Recombinant Macaca mulatta Calcium-activated chloride channel regulator 1 (CLCA1), partial

What is CLCA1 and what are its primary functions in biological systems?

CLCA1 (Calcium-activated chloride channel regulator 1) is one of the major non-mucin proteins present in intestinal mucus. Initially mischaracterized as a calcium-activated chloride channel, CLCA1 is now recognized as having multiple significant physiological functions. CLCA1 acts as a secreted metalloprotease that can cleave mucus structural components, particularly MUC2, thereby regulating mucus dynamics and structure . Additionally, CLCA1 functions as a regulatory protein that modulates calcium-dependent chloride currents in a paracrine fashion by interacting with and stabilizing TMEM16A/Anoctamin1 on the cell surface .

CLCA1 plays critical roles in multiple physiological processes including the regulation of mucus production and secretion by goblet cells, tissue inflammation in innate immune responses, and potentially as a tumor suppressor . The protein is particularly important in the context of respiratory and intestinal biology, with implications for conditions such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis .

How is CLCA1 structurally organized and how does this relate to its function?

CLCA1 belongs to the CLCR (Calcium-activated chloride channel regulator) protein family and shares domain architecture similar to ADAM (a disintegrin and metalloproteinase) family proteins . The protein undergoes self-cleavage, resulting in N-terminal and C-terminal fragments with distinct functional properties.

The structural organization of CLCA1 includes:

• An N-terminal region containing the catalytic domain and von Willebrand domain type A (VWA)

• A C-terminal self-cleavage product that forms a disulfide-linked dimer

• Ability to form complex oligomeric structures, with evidence suggesting CLCA1 can form octamers through noncovalent bonds between N-termini

This complex structural arrangement enables CLCA1 to perform its dual roles: as a metalloprotease acting on mucus components and as a regulator of chloride channels. The N-terminal fragment containing the catalytic domain and VWA domain has been characterized as more catalytically active but less stable than the full protein, and can be identified in freshly prepared mucus samples .

What experimental techniques are most effective for studying recombinant CLCA1?

Research with recombinant CLCA1 benefits from multiple complementary approaches:

When working specifically with Macaca mulatta CLCA1, researchers should consider species-specific antibodies and validation of cross-reactivity with human CLCA1 antibodies if utilizing them.

What challenges exist in expressing and purifying functional recombinant Macaca mulatta CLCA1?

Expression and purification of functional recombinant CLCA1 present several significant challenges:

• Protein instability: Research has shown that certain fragments of CLCA1, particularly the catalytically active N-terminal product encompassing the catalytic domain with its von Willebrand domain type A (VWA), are unstable . This instability complicates purification efforts and may necessitate specific stabilization strategies.

• Complex quaternary structure: CLCA1 forms complex oligomeric arrangements, including disulfide-linked dimers and non-covalent octamers . Preserving these structures during recombinant expression and purification requires careful optimization of conditions.

• Post-translational modifications: CLCA1 is glycosylated , and these modifications may be critical for proper folding, stability, and function. Expression systems must be selected that can reproduce the appropriate post-translational modifications.

• Self-processing activity: CLCA1 undergoes autoproteolytic processing, which complicates the production of full-length protein . Researchers must decide whether to produce the individual fragments or develop strategies to control the self-cleavage process.

• Functional validation: Confirming that recombinant CLCA1 retains its dual functionalities (proteolytic activity and chloride channel regulation) requires multiple specialized assays and appropriate controls.

For Macaca mulatta CLCA1 specifically, the limited availability of species-specific reagents and the potential for structural differences from human CLCA1 may introduce additional challenges requiring careful comparative analysis.

How can researchers validate that recombinant CLCA1 maintains its native functions?

Validation of functional recombinant CLCA1 requires a multi-faceted approach targeting its known activities:

• Proteolytic activity assessment:

  • Cleavage assays using natural substrates such as MUC2

  • Zymography to detect general proteolytic activity

  • Site-directed mutagenesis of catalytic residues as negative controls

• Chloride channel regulation validation:

  • Patch-clamp electrophysiology to measure calcium-dependent chloride currents in cells exposed to recombinant CLCA1

  • Surface biotinylation assays to quantify TMEM16A surface expression after CLCA1 exposure

  • Co-immunoprecipitation to confirm CLCA1-TMEM16A interaction

• Structural integrity confirmation:

  • Western blotting under reducing and non-reducing conditions to verify disulfide-linked dimer formation

  • Native PAGE and size exclusion chromatography to confirm oligomerization states

  • Domain-specific antibody reactivity to verify proper folding

• Functional comparison with native CLCA1:

  • Side-by-side comparison with CLCA1 isolated from tissue sources

  • Mass spectrometry to verify correct processing and post-translational modifications

  • Comparison of kinetic parameters for proteolytic activity

A comprehensive validation protocol should include positive controls (native CLCA1), negative controls (catalytically inactive mutants), and dose-response relationships to establish physiological relevance.

What methodological approaches can resolve contradictions in CLCA1 functional studies?

Research on CLCA1 has evolved from initial misconceptions about its function as a chloride channel to current understanding of its dual roles. Resolving contradictions in the literature requires targeted methodological approaches:

• Precise protein fragment characterization:

  • Clearly define which CLCA1 fragment is being studied (full-length, N-terminal, C-terminal, or specific domains)

  • Use domain-specific antibodies to track different fragments in experimental systems

  • Report exact amino acid ranges for recombinant fragments

• Temporal considerations:

  • The unstable nature of the catalytically active N-terminal fragment necessitates careful timing of experiments

  • Fresh preparation of mucus samples is crucial for detecting unstable fragments

  • Time-course studies can reveal dynamic processing events that might be missed at single timepoints

• Comparative species studies:

  • Direct comparison of human and Macaca mulatta CLCA1 using identical experimental conditions

  • Chimeric proteins can identify species-specific functional domains

  • Analysis of sequence conservation in key functional domains

• Contextual dependencies:

  • Investigation of CLCA1 function in different cellular environments (intestinal vs. airway)

  • Examination of the impact of the inflammatory milieu on CLCA1 function

  • Consideration of interactions with tissue-specific factors

• Comprehensive controls:

  • Clca1^−/− animals or CLCA1-knockout cell lines provide crucial negative controls

  • Complementation experiments to restore function in knockout systems

  • Dose-response relationships to establish physiological relevance

How does the proteolytic activity of CLCA1 impact mucus properties and homeostasis?

CLCA1's metalloprotease activity plays a crucial role in regulating mucus properties through several mechanisms:

• MUC2 processing:

  • CLCA1 can cleave the N-terminal part of MUC2, a key structural component of intestinal mucus

  • This cleavage likely affects the polymerization and structural arrangement of the mucus network

  • Processing of MUC2 may alter mucus viscosity, penetrability, and protective functions

• Mucus structural regulation:

  • CLCA1 contributes to the regulation of mucus processing and structural arrangement

  • The complex formed by CLCA1's N-terminal and C-terminal fragments appears to be involved in this regulatory function

  • Oligomerization of CLCA1 may create a scaffold that influences mucus architecture

• Disease relevance:

  • CLCA1 induction correlates with mucus accumulation in airway diseases such as COPD and asthma

  • In Clca1^−/− animals, compensatory protease activity maintains mucus function, suggesting redundant mechanisms for mucus processing

  • Understanding CLCA1's proteolytic activity may lead to therapeutic approaches targeting mucus hypersecretion

• Experimental approaches to study the proteolytic effects:

  • Ex vivo mucus penetrability assays comparing wild-type and Clca1^−/− samples

  • Rheological measurements of mucus viscoelasticity after exposure to recombinant CLCA1

  • Microscopic visualization of mucus network structures using fluorescently labeled MUC2 before and after CLCA1 treatment

These findings suggest that CLCA1 functions as part of a complex regulatory system that maintains proper mucus composition and structure, with implications for both normal physiology and disease states.

How can researchers effectively investigate the regulation of CLCA1 enzymatic activity?

Understanding CLCA1 enzymatic regulation requires targeted experimental approaches focusing on multiple regulatory mechanisms:

• Proteolytic processing investigation:

  • Characterization of shorter versions of CLCA1 for enhanced enzymatic activity

  • Mass spectrometry to identify precise cleavage sites and resulting fragments

  • Site-directed mutagenesis of potential autoproteolytic sites to prevent self-processing

  • In vitro processing assays to identify conditions that affect self-cleavage

• Domain contribution analysis:

  • Creation of domain deletion or swap constructs to identify regulatory domains

  • Focus on the catalytic domain and von Willebrand domain type A (VWA), which appear crucial for proteolytic activity

  • Investigation of how the C-terminal fragment interacts with and potentially regulates the N-terminal region

• Structural and biochemical approaches:

  • Analysis of disulfide bond formation and its impact on oligomerization and activity

  • Investigation of calcium dependency using calcium chelators and varying calcium concentrations

  • Examination of potential allosteric regulators through binding assays and activity measurements

• Cellular context considerations:

  • Comparison of CLCA1 activity in different cellular environments (intestinal vs. airway)

  • Investigation of tissue-specific co-factors that might modulate CLCA1 function

  • Study of inflammatory mediators that might alter CLCA1 expression or activity in disease states

• Technological approaches:

  • Development of FRET-based biosensors to monitor CLCA1 activity in real-time

  • Cryo-EM studies of CLCA1 oligomeric complexes to understand structural basis of regulation

  • High-throughput screening for small molecule modulators of CLCA1 activity

These methodological approaches would provide comprehensive insights into how CLCA1 enzymatic activity is regulated, potentially revealing therapeutic targets for conditions characterized by mucus dysfunction.

What is the evidence linking CLCA1 to respiratory diseases and what methodologies can further elucidate these connections?

CLCA1 has significant implications for respiratory diseases through multiple mechanisms:

• Disease correlations and expression patterns:

  • Strong induction of CLCA1 has been correlated with mucus accumulation in COPD and certain types of asthma

  • CLCA1 may play critical roles in goblet cell metaplasia and mucus hypersecretion, common features of respiratory diseases

  • CLCA1 is also implicated in cystic fibrosis pathophysiology

• Mechanistic contributions:

  • CLCA1 activates calcium-dependent chloride currents through TMEM16A/Anoctamin1, affecting airway fluid secretion

  • As a protease, CLCA1 can cleave MUC2 and potentially other mucins, altering mucus properties

  • CLCA1 is involved in the regulation of tissue inflammation in innate immune responses

• Methodological approaches to investigate CLCA1 in respiratory disease:

  • Analysis of CLCA1 levels in bronchial biopsies or bronchoalveolar lavage from patients with respiratory diseases

  • Airway epithelial cell culture models to study CLCA1 effects on mucus production and chloride transport

  • Animal models of asthma or COPD using Clca1^−/− mice to determine disease-modifying effects

  • Development of specific inhibitors of CLCA1 proteolytic activity to test therapeutic potential

  • Precision-cut lung slices from human or Macaca mulatta tissue to study CLCA1 function in a complex tissue environment

• Translational implications:

  • Understanding CLCA1 regulation could aid development of drugs to facilitate mucus clearance

  • CLCA1 might serve as a biomarker for specific endotypes of respiratory diseases

  • Targeting CLCA1-TMEM16A interaction could represent a novel therapeutic approach for chloride transport dysfunction

Future research should systematically evaluate how Macaca mulatta CLCA1 compares to human CLCA1 in these disease-relevant mechanisms to validate its use in translational studies.

How does CLCA1 interact with TMEM16A/Anoctamin1 and what is the significance for chloride transport?

The interaction between CLCA1 and TMEM16A represents a crucial mechanism for regulating chloride transport:

• Mechanism of interaction:

  • Secreted CLCA1 activates calcium-dependent chloride currents by engaging TMEM16A on the cell surface in a paracrine fashion

  • CLCA1 stabilizes TMEM16A on the cell surface, leading to increased surface expression of this channel protein

  • This interaction results in enhanced calcium-dependent chloride currents through TMEM16A

• Physiological significance:

  • CLCA1 acts as the first identified secreted direct modifier of TMEM16A activity

  • This interaction establishes a unique mechanism to increase chloride currents without direct channel formation

  • The CLCA1-TMEM16A relationship suggests cooperative roles in tissues where both are expressed

• Experimental approaches to study this interaction:

  • Cell surface binding assays to quantify CLCA1-TMEM16A engagement

  • Surface biotinylation experiments to measure changes in TMEM16A surface expression

  • Patch-clamp electrophysiology to measure functional changes in calcium-dependent chloride currents

  • Mutagenesis studies to identify key interaction domains on both proteins

  • Live-cell imaging with fluorescently tagged proteins to visualize the dynamics of interaction

• Disease relevance:

  • Dysregulation of chloride transport is implicated in multiple diseases including cystic fibrosis, asthma, and COPD

  • Understanding the CLCA1-TMEM16A axis could provide novel therapeutic targets for these conditions

  • The paracrine nature of this interaction suggests potential for targeted biological therapies

Investigation of species-specific differences in this interaction between human and Macaca mulatta CLCA1 would be valuable for translational research using non-human primate models.

What comparative approaches can reveal functional differences between human and Macaca mulatta CLCA1?

Understanding the similarities and differences between human and Macaca mulatta CLCA1 is crucial for translational research. Several methodological approaches can address this:

• Sequence and structural analysis:

  • Detailed bioinformatic comparison of amino acid sequences, focusing on:

    • Catalytic domain conservation

    • Von Willebrand domain type A (VWA) structure

    • Autoproteolytic cleavage sites

    • Post-translational modification sites

  • Homology modeling to predict structural differences that might impact function

  • Analysis of evolutionary conservation across domains to identify critical functional regions

• Comparative biochemical characterization:

  • Side-by-side expression and purification of recombinant proteins from both species

  • Enzymatic activity assays using identical substrates

  • Oligomerization analysis using native PAGE and size exclusion chromatography

  • Post-translational modification mapping using mass spectrometry

• Functional cross-species testing:

  • Cross-species complementation experiments in knockout systems

  • Testing human and macaque CLCA1 on both human and macaque cell lines

  • Chloride channel regulation assessment for both proteins on TMEM16A

  • Proteolytic activity comparison using MUC2 from both species as substrates

• Domain swap experiments:

  • Creation of chimeric proteins with domains exchanged between human and macaque CLCA1

  • Functional testing of chimeras to identify domains responsible for any observed differences

  • Mutagenesis of non-conserved residues to determine their functional significance

These approaches would provide comprehensive insights into functional conservation and divergence between human and Macaca mulatta CLCA1, validating the translational relevance of macaque models for CLCA1-related research.

What protocols yield optimal results for expression and purification of recombinant Macaca mulatta CLCA1?

Optimal expression and purification of recombinant Macaca mulatta CLCA1 requires careful consideration of several factors:

• Expression system selection:

  • Mammalian expression systems (HEK293, CHO cells): Preferred for full-length CLCA1 to ensure proper folding and post-translational modifications, particularly glycosylation

  • Wheat germ cell-free systems: Successfully used for specific CLCA1 fragments, offering advantages for proteins toxic to mammalian cells

  • Bacterial systems: May be suitable for non-glycosylated domains but likely inadequate for full-length functional protein

• Construct design considerations:

  • Protein fragments vs. full-length: Consider expressing specific functional domains (e.g., N-terminal catalytic region) separately

  • Fusion tags: N-terminal tags preferable to avoid interference with C-terminal interactions

  • Protease sites: Include specific protease cleavage sites for tag removal

  • Signal sequences: Retain native signal sequence for secretion into media if using mammalian systems

• Purification strategy:

  • Two-step affinity purification: Recommended for higher purity

  • Size exclusion chromatography: Critical for separating monomeric, dimeric, and oligomeric species

  • Preservation of disulfide bonds: Use non-reducing conditions during certain purification steps

  • Stability considerations: Add protease inhibitors and optimize buffer conditions to minimize degradation of unstable fragments

• Quality control methods:

  • SDS-PAGE: Under both reducing and non-reducing conditions

  • Western blot: Using domain-specific antibodies

  • Mass spectrometry: To confirm identity and detect post-translational modifications

  • Functional assays: Proteolytic activity testing or chloride channel modulation assessment

• Storage and handling:

  • Stability testing: Determine optimal temperature, buffer conditions, and additives

  • Avoid repeated freeze-thaw cycles: Aliquot purified protein

  • Consider flash-freezing in liquid nitrogen: May better preserve activity than standard freezing

These optimized protocols should yield recombinant Macaca mulatta CLCA1 suitable for structural and functional studies.

How can researchers effectively analyze CLCA1 oligomerization and complex formation?

Analysis of CLCA1's complex oligomeric structure requires a multi-technique approach:

• Native gel electrophoresis:

  • Native PAGE has successfully revealed CLCA1 in large complexes with apparent mass >1 MDa

  • Use of domain-specific antibodies (N or C terminus–recognizing) in Western blotting after native PAGE helps identify composition of complexes

  • Comparison of recombinant CLCA1 with native CLCA1 from mouse and human samples provides validation

• Size exclusion chromatography (SEC):

  • Critical for separating various oligomeric states

  • Multi-angle light scattering (MALS) coupled with SEC provides absolute molecular weight determination

  • Analyzing fractions by SDS-PAGE under reducing and non-reducing conditions reveals disulfide-linked components

• Cross-linking studies:

  • Chemical cross-linking followed by mass spectrometry can identify interaction interfaces

  • Variable-length cross-linkers help determine spatial relationships between subunits

  • Photo-activated cross-linkers offer temporal control for capturing transient interactions

• Analytical ultracentrifugation:

  • Sedimentation velocity experiments provide information on size distribution and shape

  • Sedimentation equilibrium determines absolute molecular weights of complexes

• Structural biology approaches:

  • Negative stain electron microscopy for initial visualization of complexes

  • Cryo-electron microscopy for higher resolution structural determination

  • Small-angle X-ray scattering (SAXS) for solution structure and conformational states

• Functional correlation studies:

  • Analysis of how oligomerization state correlates with proteolytic activity

  • Investigation of whether different oligomeric forms interact differently with TMEM16A

  • Mutagenesis of residues at predicted interfaces to disrupt specific oligomeric states

Based on existing data, researchers should be prepared to analyze a range of structures, including octamers formed by noncovalent bonds between N termini, disulfide-linked C-terminal dimers, and the interaction between N and C terminal fragments .

What strategies are most effective for studying the dynamic proteolytic activity of CLCA1?

Investigating the dynamic proteolytic activity of CLCA1 requires specialized approaches addressing its unique characteristics:

• Substrate identification and validation:

  • Natural substrates: MUC2 N-terminal region has been identified as a CLCA1 substrate

  • Synthetic peptide libraries: To identify cleavage site preferences

  • Fluorogenic substrates: Development of FRET-based reporters for real-time activity monitoring

  • Proteomic approaches: N-terminomics to identify additional physiological substrates

• Activity assays optimized for CLCA1 characteristics:

  • Timing considerations: Account for the instability of the catalytically active N-terminal fragment

  • Fresh sample preparation: Critical for detecting unstable fragments in mucus samples

  • Temperature and pH optimization: Determine physiological conditions for optimal activity

  • Metal ion requirements: Test dependence on calcium and other divalent cations

• Kinetic analysis methodologies:

  • Progress curve analysis: To capture initial rates before product inhibition or substrate depletion

  • Stopped-flow spectroscopy: For rapid kinetics of substrate cleavage

  • Competitive substrate assays: To determine substrate preferences

  • Inhibitor studies: Using both broad-spectrum and specific metalloprotease inhibitors

• Structure-function approaches:

  • Domain deletion constructs: To identify regions critical for catalytic activity

  • Point mutations: Of predicted catalytic residues to confirm mechanism

  • Interaction studies: Between N-terminal and C-terminal fragments to assess regulatory effects

  • Oligomerization analysis: To correlate catalytic activity with oligomeric state

• In situ activity visualization:

  • Activity-based probes: For labeling active enzyme in complex biological samples

  • Zymography: Adaptation for CLCA1-specific activity detection

  • Live-cell imaging: With clevable fluorescent reporters to visualize activity in cellular context

These methodologies would provide comprehensive insights into CLCA1's dynamic proteolytic activity and its regulation in physiological and pathological contexts.

What emerging technologies could advance understanding of CLCA1 function and regulation?

Several cutting-edge technologies could significantly enhance CLCA1 research:

• Advanced structural biology approaches:

  • Cryo-electron microscopy: For high-resolution structures of CLCA1 complexes in different functional states

  • Hydrogen-deuterium exchange mass spectrometry: To map conformational dynamics and protein-protein interaction surfaces

  • AlphaFold and other AI-based structure prediction: To model species-specific structural features and guide experimental design

• Single-molecule techniques:

  • Single-molecule FRET: To observe conformational changes during activation and substrate binding

  • Optical tweezers: To measure force generation during proteolytic activity

  • Super-resolution microscopy: To visualize CLCA1-TMEM16A interactions at nanoscale resolution

• Genetic and genomic approaches:

  • CRISPR-Cas9 genome editing: To create precise mutations in endogenous CLCA1

  • Single-cell transcriptomics: To identify cell-specific responses to CLCA1 in complex tissues

  • Spatial transcriptomics: To map CLCA1 expression patterns in relation to its substrates and targets

• Advanced tissue models:

  • Organoids: Intestinal or airway organoids for studying CLCA1 in a physiologically relevant context

  • Microfluidic organ-on-chip models: To investigate CLCA1 function under flow conditions

  • 3D bioprinting: To create complex tissue architectures with defined CLCA1 expression

• High-throughput screening platforms:

  • Automated proteolytic activity assays: For identification of modulators

  • Phenotypic screening: In disease-relevant cell models

  • Fragment-based drug discovery: To identify binding pockets for small molecule modulation

• Computational approaches:

  • Molecular dynamics simulations: To investigate the dynamics of CLCA1-substrate interactions

  • Systems biology modeling: To integrate CLCA1 into broader mucus homeostasis networks

  • Machine learning: To predict species-specific functional differences between human and Macaca mulatta CLCA1

These technologies would provide unprecedented insights into CLCA1 biology and potentially reveal new therapeutic approaches for mucus-related disorders.

What are the most promising applications of CLCA1 research for therapeutic development?

CLCA1 research holds significant promise for therapeutic applications in several disease areas:

• Respiratory disease applications:

  • Mucus hypersecretion modulation: Targeting CLCA1's proteolytic activity could help address mucus accumulation in COPD and asthma

  • Chloride transport regulation: Modulating CLCA1-TMEM16A interaction could provide a novel approach for cystic fibrosis

  • Anti-inflammatory therapy: Given CLCA1's role in tissue inflammation, it represents a potential target for inflammatory airway diseases

• Gastrointestinal disease applications:

  • Mucus barrier modulation: As CLCA1 regulates mucus structure through MUC2 cleavage, it could be targeted to enhance barrier function in inflammatory bowel disease

  • Intestinal inflammation: CLCA1's involvement in innate immune responses makes it relevant for inflammatory conditions

• Oncology applications:

  • Tumor suppression: Evidence suggests CLCA1 may function as a tumor suppressor, opening possibilities for cancer therapeutics

  • Diagnostic marker: CLCA1 expression patterns could serve as biomarkers for specific cancer types

• Therapeutic modalities:

  • Small molecule inhibitors: Targeting CLCA1's metalloprotease activity

  • Biologics: Recombinant CLCA1 fragments or antibodies targeting specific domains

  • Gene therapy: Regulation of CLCA1 expression in disease contexts

  • Peptide-based therapeutics: Designed to modulate CLCA1-TMEM16A interaction

• Precision medicine approaches:

  • Patient stratification: CLCA1 expression or variant patterns may identify responders to specific therapies

  • Combination therapies: CLCA1-targeted approaches could complement existing mucus-modifying treatments

  • Biomarker development: CLCA1 levels or processing patterns as indicators of disease activity