGLVPRGSPMGSMSRLSWG .

What is the tissue distribution pattern of CA13 in humans?

CA13 demonstrates a distinct and widespread distribution pattern compared to other cytosolic carbonic anhydrase isozymes. In human tissues, expression analysis using cDNA panels has identified positive signals in multiple organs:

This distribution pattern differs from that observed in mouse tissues, where positive expression has been detected in spleen, lung, kidney, heart, brain, skeletal muscle, and testis . Immunohistochemical staining using specific antibodies raised against a conserved 14-amino acid polypeptide has further confirmed this widespread distribution pattern .

What are the enzymatic properties of CA13?

Human CA13 functions as an active carbonic anhydrase, catalyzing the reversible hydration of carbon dioxide to bicarbonate and protons. Recombinant mouse CA13 exhibits catalytic activity comparable to mitochondrial CA V and cytosolic CA I with the following kinetic parameters:

The enzyme shows high susceptibility to inhibition by sulfonamides and anionic inhibitors, with inhibition constants of 17 nM for acetazolamide (a clinically used sulfonamide) and 0.25 μM for cyanate . This inhibition profile provides valuable insights for potential therapeutic targeting of CA13 in various conditions.

How does CA13 differ from other cytosolic carbonic anhydrases?

CA13 distinguishes itself from other cytosolic carbonic anhydrases (CA I, II, and III) in several key aspects:

• Distribution pattern: CA13 shows a unique and widespread tissue distribution compared to other cytosolic isozymes .

• Enzymatic activity: While structurally similar to cytosolic isozymes, CA13's catalytic activity is more comparable to that of mitochondrial CA V and cytosolic CA I, rather than the highly active CA II .

• Inhibition profile: CA13 demonstrates high susceptibility to sulfonamide inhibitors, with an inhibition constant for acetazolamide (17 nM) that differs from those of other isozymes .

• Physiological roles: Emerging evidence suggests unique physiological functions for CA13, including potential involvement in cancer suppression, particularly in breast cancer bone metastasis .

These distinctions highlight CA13 as a unique member of the carbonic anhydrase family with potentially specialized physiological functions.

What role does CA13 play in cancer progression and metastasis?

Recent investigations have revealed a potential tumor-suppressive role for CA13, particularly in breast cancer bone metastasis. Transcriptome analysis of bone metastatic breast cancer cells showed reduced expression of several genes, including metabolism-related CA13 . Functional studies demonstrated that overexpression of CA13 in iRFP-iCSCL-10A breast cancer cells suppressed migration, invasion, and bone metastasis capabilities .

The suppression of metastatic potential was associated with reduced expression of vascular endothelial growth factor-A (VEGF-A) and macrophage colony-stimulating factor (M-CSF), suggesting that CA13 may inhibit metastasis by modulating angiogenic and immunomodulatory factors . This finding highlights CA13 as a potential negative regulator of the metastatic cascade in breast cancer.

How does CA13 expression correlate with patient survival in cancer?

The inverse relationship between CA13 expression and poor prognosis supports the hypothesis that downregulation of CA13 might be a mechanism through which breast cancer cells acquire metastatic capabilities, particularly for bone tropism. This clinical correlation provides translational relevance to the functional studies demonstrating CA13's metastasis-suppressive effects.

What molecular mechanisms underlie CA13's suppression of metastasis?

The molecular mechanisms through which CA13 suppresses metastasis are beginning to be elucidated. Current evidence suggests several potential pathways:

• Modulation of angiogenic factors: CA13 overexpression reduces VEGF-A levels, potentially limiting the formation of new blood vessels required for metastatic colonization .

• Immunomodulatory effects: Reduced M-CSF expression following CA13 overexpression suggests potential impacts on tumor-associated macrophages, which are known to facilitate metastatic processes .

• Metabolic effects: As a carbonic anhydrase, CA13 may influence the tumor microenvironment by modulating pH and carbon dioxide/bicarbonate levels, which could affect various aspects of the metastatic cascade .

• Cell motility regulation: The observed suppression of migration and invasion suggests that CA13 may influence cytoskeletal dynamics or cell-matrix interactions, though the precise mechanisms remain to be fully elucidated .

Further research is needed to delineate the precise signaling pathways and molecular interactions through which CA13 exerts its metastasis-suppressive effects.

How do inhibitors affect CA13 function in experimental systems?

CA13 demonstrates significant susceptibility to inhibition by both sulfonamide and anionic inhibitors. The inhibition constants for CA13 are 17 nM for acetazolamide (a clinically used sulfonamide) and 0.25 μM for cyanate . These inhibition properties provide valuable tools for investigating CA13 function in experimental systems.

Inhibitor studies can help elucidate:

• The contribution of CA13's enzymatic activity to its biological functions, including its potential role in suppressing metastasis.

• Structure-activity relationships within the active site of CA13.

• Potential therapeutic strategies targeting CA13 for various conditions.

• Differential effects compared to inhibition of other carbonic anhydrase isozymes.

Understanding the inhibition profile of CA13 is particularly relevant given the clinical use of carbonic anhydrase inhibitors for conditions such as glaucoma, potentially opening avenues for drug repurposing strategies.

What is the evolutionary significance of CA13 compared to other carbonic anhydrases?

Evolutionary analysis of CA13 reveals its conservation across mammalian species, suggesting important biological functions. The human and mouse CA13 orthologs share significant sequence homology, enabling the use of common antibodies for detection . This conservation contrasts with the diverse specialization observed across different carbonic anhydrase isozymes.

Comparative genomic analyses indicate that CA13 represents a relatively recent addition to the carbonic anhydrase gene family, which now comprises at least 12 enzymatically active members . The structural similarity to cytosolic isozymes, coupled with distinct enzymatic properties and tissue distribution, suggests that CA13 may have evolved to fulfill specialized physiological roles that remain to be fully characterized.

The widespread distribution of CA13 across multiple tissues further suggests that it may serve fundamental cellular functions beyond the specialized roles of some other carbonic anhydrase isozymes.

What are optimal methods for detecting CA13 expression in experimental systems?

Several complementary approaches can be employed for reliable detection of CA13 expression:

• Transcript-level analysis:

  • RT-PCR using specific primers targeting CA13 mRNA

  • cDNA panels for multi-tissue expression analysis

  • RNA-Seq for transcriptome-wide analysis and quantification

• Protein-level detection:

  • Western blotting using specific antibodies

  • Immunohistochemistry using antibodies raised against conserved epitopes (e.g., 14-amino acid polypeptide common to human and mouse CA13)

  • Immunofluorescence for subcellular localization studies

For immunodetection, antibodies raised against specific epitopes unique to CA13 are crucial to avoid cross-reactivity with other carbonic anhydrase isozymes, given their structural similarities. The use of knockout controls or competing peptides can help validate antibody specificity.

How can recombinant CA13 be produced for experimental applications?

Recombinant CA13 production for experimental studies typically involves:

• Expression system selection:

  • Escherichia coli is commonly used for CA13 expression

  • Mammalian or insect cell systems may be considered for studies requiring mammalian post-translational modifications

• Construct design:

  • Full-length human CA13 cDNA (encoding 262 amino acids)

  • Addition of purification tags (e.g., His-tag) for simplified isolation

  • Inclusion of appropriate promoters and selection markers

• Purification strategy:

  • Metal affinity chromatography for His-tagged proteins

  • Ion exchange and size exclusion chromatography for higher purity

  • Enzyme activity testing to confirm functional integrity

For example, one approach has produced recombinant mouse CA13 as a single, non-glycosylated polypeptide chain containing 285 amino acids (including a 23-amino acid His-tag at the N-terminus) with a molecular mass of 31.8 kDa . Purified protein is typically formulated in phosphate-buffered saline (pH 7.4) with additives such as 10% glycerol and 1 mM DTT for stability .

What experimental models are appropriate for studying CA13's role in cancer?

Several experimental models can be employed to investigate CA13's functions in cancer, particularly its role in metastasis:

• Cell culture models:

  • Cancer cell lines with manipulated CA13 expression (overexpression, knockdown, knockout)

  • iRFP-labeled cancer cells for in vivo tracking

  • 3D organoid cultures for studying invasion in a more physiologically relevant context

• Animal models:

  • Orthotopic injection models (e.g., mammary fat pad for breast cancer)

  • Intracardiac or intratibial injection for bone metastasis studies

  • Transgenic models with tissue-specific CA13 modulation

• Clinical samples:

  • Patient-derived xenografts

  • Tumor tissue microarrays for correlative studies

  • Analysis of circulating tumor cells

The use of fluorescent or bioluminescent reporters (e.g., iRFP713-labeled breast cancer cells) enables non-invasive monitoring of tumor growth and metastasis in vivo, as demonstrated in studies showing bone metastasis in hind legs after 5-week post-injection .

How can CA13 enzymatic activity be accurately measured?

Accurate measurement of CA13 enzymatic activity can be achieved through several complementary approaches:

• CO₂ hydration assay:

  • Measures the rate of CO₂ conversion to bicarbonate and protons

  • Can be monitored by pH indicators or stopped-flow spectrophotometry

  • Allows determination of kinetic parameters (k(cat), K(m))

• Esterase activity assay:

  • Uses 4-nitrophenyl acetate as substrate

  • Measures hydrolysis to 4-nitrophenol (specific activity > 2,500 pmol/min/μg)

  • Can be performed at pH 7.5 and 37°C using spectrophotometric detection

• Inhibition studies:

  • Measures activity in the presence of various concentrations of inhibitors

  • Determines inhibition constants (Ki) for compounds such as acetazolamide (17 nM) and cyanate (0.25 μM)

  • Provides insights into active site properties

These methodologies enable comprehensive characterization of CA13's catalytic properties and comparative analysis with other carbonic anhydrase isozymes.

What approaches are used to study CA13's impact on cell migration and invasion?

Investigation of CA13's effects on cancer cell migration and invasion employs various complementary methodologies:

• Two-dimensional migration assays:

  • Wound healing/scratch assays

  • Single-cell tracking using time-lapse microscopy

  • Transwell migration assays

• Three-dimensional invasion assays:

  • Matrigel invasion assays

  • Spheroid invasion into collagen matrices

  • Organotypic culture models

• Molecular analyses:

  • Profiling of migration/invasion-related genes

  • Assessment of epithelial-mesenchymal transition markers

  • Analysis of secreted factors (e.g., VEGF-A, M-CSF)

• In vivo metastasis models:

  • Spontaneous metastasis models following orthotopic injection

  • Experimental metastasis via tail vein, intracardiac, or intratibial injection

  • Bioluminescent or fluorescent imaging for tracking metastatic spread

These approaches have revealed that CA13 overexpression suppresses migration and invasion capabilities of breast cancer cells, correlating with reduced metastatic potential in vivo .

What are promising therapeutic strategies targeting CA13?

Based on current understanding of CA13's functions, several therapeutic strategies warrant investigation:

• CA13 activation/restoration approaches:

  • Gene therapy to restore CA13 expression in metastatic cancers

  • Small molecules that enhance CA13 stability or activity

  • Targeting upstream regulators of CA13 expression

• Differential inhibitor development:

  • Design of isozyme-selective inhibitors that spare CA13 while targeting other carbonic anhydrases

  • Structure-based drug design utilizing CA13's unique active site properties

• Combination strategies:

  • Pairing CA13-targeted approaches with conventional anti-metastatic therapies

  • Sequential treatment strategies based on disease stage

The development of these approaches requires deeper understanding of CA13's structure-function relationships and regulatory mechanisms, highlighting the need for continued basic research alongside translational efforts.

How might CA13 serve as a biomarker in clinical settings?

CA13's potential as a biomarker in cancer and other conditions is supported by several lines of evidence:

The translation of CA13 as a clinical biomarker would require standardization of detection methods and prospective validation in large patient cohorts across different cancer types and stages.

What fundamental questions about CA13 remain unanswered?

Despite significant progress, several fundamental questions about CA13 remain to be addressed:

• Regulatory mechanisms:

  • What factors regulate CA13 expression in normal and pathological conditions?

  • How is CA13 expression lost during cancer progression?

• Physiological functions:

  • What are CA13's primary physiological roles in the diverse tissues where it is expressed?

  • How does CA13 contribute to normal cellular homeostasis?

• Structural insights:

  • What structural features distinguish CA13 from other carbonic anhydrases?

  • How do these structural elements relate to its unique functions?

• Translational aspects:

  • Can CA13 restoration effectively suppress metastasis in established tumors?

  • What patient populations would benefit most from CA13-targeted interventions?

Addressing these questions will require interdisciplinary approaches combining structural biology, molecular genetics, biochemistry, and translational research methodologies.