CLCA1 Antibody

What is CLCA1 and what are its primary functions in normal physiology?

CLCA1 (Calcium-activated chloride channel regulator 1) is a major non-mucin protein found in intestinal mucus. Despite its name suggesting it functions as an ion channel, CLCA1 is actually a secreted protein that modulates calcium-dependent chloride currents by interacting with TMEM16A (Anoctamin1) . It is primarily expressed along the gastrointestinal tract under normal conditions and is highly abundant in the intestinal mucus layer . Functionally, CLCA1 plays crucial roles in regulating ion transport across epithelial cell membranes, influencing fluid secretion and mucosal hydration, particularly in the respiratory and gastrointestinal tracts .

What are the main structural domains of CLCA1 and how do they contribute to its function?

CLCA1 contains several distinct structural domains that contribute to its self-cleavage and regulatory functions:

• N-terminal zinc-dependent metallohydrolase domain with a conserved HExxE catalytic motif (similar to matrix metalloproteases)

• CAT/Cys domain - contains the metalloprotease activity responsible for self-cleavage

• VWA (von Willebrand factor type A) domain - involved in protein-protein interactions

• C-terminal domain - forms a relatively stable structure after cleavage

After synthesis, CLCA1 undergoes intracellular autocatalytic cleavage resulting in N-terminal fragments (various sizes including 85, 68, 53, and 31 kDa) and C-terminal cleavage products (72 kDa in humans, 45 kDa in mice). The N-terminal fragment is necessary and sufficient to activate calcium-dependent chloride currents through interaction with TMEM16A .

What are the recommended methods for detecting different forms of CLCA1 in Western blot experiments?

For optimal Western blot detection of CLCA1 and its processed forms, researchers should follow these guidelines:

The detection of multiple bands is expected due to CLCA1's autocatalytic processing. Analysis of both cellular and secreted fractions (culture media) may be necessary to fully characterize CLCA1 processing in experimental systems .

What procedures are recommended for immunohistochemical detection of CLCA1?

For effective immunohistochemical detection of CLCA1 in tissues, follow these technical recommendations:

Specific staining patterns should be validated against known expression profiles, with particular attention to goblet cell cytoplasmic localization in intestinal and respiratory tissues .

How does CLCA1 interact with TMEM16A at the molecular level?

The interaction between secreted CLCA1 and TMEM16A involves several molecular mechanisms:

• Direct physical interaction: The N-terminal fragment of CLCA1 (N-CLCA1) directly engages with TMEM16A on the cell surface. Flow cytometry binding assays show that biotinylated N-CLCA1 binds to intact cells expressing TMEM16A, and this binding is significantly reduced by pre-incubating cells with antibodies against TMEM16A's last extracellular loop .

• Surface stabilization mechanism: CLCA1 increases TMEM16A protein levels at the cell surface without changing total TMEM16A expression. Immunofluorescence studies demonstrate that cells exposed to secreted CLCA1 display robust staining for TMEM16A at the cell membrane, indicating that CLCA1 enhances TMEM16A surface expression rather than increasing protein production .

• Paracrine activation: Secreted CLCA1 can activate TMEM16A-dependent chloride currents in a paracrine fashion. Patch-clamp experiments confirm that cells exposed to conditioned medium from CLCA1-expressing cells exhibit enhanced calcium-dependent chloride currents .

• Domain specificity: The N-terminal fragment of CLCA1 containing the metalloprotease domain is necessary and sufficient for TMEM16A activation. In electrophysiology experiments, application of purified N-CLCA1 robustly activates chloride currents similar to those observed with full-length CLCA1 .

This interaction provides the molecular basis for how a secreted protein (CLCA1) can modulate ion channel activity without being an ion channel itself, solving a long-standing question in the field .

What are the contradictory findings regarding CLCA1's role in mucus production and how might these be resolved?

Research on CLCA1's role in mucus production has yielded contradictory findings that require careful interpretation:

Supporting CLCA1's role in mucus regulation:

• OVA-challenged CLCA1-deficient (Clca1-/-) mice showed decreased peri-vascular tissue inflammation, goblet cell hyperplasia, mucus production, and airway hyperresponsiveness

• Anti-mCLCA1 antibody treatment reduced airway inflammation, goblet cell numbers, and promoted goblet cell apoptosis

• CLCA1 antibody treatment significantly reduced the production of MUC5AC and IL-13 in bronchoalveolar lavage fluid

Contradicting CLCA1's role:

• In different studies, allergic responses produced by acute intranasal IL-13 instillation or OVA challenge were similar in Clca1-/- mice and their wild-type littermates

• siRNA knockdown of hCLCA1 gene expression in human lung epithelial cells failed to reduce MUC5AC mRNA level or protein production

• Intratracheal administration of IL-13 generated ~30-fold up-regulation of mCLCA1 transcripts without inducing CaCCs activity in wild-type mouse airways

Methodological approaches to resolve these contradictions:

• Use standardized animal models with identical genetic backgrounds and environmental conditions

• Conduct time-course studies to determine if CLCA1's role varies at different stages of disease development

• Investigate compensatory mechanisms that might mask CLCA1 deficiency effects

• Examine strain-specific differences in CLCA1 function

• Employ tissue-specific and inducible CLCA1 knockout models to avoid developmental compensation

These methodological refinements would help clarify the specific contexts in which CLCA1 influences mucus production and airway pathophysiology .

How can researchers validate the specificity of CLCA1 antibodies?

To ensure antibody specificity in CLCA1 detection, researchers should implement this comprehensive validation approach:

This multi-layered validation strategy ensures that experimental observations are genuinely attributable to CLCA1 rather than antibody cross-reactivity or non-specific binding.

What are the key considerations when designing experiments to study CLCA1 secretion and processing?

When designing experiments to study CLCA1 secretion and processing, researchers should consider these methodological factors:

• Expression systems selection:

  • HEK293T cells are well-established for CLCA1 expression studies

  • CHO-K1 cells have been used for truncated CLCA1 expression

  • Consider physiologically relevant models like primary intestinal or airway epithelial cells

• Secreted protein detection methods:

  • Analyze conditioned media from CLCA1-expressing cells via Western blot or ELISA

  • For co-culture experiments, wash cells thoroughly after transfection to prevent media carry-over

  • Use concentrated conditioned media for enhanced detection

• Processing kinetics analysis:

  • Limited proteolysis experiments reveal CLCA1 processing over time (10-120 minutes)

  • The 85-kDa full N-terminus and 53-kDa fragment (CAT/Cys+VWA domains) form relatively stable structures

• Domain-specific construct design:

  • Create truncated constructs encompassing specific domains:

    • CAT/Cys (aa 1-300)

    • CAT/Cys + VWA (aa 1-477)

    • Full N-terminus (aa 1-695)

• Functional readout selection:

  • Patch-clamp electrophysiology to measure calcium-dependent chloride currents

  • Flow cytometry to assess binding to cell surface proteins like TMEM16A

  • Immunofluorescence microscopy for co-localization studies

This experimental framework enables comprehensive analysis of CLCA1 secretion, processing dynamics, and functional consequences in both physiological and pathological contexts.

How do different applications compare in their sensitivity and specificity for CLCA1 detection?

Different detection methods for CLCA1 vary in their sensitivity, specificity, and applications:

The optimal method selection depends on the specific research question: WB for processing studies, IHC/IF for localization studies, flow cytometry for binding studies, and ELISA for quantification in biological samples .

How do expression patterns of CLCA1 differ between normal and disease states?

CLCA1 expression shows significant tissue-specific and disease-associated patterns:

Normal expression:

• Primarily expressed along the gastrointestinal tract under normal conditions

• Highly abundant in the intestinal mucus layer

• Low expression in normal airways

Disease-associated expression:

• Significantly upregulated in respiratory diseases such as asthma and COPD

• Associated with mucus overproduction in airway diseases

• N-terminal cleavage products detected in bronchoalveolar lavage fluid from asthmatic subjects

Optimal techniques for detecting expression differences:

• qRT-PCR:

  • Most sensitive for detecting transcript-level changes

  • Can quantify fold-changes in expression (e.g., 30-fold upregulation in IL-13 stimulated airways)

• Western blot analysis:

  • Detects protein-level changes and processing differences

  • Distinguishes between full-length and cleaved forms

  • Recommended for comparing mucus vs. epithelial expression

• Immunohistochemistry:

  • Visualizes cellular and spatial distribution changes

  • Particularly useful for detecting changes in goblet cell numbers and CLCA1 localization

  • Recommended for patient tissue samples from respiratory diseases

• Bronchoalveolar lavage analysis:

  • Detects secreted forms in airways

  • Particularly useful for clinical samples from asthma patients

These expression patterns make CLCA1 a potential biomarker and therapeutic target for respiratory diseases characterized by mucus hypersecretion.

What are the functional consequences of CLCA1 knockdown in different experimental systems?

The functional consequences of CLCA1 manipulation vary across experimental systems:

In mouse models:

• Contradictory findings in asthma models:

  • Some studies show Clca1-/- mice have decreased peri-vascular inflammation, goblet cell hyperplasia, mucus production, and airway hyperresponsiveness after OVA challenge

  • Other studies report similar allergic responses to IL-13 or OVA challenge in both Clca1-/- mice and wild-type littermates

• No significant differences observed in:

  • Basal short-circuit current

  • Amiloride-sensitive Na+ absorption

  • cAMP-dependent Cl- secretion

  • Activation of Ca2+-activated Cl- secretion

• No compensatory upregulation of other chloride channel candidates in knockout mice

In cell culture models:

• siRNA-mediated knockdown of TMEM16A (but not CLCA1) in HEK293T cells reduces CLCA1-dependent calcium-activated chloride currents

• Knockdown of hCLCA1 in human lung epithelial cells (NCI-H292) does not reduce MUC5AC mRNA or protein levels

Functional readouts affected by CLCA1 manipulation:

• Electrophysiology: CLCA1 activates calcium-dependent chloride currents in a paracrine manner

• Protein localization: CLCA1 increases TMEM16A at the cell surface without changing total protein levels

• Goblet cell biology: Anti-mCLCA1 antibody treatment promotes goblet cell apoptosis with increased Bax and decreased Bcl-2 expression

These variable outcomes highlight the complexity of CLCA1 biology and the importance of experimental context when interpreting knockdown studies.

What advanced techniques can be employed to study CLCA1-protein interactions?

Several sophisticated molecular techniques are valuable for investigating CLCA1 interactions:

• Proximity Ligation Assay (PLA):

  • Detects protein-protein interactions in situ with high sensitivity

  • Useful for confirming CLCA1-TMEM16A interactions in native tissues

  • Generates fluorescent spots only when proteins are within 40nm of each other

• Co-immunoprecipitation (Co-IP):

  • Effective for pulling down protein complexes containing CLCA1

  • Western blot analysis has confirmed successful immunoprecipitation of CLCA1 from cancer lysates

  • Use antibodies against different CLCA1 domains to identify domain-specific interactions

• Tetrameric protein labeling:

  • The technique used with biotinylated N-CLCA1 coupled to streptavidin-phycoerythrin

  • Creates a fluorescent tetramer with enhanced avidity toward binding partners

  • Flow cytometry analysis can confirm binding and competition with antibodies

• Biolayer Interferometry/Surface Plasmon Resonance:

  • Measures binding kinetics between purified CLCA1 and potential partners

  • Provides quantitative binding constants (Kd, kon, koff)

  • Requires purified recombinant proteins

• Crosslinking Mass Spectrometry:

  • Identifies protein-protein interaction interfaces at amino acid resolution

  • Chemical crosslinking preserves transient interactions

  • MS/MS analysis identifies crosslinked peptides

• BioID or APEX2 proximity labeling:

  • Fusion of CLCA1 with a biotin ligase or peroxidase

  • Labels proteins in close proximity in living cells

  • Mass spectrometry identifies labeled proteins, revealing the CLCA1 "interactome"

These techniques provide complementary information about CLCA1's binding partners, interaction dynamics, and functional complexes, particularly with TMEM16A and potential novel interaction partners .

What are common challenges in detecting CLCA1 and how can they be addressed?

Researchers face several technical challenges when studying CLCA1:

Addressing these challenges ensures more reliable and reproducible results in CLCA1 research across different experimental systems and applications.