CA14 Human

What is the genomic location and structure of the human CA14 gene?

The human CA14 gene (Entrez Gene ID: 23632) is located on chromosome 1q21 . The gene encodes a full-length cDNA that is approximately 1757 bp long and encodes a 337-amino-acid polypeptide with a molecular mass of 37.6 kDa . When conducting genomic analyses, researchers should note that multiple reference assemblies are available, including:

Researchers investigating genomic variants should consult resources such as ClinVar and Variation Viewer for comprehensive variant data across different reference assemblies .

What are the key structural features of the CA14 protein?

CA14 is distinguished by its transmembrane structure. The protein features hydrophobic segments at both termini of the deduced protein structure - one serving as a putative signal sequence and the other functioning as a transmembrane domain . This classifies CA14 as a type I membrane protein .

How does CA14 expression vary across human tissues?

CA14 shows a distinctive tissue expression pattern that differs from other carbonic anhydrase isoforms. Based on multiple detection methods, the following tissue distribution has been documented:

Notably, this expression pattern contrasts with that of CA XII, which is expressed in kidney, salivary gland, and pancreas, but not in liver or spinal cord . This distinct tissue distribution suggests different physiological roles for these related transmembrane carbonic anhydrases.

What distinguishes CA14 from other carbonic anhydrase isoforms?

CA14 stands apart from other carbonic anhydrase isoforms in several key aspects:

• Enzymatic activity: CA14 demonstrates relatively low catalytic activity compared to other CA isoforms .

• Inhibitor sensitivity: It shows sensitivity to acetazolamide but is notably insensitive to sulfonamide, which is a distinguishing pharmacological feature .

• Membrane localization: As a type I membrane protein, CA14's cellular localization differs from the cytosolic, mitochondrial, and secreted CA isoforms .

• Tissue distribution: CA14 has a unique tissue expression pattern, with particularly strong expression in the brain and liver, which differs from other transmembrane CAs like CA XII .

• Evolutionary relationship: While CA14 shares highest sequence similarity with CA XII (both being transmembrane isoforms), they have genetically distinct origins and expression patterns, suggesting divergent physiological functions .

What antibodies and detection methods are available for studying CA14?

Several antibodies and detection methods have been developed for studying CA14 in experimental settings:

Antibodies:
Commercial antibodies such as the Human/Mouse/Rat Carbonic Anhydrase XIV/CA14 Antibody (e.g., AF2504) target the extracellular domain (Ala16-Met290) and have been validated for multiple applications .

Detection Methods:

• Western Blot: CA14 can be detected using PVDF membranes under reducing conditions with appropriate antibodies. The protein typically appears as a band at approximately 42 kDa .

• Simple Western™: This automated capillary-based detection method can identify CA14 at approximately 54 kDa in tissue lysates .

• RT-PCR: For transcript detection, RT-PCR protocols targeting the CA14 mRNA have been effective in distinguishing expression across tissues .

• Northern Blot Analysis: This method can detect the approximately 1.7-kb CA14 transcript in relevant tissues .

• RNA Dot-Blot Analysis: This technique allows for comparative analysis of CA14 expression across multiple tissues simultaneously .

When designing experiments, researchers should consider:

• Using both protein and transcript detection methods for comprehensive analysis

• Including appropriate tissue-specific positive controls based on the established expression pattern

• Employing reducing conditions for Western blot analysis

• Validating antibody specificity using multiple detection methods

How can CA14 enzymatic activity be measured and characterized?

Measuring CA14 enzymatic activity presents unique challenges due to its transmembrane localization and relatively low catalytic efficiency. Recommended approaches include:

• Carbonic Anhydrase Activity Assays: These typically measure the rate of CO₂ hydration or dehydration. For CA14, which has low activity, highly sensitive methods are required.

• Inhibitor Profiling: CA14 has distinctive inhibitor sensitivity, being responsive to acetazolamide but not to sulfonamide . This profile can be used to characterize CA14 activity in complex samples.

• Recombinant Enzyme Assays: Expression of the extracellular catalytic domain in appropriate systems allows for purification and direct activity measurement.

• Comparative Kinetic Analysis: When characterizing CA14 activity, it is essential to compare with other CA isoforms under identical conditions to accurately assess relative efficiency.

• pH-Dependent Activity Measurements: Since carbonic anhydrases function in acid-base balance, assessing activity across pH ranges can provide insights into physiological roles.

The development of specific inhibitors like 3,4-Dihydroisoquinoline-2(1H)-sulfonamides has facilitated more detailed characterization of CA14 activity . When designing activity assays, researchers should account for the membrane-bound nature of the enzyme and its relatively low catalytic efficiency.

What are effective approaches for CA14 cloning and expression?

Based on successful previous work with CA14, researchers should consider the following approaches for cloning and expression:

Cloning Strategies:

• Full-length vs. Catalytic Domain: Depending on the research question, either the full-length CA14 (including transmembrane domains) or just the extracellular catalytic domain (Ala16-Met290) can be cloned .

• Vector Selection: For membrane proteins like CA14, vectors designed for expression of transmembrane proteins should be considered.

• Source Material: Human brain, liver, or heart tissue serves as appropriate source material for CA14 cDNA isolation, based on expression patterns .

Expression Systems:

• Mammalian Expression: For functional studies requiring proper folding and post-translational modifications, mammalian cell lines (such as HEK293 or CHO cells) are recommended.

• Bacterial Expression: For production of the soluble catalytic domain, E. coli systems can be utilized, though refolding may be necessary.

• Cell-Free Systems: These may be suitable for the catalytic domain when rapid expression is needed for initial characterization.

When expressing CA14, researchers should monitor for proper membrane insertion when using the full-length construct, and verify enzymatic activity using the mentioned activity assays.

What model systems are most appropriate for investigating CA14 function?

Selection of appropriate model systems is critical for meaningful CA14 research. Based on expression patterns and available tools, the following models are recommended:

Cellular Models:

• Hepatocyte Cell Lines: Given CA14's expression in the liver, hepatocyte lines such as Hepa 1-6 mouse hepatoma cells are suitable models, as they naturally express CA14 .

• Neuronal Cell Lines: Given the strong expression in brain tissue, neuronal models are appropriate for investigating CA14's role in the nervous system.

• Overexpression Systems: Transfected cell lines with controlled CA14 expression can provide insights into function and localization.

Tissue/Animal Models:

• Brain Tissue (particularly cerebellum): Strong expression makes this an excellent model for studying CA14 in the context of neural function .

• Liver Tissue: High expression in the liver suggests this tissue is valuable for investigating CA14's role in hepatic physiology .

• Mouse Models: Murine CA14 shares significant homology with the human protein, making mouse models valuable for in vivo studies .

When selecting model systems, researchers should verify CA14 expression in their specific model and consider the differential expression patterns between species. For comparative studies between human and model organisms, attention should be paid to potential functional differences despite sequence conservation.

How do CA14 and CA12 differ in their tissue expression patterns?

Despite their structural similarity as transmembrane carbonic anhydrases, CA14 and CA12 exhibit distinctly different tissue expression patterns that suggest divergent physiological roles:

This complementary expression pattern suggests these enzymes may have evolved specialized functions in different organ systems . The strong expression of CA14 in the brain and liver, where CA12 is absent, points to unique roles in these tissues. Conversely, CA12's presence in secretory organs like salivary glands and pancreas suggests involvement in secretion processes.

Researchers investigating the physiological significance of these isozymes should consider these distinct patterns when designing experiments and interpreting results. The lack of overlapping strong expression further suggests these enzymes are not functionally redundant in most tissues.

What is known about CA14 inhibitors and their selectivity?

Developing selective inhibitors for specific carbonic anhydrase isoforms presents significant challenges due to the high conservation of the active site across the CA family. For CA14:

• Acetazolamide Sensitivity: CA14 demonstrates sensitivity to acetazolamide, a classical CA inhibitor, but shows resistance to sulfonamide, creating a distinctive inhibition profile .

• 3,4-Dihydroisoquinoline-2(1H)-sulfonamides: These compounds have been identified as potent CA14 inhibitors and represent promising scaffolds for developing more selective agents .

• Inhibitor Screening Considerations: When screening potential CA14 inhibitors, researchers should:

  • Compare inhibition against multiple CA isoforms to determine selectivity

  • Assess membrane permeability for accessing the extracellular catalytic domain

  • Consider the lower baseline activity of CA14 when interpreting inhibition data

• Structure-Based Design: The structural similarities between CA14 and CA12 present challenges for selective inhibitor design, necessitating exploitation of subtle differences in the active site and surrounding regions.

Researchers interested in developing CA14-selective compounds should consider rational design approaches based on the structural features that distinguish CA14 from other CA isoforms, particularly focusing on regions outside the highly conserved active site.

What challenges arise when studying membrane-bound carbonic anhydrases?

Investigating membrane-bound carbonic anhydrases like CA14 presents several unique experimental challenges:

• Protein Isolation and Purification:

  • The transmembrane domain complicates extraction and purification

  • Maintaining native conformation during solubilization is difficult

  • Detergent selection is critical for preserving structure and function

• Activity Assessment:

  • Membrane anchoring may affect enzyme kinetics

  • Standard solution-based assays may not accurately reflect in vivo activity

  • The local microenvironment created by the membrane may influence function

• Structural Analysis:

  • Crystallization of full-length membrane proteins is technically challenging

  • Alternative structural biology approaches (cryo-EM, NMR) may be required

  • The orientation of the catalytic domain relative to the membrane may be functionally important

• Localization Studies:

  • Determining precise subcellular localization requires specialized techniques

  • Antibody accessibility may differ between fixed and live-cell preparations

  • Trafficking and membrane insertion mechanisms remain poorly understood

• Physiological Context:

  • The functional relationship between membrane localization and enzymatic activity

  • Potential interactions with other membrane components

  • Integration of CA14 activity with other cellular processes

When designing experiments to address these challenges, researchers should consider using truncated constructs of the catalytic domain for biochemical studies while employing full-length constructs for cellular localization and in vivo functional analyses.

How can researchers account for species-specific differences when studying CA14?

When conducting CA14 research across different species, researchers should be aware of potential variations that may impact experimental design and interpretation:

• Sequence Divergence:

  • While the catalytic domain is generally well-conserved, species-specific variations exist

  • The Human/Mouse/Rat CA14 antibody (targeting Ala16-Met290) demonstrates cross-reactivity, suggesting conservation of key epitopes

  • Sequence alignments should be performed when designing primers or targeting strategies

• Expression Pattern Differences:

  • Expression patterns may vary between species

  • Verification of expression in the target tissue for each species is essential

  • Western blot analysis reveals slightly different molecular weights across species (42-54 kDa range)

• Functional Implications:

  • Despite sequence conservation, functional roles may differ between species

  • Environmental adaptations may influence CA14 function in different organisms

  • Regulatory mechanisms controlling expression may vary

• Experimental Considerations:

  • Use species-specific primers for RT-PCR and gene expression analysis

  • Validate antibody cross-reactivity before proceeding with interspecies comparisons

  • Consider evolutionary context when interpreting functional data

To effectively translate findings between species, researchers should perform comparative analyses of expression, activity, and inhibitor sensitivity using consistent methodologies across species-derived samples.

What are potential physiological roles of CA14 based on its expression pattern?

The distinctive tissue distribution of CA14 suggests several potential physiological roles that merit further investigation:

• Neurological Functions:

  • The strong expression throughout the brain and spinal cord suggests involvement in neural physiology

  • Potential roles include regulation of extracellular pH in neural microenvironments

  • May participate in bicarbonate transport systems important for neural signaling

  • Could influence cerebrospinal fluid composition and pH regulation

• Hepatic Functions:

  • High expression in the liver points to a role in hepatic physiology

  • May participate in acid-base balance mechanisms specific to liver metabolism

  • Potential involvement in bile production or secretion

  • Could contribute to the liver's role in systemic pH regulation

• Muscular Tissue Roles:

  • Expression in heart and skeletal muscle suggests involvement in:

  • Regulation of muscle cell pH during contraction/relaxation cycles

  • Facilitation of carbon dioxide transport in highly metabolic tissues

  • Potential contribution to intracellular or extracellular buffering systems

• Gastrointestinal Functions:

  • Weak expression in the colon and small intestine indicates possible roles in:

  • Local pH regulation in the intestinal microenvironment

  • Modulation of ion transport across intestinal epithelia

  • Contribution to digestive processes through pH homeostasis

Research strategies to elucidate these functions might include tissue-specific knockdown or knockout studies, localization analyses to determine cellular and subcellular distribution, and functional assays examining pH regulation in relevant tissues.

How might CA14 function in specialized tissues like brain and liver?

The strong expression of CA14 in brain and liver suggests specialized functions that deserve targeted investigation:

Brain Functions:

• Blood-Brain Barrier Physiology: The transmembrane nature of CA14 positions it to potentially regulate acid-base transport across the blood-brain barrier.

• Neuronal Excitability: By influencing local pH, CA14 may modulate neuron excitability, as pH affects numerous ion channels and neurotransmitter receptors.

• Glial Cell Functions: CA14 might participate in glial cell pH regulation, affecting their supportive functions for neurons.

• Cerebrospinal Fluid Homeostasis: The enzyme could contribute to the formation and pH balance of cerebrospinal fluid, which is critical for brain function .

Liver Functions:

• Bile Formation: CA14 might participate in the pH regulation necessary for proper bile formation and secretion.

• Detoxification Processes: Many hepatic detoxification reactions are pH-dependent, suggesting a potential regulatory role for CA14.

• Glucose Metabolism: pH regulation impacts enzymatic activities involved in glucose metabolism, a key hepatic function.

• Protein Synthesis: The liver's extensive protein synthesis machinery requires tight pH control, potentially involving CA14.

Methodological approaches to investigate these specialized functions could include:

• Subcellular localization studies in specific cell types within these organs

• Targeted gene silencing in relevant primary cell cultures

• Metabolic flux analysis under conditions of CA14 inhibition

• Integration of CA14 activity with known tissue-specific physiological processes

What is the potential significance of CA14 in disease contexts?

While direct associations between CA14 and specific diseases remain to be fully elucidated, its expression pattern and enzymatic function suggest several potential pathological contexts where it may play a role:

• Neurological Disorders:

  • Given its strong brain expression, CA14 may be involved in conditions where pH dysregulation contributes to pathology

  • Potential relevance to seizure disorders, as pH affects neuronal excitability

  • Possible contributions to neurodegenerative diseases where metabolic dysfunction occurs

• Liver Diseases:

  • High hepatic expression suggests potential roles in liver pathologies

  • May contribute to acid-base disturbances in hepatic encephalopathy

  • Possible involvement in conditions affecting bile production or secretion

• Metabolic Disorders:

  • As carbonic anhydrases participate in acid-base balance, CA14 may be relevant in metabolic acidosis or alkalosis

  • Potential contribution to local pH regulation in metabolically active tissues

• Cancer Biology:

  • Other carbonic anhydrases have established roles in tumor pH regulation

  • Altered expression in tumors of tissues normally expressing CA14 might have prognostic or therapeutic implications

Research approaches to investigate these disease associations could include:

• Expression analyses in patient-derived samples

• Genetic association studies looking for CA14 variants in relevant conditions

• Functional studies examining CA14 activity in disease models

• Therapeutic exploration of selective CA14 inhibitors in conditions where its activity may be detrimental

What are promising approaches for developing selective CA14 modulators?

Developing selective modulators for CA14 represents an important research direction with potential therapeutic applications. Several strategies show promise:

• Structure-Based Design:

  • Targeting unique features of the CA14 active site or adjacent regions

  • Leveraging structural differences between CA14 and other CA isoforms

  • Computational docking studies to identify selective binding pockets

  • Fragment-based drug discovery approaches focused on CA14-specific regions

• Exploiting Unique Inhibitor Sensitivity:

  • Building upon CA14's sensitivity to acetazolamide but resistance to sulfonamide

  • Using 3,4-Dihydroisoquinoline-2(1H)-sulfonamides as starting scaffolds for optimization

  • Developing hybrid molecules incorporating features of known selective inhibitors

• Targeting Membrane Localization:

  • Designing compounds that specifically access the extracellular catalytic domain

  • Exploiting the membrane microenvironment to enhance selectivity

  • Developing modulators that affect CA14 membrane insertion or trafficking

• Alternative Modulation Strategies:

  • Allosteric modulators that bind outside the conserved active site

  • Compounds affecting CA14 expression rather than direct activity

  • Peptide-based approaches targeting unique surface epitopes

• Screening Methodologies:

  • Differential screening against multiple CA isoforms to identify selective hits

  • Cell-based assays incorporating membrane localization

  • Activity-based assays under conditions optimized for CA14