AMICA1 Antibody

What is AMICA1 and what cellular functions does it regulate in research models?

AMICA1 (Adhesion Molecule Interacting with CXADR Antigen 1), also known as JAML (Junction Adhesion Molecule Like), is a transmembrane protein belonging to the junctional adhesion molecule (JAM) subset of the immunoglobulin superfamily . The human AMICA protein consists of a 384 amino acid precursor that includes a 19 amino acid signal sequence, a 256 amino acid extracellular domain with two Ig-like domains, a 21 amino acid transmembrane segment, and a 98 amino acid cytoplasmic domain .

Unlike other JAM family proteins, AMICA1 does not contain a cytoplasmic PDZ-binding motif . It functions primarily in:

• Controlling leukocyte migration and activation through interaction with CXADR (Coxsackievirus and Adenovirus Receptor)

• Mediating the activation of gamma-delta T-cells in epithelial tissues

• Inducing downstream cell signaling through PI3-kinase and MAP kinases

• Promoting proliferation and production of cytokines and growth factors by T-cells

• Facilitating tissue repair through epithelial-immune cell interactions

AMICA1 is predominantly expressed on the surface of granulocytes and monocytes, with expression increasing during myeloid leukemia cell differentiation .

What are the optimal methodological approaches for detecting AMICA1 expression in different experimental systems?

The detection of AMICA1 expression can be accomplished through several methodological approaches, each with specific optimization requirements:

Western Blot (WB)

• Recommended dilutions: 1:1000-1:4000

• Positive detection in: HL-60 cells, Jurkat cells, L02 cells

• Observed molecular weight: 44 kDa (theoretical) or 50-70 kDa (observed due to glycosylation)

Immunohistochemistry (IHC)

• Recommended dilutions: 1:20-1:200

• Antigen retrieval: TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Positive detection in: human spleen tissue

Flow Cytometry

• Successfully detects AMICA1 in human monocytes

• Requires proper controls: isotype control antibody (e.g., IC003A) compared to specific AMICA1 antibody

• Protocol reference: "Staining Membrane-associated Proteins"

ELISA

• Direct ELISA methods show high specificity

• Cross-reactivity should be assessed (e.g., testing against recombinant mouse AMICA)

Researchers should titrate antibodies in their specific testing systems to achieve optimal results, as sample-dependent variations may occur .

How does AMICA1 structurally and functionally relate to other JAM family proteins in cellular adhesion studies?

AMICA1/JAML shares several structural and functional characteristics with the JAM family but also exhibits important differences:

Structural Comparison:

• Contains two extracellular immunoglobulin-like domains similar to other JAMs

• Lacks the cytoplasmic PDZ-binding motif that is characteristic of other JAM family proteins

• Shares only 18-20% amino acid sequence identity with the extracellular domains of human JAM-A, -B, -C, and JAM4

• Contains a motif in the extracellular domain that promotes dimerization, which is required for surface localization

Functional Differences:

• Unlike other JAMs, AMICA1 does not appear to interact homophilically (neutrophils adhere to immobilized CXADR but not to immobilized AMICA1)

• While other JAMs primarily mediate epithelial and endothelial cell layer interactions, AMICA1 specifically controls interactions between these layers and hemopoietic cells

• The membrane-proximal Ig-like domain of AMICA1 binds specifically to the membrane-distal Ig-like domain of CXADR, creating a unique binding interface

These structural and functional distinctions make AMICA1 a specialized member of the JAM family, particularly important in leukocyte-epithelial/endothelial interactions rather than in homotypic cell junctions.

How does AMICA1 expression correlate with cancer prognosis, particularly in lung adenocarcinoma (LUAD)?

Research demonstrates that AMICA1 expression has significant prognostic value in lung adenocarcinoma:

Expression Pattern and Diagnostic Value:

• AMICA1 is significantly downregulated in LUAD compared to normal tissues across multiple datasets (GSE116959, GSE32867, GSE43458, TCGA)

• ROC curve analysis revealed high diagnostic accuracy with AUC values of:

  • GSE116959: 0.927 (sensitivity 0.825, specificity 1.000)

  • GSE32867: 0.982 (sensitivity 0.948, specificity 0.914)

  • GSE43458: 0.797 (sensitivity 0.867, specificity 0.713)

  • TCGA database: 0.956 (sensitivity 0.932, specificity 0.907)

Clinical Correlation:

• Decreased AMICA1 expression correlates with higher T stage, M stage, and pathological stage in LUAD

• Kaplan-Meier survival analysis showed that patients with low AMICA1 expression had worse prognosis

• Multivariate Cox analysis confirmed AMICA1 expression level as an independent prognostic factor for LUAD patients

Functional Significance:

• In vitro experiments demonstrated that AMICA1 significantly suppresses LUAD cell proliferation

• AMICA1 expression was found to positively correlate with immune cell infiltration in LUAD

• Mechanistically, AMICA1 appears to play an important role in activating cGAS-STING signaling in LUAD

These findings collectively support AMICA1's potential as both a diagnostic and prognostic biomarker in LUAD, with functional implications for tumor suppression and immune response.

What mechanisms underlie AMICA1's role in immune cell transmigration across epithelial and endothelial barriers?

AMICA1/JAML facilitates immune cell transmigration through several specific molecular mechanisms:

Receptor-Ligand Interaction:

• AMICA1 on leukocytes directly interacts with CXADR (Coxsackievirus and Adenovirus Receptor) present in epithelial tight junctions and on endothelial cells

• This specific interaction is structurally defined: the membrane-proximal Ig-like domain of AMICA1 binds to the membrane-distal Ig-like domain of CXADR

• Unlike other adhesion molecules, AMICA1 does not interact homophilically with itself on other cells

Signaling Cascade:

• Upon binding to epithelial CXADR, AMICA1 triggers downstream cell signaling in leukocytes

• This signaling cascade involves:

  • Activation of PI3-kinase pathway

  • Engagement of MAP kinases

  • Subsequent cellular responses including proliferation and cytokine production

Gamma-Delta T-Cell Specific Functions:

• In epithelial tissues, AMICA1-CXADR interaction specifically mediates the activation of gamma-delta T-cells, which are crucial for tissue homeostasis and repair

• This activation results in:

  • T-cell proliferation

  • Production of cytokines and growth factors

  • Stimulation of epithelial tissue repair processes

Adhesion and Migration:

• AMICA1 mediates the adhesion of monocytes to endothelial cells

• It facilitates neutrophil migration across epithelial cell monolayers through CXADR binding

• The molecule contributes to the controlled transmigration of leukocytes within epithelial and endothelial tissues

These mechanisms collectively enable AMICA1 to regulate immune cell movement across tissue barriers, playing a critical role in immune surveillance and response to tissue damage.

How can researchers validate the specificity of AMICA1 antibodies for experimental applications?

Validating AMICA1 antibody specificity requires multiple complementary approaches:

Cross-Reactivity Testing:

• Perform direct ELISAs with recombinant proteins from multiple species

• Example: Testing human AMICA1 antibodies against recombinant mouse AMICA1 to confirm specificity

• Test against related JAM family proteins to ensure no cross-reactivity within the family

Multiple Detection Methods:

• Employ at least two different techniques (e.g., Western blot and flow cytometry)

• For Western blot: Confirm band size matches predicted molecular weight (44.3 kDa theoretical, though often 50-70 kDa due to glycosylation)

• For flow cytometry: Compare with proper isotype control antibodies

Positive and Negative Controls:

• Positive controls: Use cell lines known to express AMICA1 (e.g., HL-60 cells, Jurkat cells, and L02 cells for Western blot)

• Negative controls: Test in tissues or cells known not to express AMICA1

• Include knockout/knockdown validation when possible

Monoclonal Antibody Characterization:

• For strain-specific antibodies, test against multiple variants

• Example from research: mAb 5A6 (strain-specific, inhibitory) compared with mAb MRA-479A (N3-2D9) and MRA-480A (N3-1D7) showed different binding patterns to AMA1 variants

• Correlation analysis between different antibodies can reveal binding characteristics (e.g., Pearson's r = 0.89, r² = 0.79 between certain antibodies)

Application-Specific Optimization:

• Titrate antibody dilutions for each application:

  • Western Blot: 1:1000-1:4000

  • IHC: 1:20-1:200

  • Flow cytometry: Follow manufacturer protocols for conjugated antibodies

• Optimize antigen retrieval methods for IHC (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

This systematic validation approach ensures reliable and reproducible results in AMICA1 research applications.

What is currently known about AMICA1's interaction with the cGAS-STING signaling pathway in immunological research?

Research investigating AMICA1's interaction with the cGAS-STING pathway reveals emerging mechanisms in immune signaling and cancer biology:

AMICA1 and cGAS-STING in LUAD:

• In vitro experiments suggest that AMICA1 plays an important role in activating the cGAS-STING signaling pathway in lung adenocarcinoma (LUAD)

• This activation appears to contribute to AMICA1's tumor-suppressive effects in LUAD cells

Functional Significance:

• The cGAS-STING pathway is a critical innate immune sensor system that detects cytosolic DNA and triggers type I interferon responses

• AMICA1's involvement suggests it may link cell adhesion/migration functions with innate immune sensing

• This connection potentially explains how AMICA1 expression correlates with immune cell infiltration in LUAD tissues

Experimental Evidence:

• Research has demonstrated that AMICA1 "significantly suppressed the proliferation of LUAD cells and played an important role in activating cGAS-STING signaling"

• This finding connects AMICA1's expression levels with both immune function and tumor suppression mechanisms

Implications for Immunotherapy:

• Previous studies have shown that "treatment with anti-AMICA1 antibodies or AMICA1 knockdown can reduce the effectiveness of dendritic cell-based cancer immunotherapy"

• The interaction with cGAS-STING may partially explain this effect, as this pathway is increasingly recognized as important in anti-tumor immunity

• These findings suggest AMICA1 may function as a "potential novel immunotherapy target"

While the molecular details of how AMICA1 activates the cGAS-STING pathway remain to be fully elucidated, this interaction represents an important area for future research, particularly in understanding immune responses in cancer and potential therapeutic applications.

How do antibody design considerations address strain-specific epitope variations in AMICA1 research?

Addressing strain-specific variations in AMICA1 research requires sophisticated antibody design strategies:

Epitope Mapping and Strain Specificity:

• Research using protein microarrays has demonstrated that monoclonal antibodies (mAbs) show varying binding patterns to different AMA1 variants

• Example: mAb 5A6, a strain-specific inhibitory antibody, bound significantly to only five AMA1 variants above control levels

• Other mAbs like MRA-479A and MRA-480A showed highly correlated binding patterns (Pearson's r = 0.89)

High-Density Peptide Array Approach:

• Advanced peptide microarrays can be designed to represent diverse epitopes from multiple strains

• Example methodology: "331 full-length AMA1 sequences were derived from field samples (263 sequences) and publicly available sequences (68 sequences) and used to generate 16 amino acid long peptides which overlapped by 15 amino acids tiled across the length of AMA1"

• This approach allows for precise mapping of strain-specific antibody responses

Single Amino Acid Resolution:

• Mutation scan peptide microarrays can identify critical residues for antibody binding

• Example: "An additional peptide array was created to perform a pilot mutation scan analysis on the cluster 1 loop (c1L): 3D7 AMA1 positions 190–206 (MSPMTLDEMRHFYKDNK)"

• This approach revealed that "a single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy"

Practical Considerations for Researchers:

• For broad detection across variants: Target conserved epitopes in AMICA1

• For strain-specific studies: Use monoclonal antibodies with characterized epitope specificity

• For comprehensive analysis: Employ multiple antibodies targeting different epitopes

• For novel variant detection: Consider using antibody cocktails or developing custom antibodies based on sequence analysis

Understanding these strain-specific considerations is critical for accurate interpretation of experimental results, particularly in studies involving diverse patient populations or when comparing results across different research groups.

What are the critical parameters for optimizing immunoprecipitation protocols using AMICA1 antibodies?

Optimizing immunoprecipitation (IP) protocols for AMICA1 requires attention to several critical parameters:

Antibody Selection:

• Choose antibodies specifically validated for IP applications

• Consider the epitope location: antibodies targeting the extracellular domain may be more effective for IP of membrane-bound AMICA1

• Monoclonal antibodies often provide more consistent results, though polyclonal antibodies may capture more protein variants

Cell Lysis Conditions:

• AMICA1 is a transmembrane protein (44.3 kDa theoretical weight) requiring effective membrane solubilization

• Recommended lysis buffers:

  • For native conditions: Non-ionic detergents (0.5-1% NP-40 or Triton X-100)

  • For denaturing conditions: RIPA buffer with protease inhibitors

• Include appropriate protease inhibitor cocktails to prevent degradation

Antibody Binding Optimization:

• Incubation time: 2-4 hours at 4°C for optimal binding

• Antibody amount: Typically 2-5 μg per IP reaction (titrate for best results)

• Consider pre-clearing lysates with control IgG and protein A/G beads to reduce non-specific binding

Washing Stringency:

• Balance between maintaining specific interactions and reducing background

• Recommended washing buffer progression:

  • High-salt wash (500 mM NaCl)

  • Medium-salt wash (250 mM NaCl)

  • Low-salt wash (150 mM NaCl)

• Include 0.1% detergent in wash buffers to reduce non-specific interactions

Elution and Detection Considerations:

• For heavily glycosylated AMICA1 (observed at 50-70 kDa range), consider:

  • Using gradient gels (4-15%) for better separation

  • Including deglycosylation steps if studying core protein interactions

• Western blot detection should use antibodies targeting different epitopes than the IP antibody

Quality Control Measures:

• Always include negative controls (isotype-matched irrelevant antibody)

• Include positive controls (known AMICA1-expressing cells like HL-60 or Jurkat)

• Verify results with reciprocal IP if studying protein-protein interactions

These optimization parameters should be systematically evaluated to develop a robust IP protocol specific to your experimental system and research questions.

How does AMICA1 expression and function differ between tissue types in normal versus disease states?

AMICA1 exhibits distinct expression patterns and functional characteristics across different tissues and disease states:

Normal Tissue Expression:

• Primarily expressed on the surface of granulocytes and monocytes

• Found on polymorphonuclear leukocytes (PMN)

• Expressed in gamma-delta T-cells residing in epithelia

• Detected in human spleen tissue by immunohistochemistry

Expression in Cancer:

• Significantly downregulated in lung adenocarcinoma (LUAD) compared to normal lung tissue

• Expression decreases with progression of cancer, correlating with:

  • Higher T stage

  • Higher M stage

  • Advanced pathological stage

Functional Differences in Normal vs. Disease States:

Molecular Interaction Differences:

• In normal tissues: AMICA1-CXADR interaction promotes controlled immune cell migration and activation

• In cancer: Decreased AMICA1 expression appears to reduce immune cell infiltration

• In immune responses: AMICA1 activates cGAS-STING signaling, which may be impaired in disease states

Diagnostic and Prognostic Implications:

• AMICA1 expression has high diagnostic accuracy for LUAD with AUC values ranging from 0.797 to 0.982

• Expression levels serve as an independent prognostic factor in LUAD, with lower expression predicting worse outcomes

• Differential expression in immune cell subtypes suggests context-dependent functions