CLE16 Antibody

What is CLEC16A and why is it significant for autoimmune research?

CLEC16A (C-type lectin-like domain family 16A) is a protein that has been identified as a significant risk locus for multiple autoimmune diseases. Unlike other C-type lectin proteins, CLEC16A lacks an active carbohydrate recognition domain and instead functions as an E3-ubiquitin ligase involved in regulating autophagy and mitophagy . Genome-wide association studies have demonstrated that CLEC16A is associated with numerous autoimmune conditions including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, primary adrenal insufficiency, Crohn's disease, selective IgA deficiency, and rheumatoid arthritis . Additionally, CLEC16A has been linked to Common Variable Immunodeficiency Disorder (CVID), with the most strongly associated SNP (rs17806056) located in intron 19 of the CLEC16A gene . This widespread association with different autoimmune conditions suggests CLEC16A plays a fundamental role in immune regulation.

How does CLEC16A function in cellular processes?

CLEC16A plays critical roles in several essential cellular processes. Research has demonstrated that CLEC16A is involved in:

• Regulation of autophagy pathways

• Mitophagy (selective degradation of mitochondria)

• Endocytosis and intracellular trafficking

• Immune function regulation, particularly in B cells

• Biological processes including insulin secretion

Studies with Clec16a knock-down mice have shown a 54.4% reduction in total CD19+ B cells compared to control groups, suggesting a significant role in B cell development or maintenance . Additionally, CLEC16A localizes to endosomal membranes and forms a protein complex with Nrdp1 (an E3 ubiquitin-protein ligase), regulating mitophagy through the Nrdp1/Parkin pathway . The Drosophila homologue of CLEC16A (Ema) has been found to localize to endosomal and Golgi membranes, with Ema mutants showing defects in lysosomal degradation and protein trafficking .

What are the primary research applications for CLEC16A antibodies?

CLEC16A antibodies serve multiple research purposes in investigating autoimmune and neurodegenerative conditions:

• Detection and quantification of CLEC16A protein expression in different cell types and tissues

• Immunoprecipitation studies to identify CLEC16A protein interactions

• Immunohistochemistry/immunofluorescence to visualize CLEC16A cellular localization

• Western blotting to assess CLEC16A expression in various experimental conditions

• Flow cytometry to analyze CLEC16A expression in immune cell populations

• Validation of genetic knockdown or knockout models

These applications are particularly valuable for studying the relationship between CLEC16A variants and disease pathogenesis in conditions like CVID, where a genetic association has been established .

What protocols should be optimized when using CLEC16A antibodies for immunoprecipitation studies?

When conducting immunoprecipitation studies with CLEC16A antibodies, researchers should consider the following methodological aspects:

• Antibody selection: Choose antibodies that recognize native protein conformations and have been validated for immunoprecipitation applications.

• Cell lysis optimization: Since CLEC16A is associated with endosomal and Golgi membranes, use lysis buffers that efficiently solubilize membrane proteins while preserving protein-protein interactions. Buffers containing 1% NP-40 or 0.5% Triton X-100 with protease inhibitors are often suitable starting points.

• Pre-clearing lysates: Implement a pre-clearing step using protein A/G beads to reduce non-specific binding.

• Controls design: Include appropriate controls:

  • IgG control (same species as the CLEC16A antibody)

  • Input sample (pre-immunoprecipitation lysate)

  • Negative control (cells with CLEC16A knockdown)

• Co-immunoprecipitation partners: Based on current knowledge, optimize protocols to detect known CLEC16A interaction partners like Nrdp1, as CLEC16A forms a protein complex with this E3 ubiquitin-protein ligase to regulate mitophagy .

• Validation strategy: Confirm results using reciprocal immunoprecipitation with antibodies against the detected interaction partners.

How should researchers approach CLEC16A antibody validation for experimental use?

Proper validation of CLEC16A antibodies is crucial for experimental reliability:

• Specificity testing:

  • Western blot analysis using positive controls (tissues/cells with known CLEC16A expression)

  • Negative controls (CLEC16A knockout or knockdown samples)

  • Peptide competition assays to confirm binding specificity

• Cross-reactivity assessment:

  • Test antibody performance across relevant species if conducting comparative studies

  • Validate in tissues where CLEC16A is highly expressed (dendritic cells, natural killer cells, and B cells)

• Application-specific validation:

  • Perform separate validations for each application (Western blot, immunoprecipitation, immunohistochemistry, flow cytometry)

  • Optimize antibody concentrations for each application

• Reproducibility verification:

  • Compare results with different antibody clones targeting distinct CLEC16A epitopes

  • Document lot-to-lot variation if using polyclonal antibodies

• Functional validation:

  • Correlate antibody staining patterns with functional data from genetic models

  • Compare with published CLEC16A localization patterns (endosomal and Golgi membranes)

What are the optimal protocols for detecting CLEC16A in different immune cell populations?

Detecting CLEC16A in immune cells requires tailored approaches:

• B cell analysis: Since CLEC16A is highly expressed in B cells and has been implicated in B cell function , protocols should:

  • Include CD19 or CD20 co-staining for population identification

  • Incorporate fixation and permeabilization steps for intracellular CLEC16A detection

  • Consider cell subset analysis based on differentiation markers (naive vs. memory B cells)

• Dendritic cells and NK cells:

  • Use appropriate surface markers for population identification before CLEC16A staining

  • Optimize permeabilization conditions to preserve dendritic cell morphology

• Flow cytometry protocol:

  • Recommended fixation: 4% paraformaldehyde for 15 minutes

  • Permeabilization: 0.1% saponin or commercial permeabilization buffers

  • Blocking: 10% serum from the same species as the secondary antibody

  • Primary incubation: CLEC16A antibody (1:100-1:500 dilution, optimized)

  • Secondary detection: Fluorophore-conjugated secondary or directly conjugated primary

• Immunofluorescence microscopy:

  • Co-stain with organelle markers (endosomal, Golgi) to verify subcellular localization

  • Include Z-stack imaging to fully capture distribution patterns

• Controls:

  • Include isotype controls matched to CLEC16A antibody

  • Use CLEC16A knockdown samples as negative controls

How can CLEC16A antibodies be utilized to investigate the relationship between CLEC16A variants and autoimmune disease mechanisms?

Investigating the functional consequences of CLEC16A genetic variants requires sophisticated experimental approaches:

• Genotype-phenotype correlation studies:

  • Generate patient-derived cell lines with different CLEC16A risk variants

  • Use CLEC16A antibodies to quantify expression levels and localization patterns

  • Compare CLEC16A protein levels across different genotype groups

• Protein interaction analysis:

  • Employ co-immunoprecipitation with CLEC16A antibodies to identify differential protein interactions between wild-type and variant CLEC16A

  • Use proximity ligation assays to visualize and quantify CLEC16A interactions in situ

• Functional pathway analysis:

  • Assess autophagy and mitophagy efficiency in cells with different CLEC16A variants

  • Measure CLEC16A-dependent endosomal trafficking in variant vs. wild-type cells

  • Evaluate B cell development and function in the context of CLEC16A variants

• Structure-function studies:

  • Use epitope-specific antibodies to determine if CLEC16A variants affect protein conformation

  • Evaluate post-translational modifications across different variants

• Clinical correlation:

  • Stratify patient samples by CLEC16A genotype and correlate with:

    • CLEC16A protein expression levels

    • B cell counts and immunoglobulin profiles

    • Autoimmune comorbidities

This integrated approach can help elucidate how specific CLEC16A variants (such as rs17806056) contribute to disease pathogenesis in conditions like CVID .

What challenges might researchers face when interpreting CLEC16A antibody results in the context of B cell abnormalities?

Interpreting CLEC16A antibody results in B cell research presents several challenges:

• Distinguishing direct and indirect effects:

  • CLEC16A affects both B cell numbers and immunoglobulin profiles

  • Challenge: Determining whether alterations in B cell function are directly caused by CLEC16A dysfunction or secondary to other cellular processes

• Heterogeneity of B cell populations:

  • Different B cell subsets may express varying levels of CLEC16A

  • Challenge: Ensuring appropriate gating strategies when using flow cytometry for CLEC16A detection

• Context-dependent expression:

  • CLEC16A expression may change during B cell activation or differentiation

  • Challenge: Accounting for dynamic changes in expression levels during experimental design

• Correlation with clinical phenotypes:

  • In CVID patients, B cell abnormalities vary widely

  • Challenge: Correlating CLEC16A expression patterns with specific clinical subtypes

• Technical considerations:

  • Intracellular staining for CLEC16A requires cell permeabilization, which may affect B cell markers

  • Challenge: Optimizing protocols to preserve both surface marker and intracellular CLEC16A detection

How can CLEC16A antibodies be employed in mechanistic studies of autophagy and mitophagy dysregulation?

CLEC16A antibodies can be powerful tools for investigating autophagy and mitophagy mechanisms:

• Co-localization studies:

  • Use CLEC16A antibodies alongside markers for:

    • Autophagosomes (LC3B)

    • Mitochondria (TOM20, COXIV)

    • Endosomes (Rab5, Rab7)

    • Ubiquitinated proteins (FK1, FK2)

  • Quantify co-localization coefficients under different cellular conditions

• Mitophagy pathway analysis:

  • Investigate CLEC16A interactions with Nrdp1 and Parkin using:

    • Proximity ligation assays

    • FRET-based interaction studies

    • Co-immunoprecipitation followed by western blotting

• Dynamic trafficking studies:

  • Use live-cell imaging with fluorescently tagged CLEC16A antibody fragments

  • Track CLEC16A recruitment to damaged mitochondria following mitophagy induction

• Quantitative autophagy assays:

  • Measure LC3-I to LC3-II conversion in the presence of CLEC16A variants

  • Assess autophagic flux using chloroquine or bafilomycin A1 to block lysosomal degradation

  • Correlate changes with CLEC16A expression levels or localization patterns

• Therapeutic intervention assessment:

  • Use CLEC16A antibodies to monitor protein levels and localization following treatment with:

    • Mitophagy-inducing drugs

    • Autophagy modulators

    • Anti-inflammatory compounds

These approaches can help elucidate how CLEC16A dysfunction leads to the "attenuated CLEC16A activity" that has been implicated in both autoimmune and neurodegenerative disorders .

How should researchers design experiments to investigate CLEC16A's role in Common Variable Immunodeficiency (CVID)?

CVID research with CLEC16A antibodies requires specific experimental considerations:

• Patient stratification approach:

  • Genotype patients for the significant CLEC16A SNP (rs17806056)

  • Collect peripheral blood for B cell isolation and CLEC16A expression analysis

  • Categorize patients based on clinical subtypes (e.g., with/without autoimmunity, lymphoid hyperplasia)

• B cell analysis protocol:

  • Isolate peripheral blood B cells using magnetic separation or flow cytometry

  • Assess B cell subpopulations (naive, memory, transitional) using surface markers

  • Quantify CLEC16A expression levels in each subpopulation

  • Correlate expression with B cell function measurements (proliferation, antibody production)

• Functional assays:

  • Measure immunoglobulin production in response to stimulation

  • Assess autophagy and mitophagy efficiency in patient B cells

  • Evaluate B cell receptor signaling pathways

• Mechanistic investigations:

  • Transfect patient B cells with wild-type CLEC16A to assess rescue of phenotype

  • Use CRISPR-Cas9 to introduce CVID-associated CLEC16A variants in healthy B cells

  • Evaluate changes in B cell development and function

• Translational research:

  • Test mitophagy-inducing compounds on patient-derived B cells

  • Monitor CLEC16A expression and localization before and after treatment

  • Assess normalization of B cell functions following intervention

This comprehensive approach can help establish the mechanistic link between CLEC16A variants and the B cell abnormalities characteristic of CVID .

What controls and experimental design considerations are crucial when studying CLEC16A in autoimmune disease models?

Robust experimental design for CLEC16A studies in autoimmune models requires careful consideration of controls:

• Genetic control selection:

  • Include subjects with different CLEC16A genotypes:

    • Homozygous risk allele carriers

    • Heterozygous individuals

    • Non-risk allele carriers

  • Match controls for age, sex, and ethnicity

• Disease-specific considerations:

  • For multiple sclerosis studies: Include both relapsing-remitting and progressive MS patients

  • For type 1 diabetes: Stratify by disease duration and autoantibody status

  • For CVID: Categorize by clinical subtypes and immunological phenotypes

• Cellular controls:

  • Positive control: Cell types with known high CLEC16A expression (B cells, dendritic cells, NK cells)

  • Negative control: CLEC16A knockdown or knockout cells

  • Isotype controls for antibody specificity

• Experimental validation approaches:

  • Use multiple antibody clones targeting different CLEC16A epitopes

  • Confirm protein-level findings with mRNA expression analysis

  • Validate in both human samples and animal models

• Animal model selection:

  • Consider Clec16a conditional knockout models for tissue-specific studies

  • Use inducible knockdown systems to avoid developmental compensation

  • Include heterozygous models to mirror human genetic variation

• Pathway validation:

  • Include controls for autophagy/mitophagy pathway activity

  • Monitor both CLEC16A expression and its downstream effects

  • Assess potential compensatory mechanisms

How can CLEC16A antibodies be used to investigate the link between autoimmunity and neurodegeneration?

CLEC16A's emerging role in both autoimmune disorders and neurodegeneration presents unique research opportunities:

• Cross-disease comparison studies:

  • Use CLEC16A antibodies to compare expression and localization patterns in:

    • Autoimmune disease samples (MS, T1D, SLE)

    • Neurodegenerative disease samples (Parkinson's disease)

    • Matched healthy controls

  • Identify common and distinct CLEC16A-related mechanisms

• Mitochondrial dysfunction analysis:

  • Assess CLEC16A-dependent mitophagy in neural and immune cells

  • Measure mitochondrial health parameters in cells with different CLEC16A variants

  • Correlate findings with cellular stress responses and inflammatory markers

• Inflammatory signaling investigation:

  • Use CLEC16A antibodies to study interactions with SOCS1 (suppressor of cytokine signaling 1)

  • Evaluate how these interactions affect inflammatory signaling in different cell types

  • Test anti-inflammatory interventions on CLEC16A-dependent pathways

• Translational research approach:

  • Test mitophagy-inducing drugs in both immune and neuronal cells

  • Monitor CLEC16A expression, localization, and function

  • Assess improvement in cellular phenotypes relevant to both disease categories

• Biomarker development:

  • Develop assays to measure CLEC16A protein levels in accessible biospecimens

  • Correlate CLEC16A expression with disease progression in longitudinal studies

  • Evaluate CLEC16A as a potential predictive marker for treatment response

This integrated approach can help elucidate how CLEC16A dysfunction contributes to the shared pathological mechanisms underlying both autoimmunity and neurodegeneration .

What emerging technologies can enhance CLEC16A antibody-based research?

Several cutting-edge technologies can advance CLEC16A research:

• Mass cytometry (CyTOF):

  • Enables simultaneous detection of CLEC16A alongside dozens of other markers

  • Allows comprehensive immune cell phenotyping in patient samples

  • Can correlate CLEC16A expression with multiple functional parameters

• Super-resolution microscopy:

  • Provides nanoscale visualization of CLEC16A localization

  • Enables precise mapping of CLEC16A in relation to subcellular structures

  • Can detect subtle changes in localization patterns caused by disease-associated variants

• Single-cell proteomics:

  • Measures CLEC16A expression at single-cell resolution

  • Identifies rare cell populations with altered CLEC16A expression

  • Captures cellular heterogeneity missed by bulk analysis

• Proximity labeling techniques:

  • BioID or APEX2 fusion with CLEC16A to identify proximal proteins

  • Maps the complete CLEC16A interactome under different conditions

  • Discovers novel interaction partners in disease-relevant contexts

• CRISPR-based screening:

  • Identifies genes that modify CLEC16A expression or function

  • Discovers synthetic lethal interactions with CLEC16A variants

  • Uncovers potential therapeutic targets

• Patient-derived cellular models:

  • iPSC-derived immune cells or neurons with native CLEC16A variants

  • Organoid systems to study CLEC16A in complex tissue environments

  • Disease-specific cellular phenotypes for drug screening

How should researchers interpret contradictory findings in CLEC16A expression studies?

Resolving contradictory findings in CLEC16A research requires systematic analysis:

• Antibody-related variables:

  • Different antibodies may recognize distinct CLEC16A epitopes or isoforms

  • Solution: Use multiple validated antibodies targeting different regions

  • Document complete antibody information (clone, lot, validation method)

• Cell type and context differences:

  • CLEC16A expression and function may vary across cell types

  • Solution: Clearly define cell populations and activation states

  • Account for potential compensatory mechanisms in different tissues

• Genetic variation impact:

  • CLEC16A variants may affect expression in tissue-specific ways

  • Solution: Document subject genotypes and correlate with expression data

  • Consider both cis and trans genetic effects on CLEC16A expression

• Technical considerations:

  • Sample preparation methods may affect CLEC16A detection

  • Solution: Standardize protocols and include technical replicates

  • Use complementary techniques (protein and mRNA analysis)

• Biological complexity:

  • CLEC16A function involves multiple pathways with potential feedback mechanisms

  • Solution: Design time-course experiments to capture dynamic changes

  • Use systems biology approaches to model pathway interactions

• Analysis framework:

  • Create a structured evaluation of contradictory findings:

    • Document methodological differences between studies

    • Assess sample sizes and statistical power

    • Consider publication bias and replication attempts

    • Evaluate biological plausibility of different interpretations

What standards should be established for CLEC16A antibody-based research to ensure reproducibility?

To enhance reproducibility in CLEC16A research, the following standards should be implemented:

• Antibody reporting standards:

  • Document complete antibody information:

    • Clone/catalog number

    • Lot number

    • Validation method and results

    • Species reactivity

    • Epitope information

  • Include all antibody dilutions and incubation conditions

• Experimental design requirements:

  • Pre-register research protocols when possible

  • Include both technical and biological replicates

  • Document sample sizes with power calculations

  • Use randomization and blinding where appropriate

• Validation criteria:

  • Establish minimum validation requirements for CLEC16A antibodies

  • Include positive and negative controls in all experiments

  • Verify findings with complementary techniques

  • Test for cross-reactivity with related proteins

• Data sharing practices:

  • Share raw data and analysis code in public repositories

  • Document exact image acquisition parameters

  • Provide complete protocols with all buffer compositions

  • Include representative images of all experimental conditions

• Methodological transparency:

  • Report both positive and negative results

  • Document all tested conditions, including unsuccessful approaches

  • Clearly state limitations of the study

  • Discuss alternative interpretations of the data

• Quantification standards:

  • Use appropriate statistical tests with correction for multiple comparisons

  • Report effect sizes alongside p-values

  • Document image analysis parameters and thresholds

  • Include objective quantification methods for microscopy data

By adhering to these standards, researchers can build a more reliable knowledge base about CLEC16A function and its role in disease pathogenesis.