WFDC2 Antibody

What is WFDC2 and what is its biological significance?

WFDC2 is a member of the Whey Acidic Protein (WAP) domain family of proteins, containing two evolutionarily conserved WAP domains connected by a short loop. Each WAP domain includes eight cysteine residues forming four disulfide bonds that are critical to the protein's structure and function . Initially identified as a transcript exclusively expressed in the epididymis and proposed as a specific marker for this tissue, WFDC2 has since been found to be highly expressed in multiple tissues including the respiratory tract and salivary glands .

The biological significance of WFDC2 appears to be related to host defense mechanisms. Similar to other WAP domain-containing proteins like Secretory Leukocyte Protease Inhibitor (SLPI) and elafin, WFDC2 is hypothesized to function as an antiproteinase involved in the innate immune defense of multiple epithelia . While its precise function remains under investigation, evidence suggests WFDC2 works in concert with related WAP domain-containing proteins to protect epithelial tissues against proteolytic enzymes released during inflammation .

Where is WFDC2 expressed in normal human tissues?

WFDC2 displays a distinctive expression pattern across various human tissues:

This distribution pattern suggests a role in the protection of mucosal surfaces against infection and inflammation, particularly in the respiratory and oral cavities .

How does WFDC2 differ from other WAP domain proteins?

While WFDC2 shares structural similarities with other WAP domain proteins, particularly SLPI, their tissue localization patterns reveal important differences. In the submandibular gland, WFDC2 and SLPI demonstrate almost mutually exclusive expression patterns - WFDC2 is predominantly found in ducts whereas SLPI is expressed in serous and mucous cells of the gland . This complementary distribution suggests different roles in innate defense mechanisms.

Both WFDC2 and SLPI are expressed in respiratory tissues, but their precise cellular localization differs, supporting the hypothesis that these proteins function in concert rather than redundantly in epithelial host defense . WFDC2 contains two WAP domains while the related protein elafin contains only one, which may contribute to functional differences in their antiproteinase activities or other immune functions .

What are optimal protocols for WFDC2 immunohistochemistry?

For effective immunohistochemical staining of WFDC2 in tissue sections, researchers should consider the following protocol based on validated approaches:

• Tissue Preparation: Fix tissues in formalin and embed in paraffin using standard procedures.

• Antigen Retrieval: Treat sections with 2% hydrogen peroxide in methanol to quench endogenous peroxidase activity .

• Primary Antibody: Use a well-characterized monoclonal antibody against human WFDC2, such as clone 12H5 at a dilution of 1:500 . This antibody has been validated for immunohistochemical analysis in multiple tissues.

• Controls: Include appropriate positive controls (e.g., submandibular gland sections) and negative controls (peripheral lung sections) based on known WFDC2 expression patterns .

• Detection System: Employ a sensitive detection system suitable for your specific research requirements.

It's important to note that staining intensity and distribution can vary between samples and tissue types. When examining respiratory epithelia, expect variability in both staining intensity and the number of positive cells .

How can I validate WFDC2 antibody specificity?

Validating antibody specificity is crucial for meaningful research results. For WFDC2 antibodies, consider these validation approaches:

• Western Blotting: Confirm that the antibody detects a band of the expected size (approximately 13 kDa for unmodified WFDC2, though glycosylated forms may appear around 24 kDa in cell lysates) .

• In Vitro Translation: Test the antibody against in vitro translated WFDC2 protein to confirm specificity .

• Expression Systems: Use HEK293T cells overexpressing wild-type and mutant WFDC2 to verify antibody recognition patterns .

• Glycosylation Analysis: Since WFDC2 is glycosylated, validate antibody recognition using treatments like tunicamycin (inhibits N-linked glycosylation) or digestion with glycosidases such as PNGase F or Endo H .

• Comparative Analysis: Compare staining patterns with other established WFDC2 antibodies and with known expression patterns from mRNA studies.

• Knockout/Knockdown Controls: If available, use WFDC2 knockout or knockdown samples as negative controls.

What considerations are important when designing experiments with WFDC2 antibodies?

When designing experiments involving WFDC2 antibodies, researchers should consider:

• Antibody Selection: Choose antibodies based on application needs - monoclonal antibodies like clone 12H5 have been validated for immunohistochemistry, western blotting, and ELISA applications .

• Species Reactivity: Confirm reactivity with your species of interest. Orthologs of WFDC2 exist in various vertebrates including canine, porcine, monkey, mouse, and rat models .

• Tissue-Specific Expression: Account for the differential expression of WFDC2 across tissues. For respiratory studies, focus on bronchial epithelium and submucosal glands rather than peripheral lung tissue .

• Isoform Recognition: Consider whether your antibody recognizes all relevant WFDC2 isoforms, as multiple transcripts may exist (e.g., mouse has at least two isoforms: NM_026323.2 and NM_001374655.1) .

• Disease State Influences: Be aware that WFDC2 expression patterns can change dramatically in disease states such as cystic fibrosis or cancer .

• Secretion Analysis: For studies of secreted WFDC2, design experiments to analyze both cell lysates and culture media, as secretion can be affected by mutations or disease states .

How is WFDC2 expression altered in respiratory diseases?

WFDC2 expression shows significant alterations in certain respiratory conditions, particularly in cystic fibrosis (CF). Research has revealed several key changes:

• Distribution Pattern: In small airways of CF patients, WFDC2 staining is more generally distributed throughout the hyperplastic epithelium compared to the more restricted pattern seen in non-CF airways .

• Inflammatory Exudates: The inflammatory mass within airway lumens of CF patients shows strong WFDC2 staining, suggesting potential involvement in the inflammatory response .

• Relationship to SLPI: Interestingly, while WFDC2 expression increases in CF tissues, SLPI expression is greatly reduced in the same samples, indicating a potential compensatory relationship between these related proteins .

• Genetic Variants: A homozygous missense variant (c.291C>G, p.C97W) in WFDC2 has been identified in families affected by respiratory conditions resembling primary ciliary dyskinesia (PCD) . This variant is associated with chronic inflammation, bronchiectasis, interstitial fibrosis, and Pseudomonas aeruginosa infections .

These alterations suggest that WFDC2 may play a role in the host defense mechanisms of the respiratory system, and dysregulation of its expression or function may contribute to the pathophysiology of respiratory diseases .

What is known about the p.C97W variant and its effect on WFDC2 function?

The p.C97W variant represents a significant mutation that impacts WFDC2 structure and function:

• Structural Impact: C97 is located in the second WAP domain and normally forms a disulfide bond with C109. The p.C97W mutation disrupts this critical disulfide bond formation .

• Evolutionary Conservation: Both C97 and C109 are highly conserved across vertebrates, suggesting functional importance .

• Protein Folding: The bulky side chain of the tryptophan (W) residue disrupts hydrogen bond formation near position 97 in the second WAP domain .

• Protein Dimerization: While wild-type WFDC2 dimers tend to exist in a 'cis' configuration, the p.C97W mutant predominantly exists in a 'trans-trans' configuration, where the second WAP domain is untangled and interacts with the other subunit .

• Secretion Defect: Most critically, while wild-type WFDC2 is readily secreted, the p.C97W variant is not secreted and shows reduced expression in cell lysates .

• Glycosylation Status: Despite secretion defects, both wild-type and p.C97W WFDC2 are glycosylated, indicating that the mutation does not impair this post-translational modification .

This variant appears to be a founder mutation in Korean populations and is associated with respiratory distress conditions that resemble PCD or cystic fibrosis .

What is the significance of WFDC2 in cancer research?

WFDC2 has emerged as an important marker and potential functional player in several cancer types:

• Ovarian Cancer: Multiple studies have reported that WFDC2 RNA and protein are overexpressed in ovarian tumors, particularly serous and endometrioid subtypes . Serum WFDC2 levels have been suggested as a sensitive marker for ovarian cancer .

• Lung Adenocarcinoma: WFDC2 is overexpressed in subgroups of lung adenocarcinomas . The majority of adenocarcinomas stain positively for WFDC2, suggesting potential diagnostic value .

• Other Lung Cancers: A significant minority of squamous cell, small cell, and large cell carcinomas exhibit focal WFDC2 staining, though there is no clear association with tumor grade .

• Re-expression in Carcinomas: WFDC2 re-expression in lung carcinomas may be associated with tumor type and warrants further investigation .

These findings suggest that WFDC2 may play an undefined role in carcinogenesis and/or tumor progression and could have utility as both a histological and serum marker for certain cancer types .

How can researchers study the relationship between WFDC2 structure and function?

Investigating structure-function relationships in WFDC2 requires sophisticated approaches:

• Computational Structural Analysis: Use prediction tools like AlphaFold2 or ColabFold to model the structures of wild-type and mutant WFDC2 . These models can reveal how mutations might disrupt disulfide bond formation and protein folding.

• Mutagenesis Studies: Employ site-directed mutagenesis to systematically alter key residues, particularly the conserved cysteines that form disulfide bonds. The QuikChange mutagenesis method has been successfully used for introducing mutations into WFDC2 .

• Dimerization Analysis: Investigate WFDC2 dimer formation using techniques like size exclusion chromatography or analytical ultracentrifugation, supported by computational predictions of dimeric structures .

• Structural Clustering: Use principal component analysis (PCA) and K-means clustering to analyze different potential conformations of WFDC2, as has been done to compare wild-type and p.C97W variants .

• Protease Inhibition Assays: Given that related WAP proteins function as protease inhibitors, design biochemical assays to test whether WFDC2 inhibits specific proteases relevant to respiratory inflammation.

• Glycosylation Analysis: Investigate the role of glycosylation in WFDC2 function using glycosidase treatments (PNGase F, Endo H) or inhibitors like tunicamycin .

What methodologies are most effective for studying WFDC2 in primary cells and tissues?

Studying WFDC2 in primary cells and tissues requires specialized approaches:

• Primary Cell Cultures: Establish primary human lung-derived epithelial cell cultures to study WFDC2 expression and regulation in a physiologically relevant context .

• Air-Liquid Interface Cultures: For respiratory studies, air-liquid interface cultures of tracheobronchial epithelial cells can be used to investigate WFDC2 expression during cell differentiation .

• Dual Immunostaining: Use immunohistochemistry with multiple markers on serial sections to understand the relationship between WFDC2 and other proteins of interest (like SLPI, MUC5AC, or SPLUNC1) .

• Proinflammatory Stimulation: Examine if WFDC2 expression, like SLPI and elafin, is regulated by proinflammatory stimuli in primary cell cultures .

• Ex Vivo Tissue Explants: Utilize ex vivo explant cultures from different tissue sources to maintain the structural complexity of the native tissue while allowing experimental manipulation.

• Tissue Microarrays: For comparing WFDC2 expression across multiple samples, consider techniques like the tissue microarray approach that has been used to examine WFDC2 in lung cancer specimens .

How can researchers investigate potential interactions between WFDC2 and other innate immunity proteins?

To explore the functional network of WFDC2 in innate immunity:

• Co-Immunoprecipitation: Use antibodies against WFDC2 to pull down potential interacting partners from tissue or cell lysates, followed by mass spectrometry identification.

• Proximity Ligation Assays: Apply this technique to detect protein-protein interactions in situ in tissue sections, which could reveal physiologically relevant associations between WFDC2 and other proteins.

• Comparative Expression Studies: Analyze the expression patterns of WFDC2 alongside other WAP domain proteins like SLPI in both normal and diseased tissues .

• Functional Complementation: In cells or tissues where SLPI expression is reduced (such as in CF), investigate whether WFDC2 upregulation compensates functionally .

• Microbial Challenge Models: Develop models where tissues or cells are challenged with relevant respiratory pathogens to determine if WFDC2 participates in antimicrobial defense, potentially in concert with other innate immunity proteins.

• Receptor Identification: Investigate whether WFDC2 interacts with specific cellular receptors, as has been found for some other innate immunity proteins.

Why might I observe variable WFDC2 staining patterns across samples?

Variability in WFDC2 staining can be attributed to several factors:

• Biological Variation: Even within the same tissue type, the intensity of WFDC2 staining and the number of positive cells can naturally vary between samples . This has been observed particularly in bronchial epithelium.

• Tissue-Specific Expression: WFDC2 expression is highly tissue-specific, with prominent expression in submucosal glands and ductal epithelia but minimal expression in peripheral lung .

• Disease State: Conditions like cystic fibrosis can dramatically alter WFDC2 expression patterns, causing more generalized distribution in the epithelium .

• Fixation Differences: Variations in tissue fixation times and conditions can affect antigen preservation and antibody accessibility.

• Antibody Specificity: Ensure your antibody recognizes the appropriate isoform and is not affected by post-translational modifications like glycosylation .

• Glandular Heterogeneity: Within individual glands, some regions may be negative for WFDC2 while others show positive staining .

What controls should be included in WFDC2 antibody experiments?

Proper experimental controls are essential for reliable WFDC2 research:

• Positive Tissue Controls: Include tissues known to express WFDC2, such as:

  • Submandibular or parotid salivary glands (ductal epithelium)

  • Bronchial submucosal glands (serous cells)

  • Nasal minor glands (ductular epithelium)

• Negative Tissue Controls: Include peripheral lung sections, which typically show no WFDC2 staining in alveolar epithelium .

• Cellular Expression Controls: Use HEK293T cells overexpressing wild-type WFDC2 as a positive control and untransfected cells as a negative control .

• Mutation Controls: When studying functional aspects, compare wild-type WFDC2 with variant forms like p.C97W to demonstrate functional differences .

• Comparative Protein Controls: When possible, include staining for related proteins like SLPI on serial sections to demonstrate specificity and relative distribution patterns .

• Secretion Controls: For studies of secreted protein, analyze both cell lysates and culture media to assess proper protein processing and secretion .

How can I optimize protocols for detecting mutant forms of WFDC2?

When working with mutant forms of WFDC2, consider these optimization strategies:

• Expression System Selection: HEK293T cells have been successfully used to express both wild-type and mutant WFDC2 for functional studies .

• Protein Tag Selection: Adding a tag (such as a 10xHIS tag) to the C-terminus of the protein can facilitate detection and purification without interfering with the signal peptide or WAP domains .

• Intracellular Localization: Use co-localization studies with markers like pEYFP-ER to determine if mutant proteins show altered subcellular distribution .

• Secretion Analysis: For mutants with potential secretion defects, analyze both cell lysates and culture media, with particularly careful handling of media samples to avoid protein degradation .

• Glycosylation Assessment: Use glycosidase digestion (PNGase F, Endo H) to determine if mutations affect glycosylation patterns .

• Antibody Selection: Choose antibodies that recognize epitopes unlikely to be affected by the mutation of interest. For the p.C97W variant, antibodies targeting the N-terminal region might be more reliable than those targeting the second WAP domain .