BPIFA1 Antibody

What is BPIFA1 and where is it primarily expressed?

BPIFA1 is a glycoprotein highly expressed in the respiratory epithelium and submucosal glands of the upper airways in both mice and humans. It is secreted by the airway epithelium and functions in innate defense against pathogens. Specifically, BPIFA1 is abundantly expressed in the respiratory epithelium and Bowman's glands of the nasal passages in mice. In humans, it is primarily found in the epithelium of proximal airways . The protein plays a critical role in mucosal defense mechanisms and has been identified as part of the initial response to both bacterial and viral pathogens .

How does BPIFA1 function in airway defense?

BPIFA1 demonstrates multiple mechanisms of action in airway defense:

• Antimicrobial activity: BPIFA1 binds to both Gram-negative and Gram-positive bacteria, potentially limiting bacterial colonization of the airways .

• Antiviral function: BPIFA1 restricts influenza A virus (IAV) infection by inhibiting the binding and entry of viral particles into airway epithelial cells .

• Smooth muscle regulation: BPIFA1 suppresses airway smooth muscle (ASM) contractility by binding to and inhibiting calcium channels (specifically Orai1), which affects calcium influx and subsequent muscle contraction .

• Surfactant properties: The protein acts as a surfactant in the airways, potentially helping to maintain airway surface liquid homeostasis .

What is the clinical significance of BPIFA1 in respiratory diseases?

BPIFA1 levels are significantly reduced in sputum samples from asthmatic patients compared to healthy controls. This reduction correlates with asthma pathophysiology, as BPIFA1 normally suppresses airway smooth muscle contractility. In asthmatic conditions, the reduction of BPIFA1 may contribute to increased airway hyperresponsiveness .

Additionally, BPIFA1 secretion is highly modulated after influenza A virus infection. Mice deficient in BPIFA1 demonstrate more severe weight loss after infection, support higher viral loads, and show earlier virus spread to the peripheral lung, indicating a critical role in the initial phase of viral infection control .

What are the recommended approaches for generating BPIFA1-specific antibodies?

Based on the research literature, successful BPIFA1-specific antibodies have been generated using the following approach:

• Peptide selection: Choose peptide sequences with minimal sequence conservation between species (e.g., human and mouse) and no similarity with other BPIF family members.

• Multiple-epitope targeting: Generate two anti-peptide antibodies for each protein to ensure specificity and validation.

• For mouse BPIFA1: Effective epitopes include:

  • Peptide sequence 189-199: (AVKDNQGRIHL)

  • Peptide sequence 31-46: (GPPLPLNQGPPLPLNQ) - unique for mouse protein

• Affinity purification: Each individual peptide should be used to affinity purify the final antibodies to ensure specificity .

• Validation: Confirm antibody specificity through Western blotting and immunohistochemistry using appropriate positive and negative controls.

What are the optimal immunohistochemistry protocols for BPIFA1 detection?

The following immunohistochemistry protocol has been validated for BPIFA1 detection:

• Section preparation: Use 4-μm paraffin sections mounted onto SuperFrost Plus glass slides.

• Deparaffinization and rehydration: Standard protocols for deparaffinization followed by rehydration.

• Endogenous peroxidase blocking: Quench sections in 3% H₂O₂ in methanol for 20 minutes.

• Antigen retrieval: For BPIFA1, antigen retrieval may not be necessary, unlike for BPIFB1 which requires tri-sodium citrate buffer treatment.

• Blocking: Incubate sections in 100% normal goat serum for 30 minutes at room temperature.

• Primary antibody: Apply BPIFA1 antibody diluted 1:750 in normal goat serum and incubate overnight at 4°C.

• Secondary antibody: Use a biotin-labeled secondary antibody (e.g., goat anti-rabbit) followed by peroxidase enzymatic development.

• Development and counterstaining: Develop with NovaRed substrate (resulting in red staining) and counterstain with hematoxylin .

This protocol can be combined with Alcian blue staining for the detection of acidic mucus when studying BPIFA1 in relation to mucous cells.

How can researchers study BPIFA1's interactions with bacteria?

To investigate BPIFA1's interactions with bacteria, researchers can employ the following methods:

• BPIFA1-tagged protein binding assays: Generate tagged recombinant BPIFA1 proteins (full-length and various mutants) to visualize binding to different bacterial species.

• Bacterial binding comparison: Both human and mouse BPIFA1 proteins have been shown to bind to Gram-negative and Gram-positive bacteria, with no apparent differences between species in binding capability .

• Mutational analysis: Human BPIFA1 lacking residues 22-42 (the S18 region) shows impaired bacterial binding, suggesting this region is important for the binding activity of the protein .

• Disulfide bond analysis: Experiments with disulfide-bond mutant BPIFA1 proteins indicate that the disulfide bond is not critical for bacterial binding .

What cell culture models are appropriate for studying BPIFA1 function?

Several validated cell culture models for BPIFA1 research include:

• Human bronchial epithelial cultures (HBECs): Primary HBECs from healthy and asthmatic donors allow comparison of BPIFA1 expression and secretion in normal versus disease states .

• Mouse tracheal epithelial cell (mTEC) cultures: In vitro differentiated mTECs from wild-type and Bpifa1-/- mice provide an excellent model for studying BPIFA1's role in host response to pathogens such as nontypeable Haemophilus influenzae (NTHi) .

• Airway smooth muscle cells (ASMCs): These cells can be used to study the effects of BPIFA1 on calcium signaling and muscle contractility .

• IL-13 exposure model: Normal HBECs exposed to the asthma-associated Th2 cytokine IL-13 show decreased BPIFA1 levels, mimicking the asthmatic condition .

How can researchers effectively evaluate BPIFA1's role in viral infections?

To study BPIFA1's antiviral functions, researchers should consider:

• Transgenic mouse models: Develop and utilize BPIFA1-deficient mouse models to assess susceptibility to viral infection, weight loss, viral load, and virus spread to the peripheral lung .

• Viral binding assays: Compare viral particle binding to wild-type versus BPIFA1-deficient cells to determine if BPIFA1 inhibits the initial attachment of virus to epithelial cells .

• Nuclear import analysis: Assess nuclear import of viral ribonucleoprotein complexes in the presence or absence of BPIFA1 to understand its effect on viral entry mechanisms .

• Replication assessment: Measure viral replication levels in control versus BPIFA1-deficient cells to quantify the antiviral effect .

What is known about the directional secretion of BPIFA1?

Interestingly, BPIFA1 demonstrates bidirectional secretion from airway epithelial cells:

• Apical secretion: BPIFA1 is secreted into the airway lumen, where it can interact with inhaled pathogens and contributes to mucosal defense.

• Basolateral secretion: Research has shown that BPIFA1 is also secreted basolaterally from normal human bronchial epithelial cultures (HBECs). This basolateral secretion is significantly reduced in asthma-derived HBECs .

• Functional significance: Basolaterally secreted BPIFA1 suppresses airway smooth muscle contractility by binding to and inhibiting the Orai1 calcium channel, affecting calcium influx and subsequent muscle contraction .

This dual secretion pattern suggests that BPIFA1 has distinct roles depending on which side of the epithelium it is secreted.

How does BPIFA1 expression differ across tissue types?

BPIFA1 shows distinct localization patterns:

• Nasal passages: Highly expressed in the respiratory epithelium and Bowman's glands in mice .

• Proximal airways: Present in the epithelium of the proximal airways in both mice and humans .

• Cell-type specificity: BPIFA1 is primarily expressed in non-ciliated cells in the respiratory epithelium. Interestingly, during bacterial infection with NTHi, the pathogen appears to associate with multiple cell types of tracheal epithelium but not with BPIFA1-positive cells, suggesting these cells may have enhanced protection .

• Limited expression elsewhere: BPIFA1 exhibits limited expression outside of the respiratory tract .

How does BPIFA1 interact with calcium channels to affect smooth muscle contraction?

BPIFA1 regulates airway smooth muscle contractility through the following mechanisms:

• Orai1 binding: BPIFA1 directly binds to the Orai1 calcium channel, as demonstrated by co-immunoprecipitation experiments. It does not interact with other calcium channels such as TRPC3 .

• Co-localization: Using ground state depletion (GSD) super resolution microscopy, BPIFA1 and Orai1 have been shown to co-localize in ASMC plasma membranes after 1 hour of co-incubation .

• Molecular docking: Structural analysis suggests that histidines in BPIFA1's α6 helix fit into the highly conserved negatively charged regions of Orai1 .

• Calcium influx inhibition: BPIFA1 binding to Orai1 inhibits calcium influx in ASMCs, which results in decreased phosphorylation of myosin light chain and reduced smooth muscle contractility .

• Receptor internalization: Extended exposure to BPIFA1 (4 hours) decreases plasma membrane Orai1 levels by approximately 50%, suggesting BPIFA1 may also promote Orai1 internalization .

What molecular regions of BPIFA1 are critical for its various functions?

Several regions of BPIFA1 have been identified as functionally important:

• S18 region (G22-L42): This region of human BPIFA1 appears crucial for bacterial binding. When these residues are deleted, the protein loses its ability to bind to bacteria .

• Disulfide bond: Unlike the S18 region, the disulfide bond in BPIFA1 is not critical for bacterial binding, as disulfide-bond mutant proteins maintain binding capability .

• α6 helix: The histidines in this helix are believed to interact with Orai1 calcium channels, mediating BPIFA1's effect on calcium influx and smooth muscle contractility .

• Species-specific insertions: Mouse BPIFA1 contains a mouse-specific insertion in exon 2 (residues 31-46: GPPLPLNQGPPLPLNQ) that is not present in the human protein. This insertion can be used to generate mouse-specific antibodies .

What are the key challenges in studying BPIFA1's multiple functions?

Researchers face several challenges when investigating BPIFA1:

• Pleiotropic functions: BPIFA1 demonstrates multiple functions (antimicrobial, antiviral, surfactant, smooth muscle regulation), making it difficult to isolate and study individual mechanisms.

• Directional secretion complexity: The bidirectional secretion of BPIFA1 (apical and basolateral) adds complexity to understanding its complete physiological role .

• Species differences: While mouse and human BPIFA1 share homology, there are species-specific insertions and potential functional differences that must be considered when translating findings between models .

• Cell-type specific effects: BPIFA1 appears to have different effects on different cell types, and certain cells (BPIFA1-positive cells) may be more resistant to pathogen invasion .

How should researchers approach contradictory findings about BPIFA1?

When confronted with seemingly contradictory results regarding BPIFA1 function:

• Consider context-dependency: BPIFA1's effects may vary based on:

  • The specific pathogen being studied

  • The cell or tissue type examined

  • The presence of inflammatory mediators (e.g., IL-13)

  • The model system (in vitro vs. in vivo)

• Examine methodological differences: Variations in experimental approaches may explain disparate findings, including:

  • Different antibodies with potentially different epitope specificities

  • Various recombinant protein production methods

  • Different cell culture conditions

• Temporal considerations: BPIFA1's effects may change over time, as seen with the delayed internalization of Orai1 after prolonged BPIFA1 exposure .

• Concentration effects: The local concentration of BPIFA1 may significantly affect its biological activity, especially in different compartments (apical vs. basolateral) .