ATP6V0A2 Antibody

What is ATP6V0A2 and what cellular functions does it perform?

ATP6V0A2 (ATPase, H+ Transporting, Lysosomal V0 Subunit A2) is a component of the V0 domain of the multisubunit vacuolar-type proton pump (H+-ATPase or V-ATPase). This protein is essential for acidification of diverse cellular components, including endosomes, lysosomes, clathrin-coated vesicles, secretory vesicles, and chromaffin granules. It is also found at high density in the plasma membrane of certain specialized cells . The V-ATPase consists of two domains: a peripheral V1 domain and an integral membrane V0 domain, with ATP6V0A2 being a critical component of the latter .

Research has demonstrated that ATP6V0A2 plays important roles in vesicular trafficking and secretory pathways. Loss-of-function mutations in ATP6V0A2 have been associated with autosomal recessive cutis laxa type 2 (ARCL2), a syndrome characterized by growth and developmental delay and redundant, inelastic skin . These mutations impair vesicular trafficking, tropoelastin secretion, and ultimately affect cell survival mechanisms .

What species reactivity is observed with commercially available ATP6V0A2 antibodies?

ATP6V0A2 antibodies exhibit varying degrees of species reactivity depending on the specific product and manufacturer. Based on available research resources, the following reactivity patterns have been observed:

When selecting an ATP6V0A2 antibody for research, it's important to verify the sequence homology across species. For example, the antibody ABIN6741595 recognizes an epitope located between amino acids 72-121 of human ATP6V0A2, which shows 100% identity in human, rat, and mouse, and 92% identity in dog and horse species . This high degree of conservation makes such antibodies valuable for comparative studies across multiple model organisms.

What applications are ATP6V0A2 antibodies validated for in research settings?

ATP6V0A2 antibodies have been validated for multiple research applications, with varying degrees of optimization for different experimental techniques:

For optimal results, researchers should follow manufacturer-recommended protocols and dilutions. Most ATP6V0A2 antibodies detect the full-length protein with an expected molecular weight of approximately 98 kDa . When using these antibodies for the first time in a new experimental system, appropriate validation through positive and negative controls is strongly recommended.

How do loss-of-function mutations in ATP6V0A2 affect vesicular trafficking and cellular morphology?

Loss-of-function mutations in ATP6V0A2 significantly impact vesicular trafficking pathways, leading to multiple cellular abnormalities. Research has demonstrated several key consequences:

First, ATP6V0A2 deficiency, either through siRNA knockdown or in ARCL2 patient-derived cells, results in distended Golgi cisternae as observed by electron microscopy . Confocal microscopy has revealed that cells with reduced ATP6V0A2 expression show dispersed, vesicular morphology of the Golgi apparatus compared to the continuous membrane structures in control cells .

Second, there is an accumulation of abnormal lysosomes and multivesicular bodies in ATP6V0A2-deficient cells . This suggests impaired degradative pathways and potential dysfunction in autophagy processes, which could contribute to the observed pathology in ARCL2.

Third, the loss of ATP6V0A2 function leads to abnormal protein trafficking, particularly affecting secretory proteins like tropoelastin (TE). Immunostaining of ARCL2 cells shows accumulation of TE in the Golgi and in large, abnormal intracellular and extracellular aggregates . This impaired secretion likely contributes to the connective tissue abnormalities observed in ARCL2 patients.

What mechanisms underlie decreased ATP6V0A2 mRNA expression in disease-associated mutations?

Research into ARCL2 has revealed important insights into how mutations affect ATP6V0A2 mRNA expression. The mechanisms depend largely on the type of mutation:

Premature termination codon (PTC) mutations in ATP6V0A2 lead to significantly reduced mRNA levels through the nonsense-mediated decay (NMD) pathway . This surveillance mechanism degrades mRNAs containing PTCs to prevent the production of truncated proteins. In studies of patient-derived fibroblasts, those with at least one mutation predicted to introduce a PTC had significantly reduced steady-state ATP6V0A2 mRNA levels compared to controls .

This NMD-mediated degradation was confirmed through cycloheximide (CHX) treatment experiments. When patient cells carrying mutations p.R133fsX135 and p.Q765X were treated with CHX, which blocks nonsense-mediated decay, the expression of these mutant mRNAs increased . This provides direct evidence that NMD contributes to the reduced mRNA levels observed in these cases.

Interestingly, not all frameshift mutations follow the same pattern. The mutation S27fsX54 was not expressed at the mRNA level even after CHX treatment, suggesting this particular mutation interferes with either transcription or RNA stability through a mechanism distinct from NMD .

In contrast, cells with missense mutations (such as p.P405L and p.R501I) showed ATP6V0A2 mRNA levels comparable to controls , indicating these mutations primarily affect protein function rather than mRNA stability.

How does ATP6V0A2 deficiency impact elastin processing and deposition?

ATP6V0A2 deficiency significantly disrupts elastin processing and deposition, which explains many of the connective tissue abnormalities observed in ARCL2 patients. The following mechanisms have been identified:

Immunostaining of ARCL2 patient-derived cells shows accumulation of tropoelastin (TE) in the Golgi apparatus and in large, abnormal intracellular and extracellular aggregates . This suggests a defect in the normal processing and secretion of this essential extracellular matrix protein.

Pulse-chase studies have confirmed impaired secretion and increased intracellular retention of tropoelastin in ATP6V0A2-deficient cells . This indicates that the protein is synthesized normally but becomes trapped within the cell's secretory pathway.

Insoluble elastin assays have demonstrated significantly reduced extracellular deposition of mature elastin in ARCL2 cells . This provides direct evidence linking ATP6V0A2 deficiency to abnormal elastic fiber formation, which would contribute to the inelastic skin phenotype characteristic of ARCL2.

Interestingly, while elastin processing is severely affected, fibrillin-1 microfibril assembly and secreted lysyl oxidase activity remain normal in ARCL2 cells . This suggests that ATP6V0A2 deficiency selectively affects specific components of the elastic fiber assembly pathway rather than causing a general secretory defect.

What are the optimal protocols for using ATP6V0A2 antibodies in various experimental techniques?

For Western Blotting:

• Recommended dilution: Typically 1:500 to 1:2000, though researcher should optimize for their specific sample and antibody

• Expected molecular weight: 98 kDa

• Sample preparation: Standard lysis buffers containing protease inhibitors

• Controls: Include positive control (tissues with known ATP6V0A2 expression) and negative control (ATP6V0A2 knockdown samples if available)

• Detection: Both chemiluminescence and fluorescence-based detection methods are suitable

For Immunohistochemistry:

• Recommended dilution: Typically 1:100 to 1:500 for paraffin-embedded sections

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is commonly effective

• Blocking: 5-10% normal serum from the species of the secondary antibody

• Incubation: Overnight at 4°C for primary antibody

• Detection system: Compatible with standard avidin-biotin or polymer-based detection systems

For ELISA:

• Coating concentration: 1-10 μg/ml of capture antibody

• Sample dilution: Serial dilutions recommended for unknown samples

• Standard curve: Recombinant ATP6V0A2 protein if available

• Incubation times: Typically 1-2 hours at room temperature or overnight at 4°C

How can researchers validate ATP6V0A2 antibody specificity in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research results. For ATP6V0A2 antibodies, consider these approaches:

• Peptide competition assay: Pre-incubate the ATP6V0A2 antibody with excess immunizing peptide (if available) before application to the sample. Specific staining should be blocked.

• Genetic knockdown/knockout validation: Use siRNA knockdown of ATP6V0A2 (as demonstrated in research studies ) or CRISPR-Cas9 mediated knockout, followed by Western blotting to confirm reduction or absence of the detected band.

• Multiple antibody validation: Use two or more antibodies targeting different epitopes of ATP6V0A2 to confirm consistent staining patterns.

• Cross-species reactivity assessment: If the antibody claims reactivity across multiple species, test samples from different species to confirm the expected molecular weight and expression pattern based on sequence homology.

• Immunoprecipitation followed by mass spectrometry: For definitive validation, immunoprecipitate ATP6V0A2 using the antibody and confirm the identity of the precipitated protein by mass spectrometry.

What experimental approaches can be used to study ATP6V0A2 function in vesicular trafficking?

Several experimental approaches have proven valuable for investigating ATP6V0A2 function:

• siRNA knockdown: Transient reduction of ATP6V0A2 expression can reveal immediate effects on cellular morphology and function. This approach has successfully demonstrated Golgi morphology changes in HeLa cells .

• Patient-derived cell studies: Fibroblasts from ARCL2 patients provide a physiologically relevant model to study long-term consequences of ATP6V0A2 deficiency .

• Confocal microscopy: Used to visualize Golgi morphology and protein localization through markers such as Giantin and GM130 .

• Electron microscopy: Provides ultrastructural details of organelle morphology, particularly useful for examining Golgi cisternae swelling and formation of abnormal vesicular structures .

• Pulse-chase experiments: Valuable for tracking protein synthesis, trafficking, and secretion. This method has demonstrated impaired tropoelastin secretion in ATP6V0A2-deficient cells .

• Insoluble matrix assays: Quantify extracellular deposition of matrix proteins like elastin to assess secretory pathway function .

• TUNEL staining: Detects apoptotic cells, useful for evaluating cell survival consequences of ATP6V0A2 deficiency. Increased apoptosis has been observed in ARCL2 cell cultures .

How do ATP6V0A2 mutations contribute to pathogenesis in autosomal recessive cutis laxa type 2?

ATP6V0A2 mutations cause ARCL2 through multiple cellular mechanisms that ultimately affect connective tissue development and maintenance:

Research has identified considerable allelic and phenotypic heterogeneity in ARCL2 patients. A cohort study of 17 patients found both homozygous and compound heterozygous mutations, with varying clinical severity . Notably, a missense mutation of a moderately conserved residue (p.P87L) led to unusually mild disease .

At the cellular level, loss of ATP6V0A2 function impairs proper acidification of cellular compartments, which disrupts normal processing and trafficking of secretory proteins. This is particularly evident in the abnormal glycosylation patterns observed in ARCL2 patients, with abnormal N- and/or mucin type O-glycosylation detected in all patients tested .

The most directly relevant mechanism to the cutis laxa phenotype involves tropoelastin processing. ATP6V0A2 deficiency leads to TE aggregation in the Golgi, impaired secretion, and reduced extracellular deposition of mature elastin . Since elastin provides skin elasticity, this explains the inelastic, redundant skin characteristic of ARCL2.

Additionally, increased apoptosis in elastogenic cells, demonstrated by TUNEL staining in ARCL2 cell cultures , further contributes to reduced elastin production and tissue integrity.

What research questions remain unanswered regarding ATP6V0A2 biology and pathology?

Despite significant advances in understanding ATP6V0A2 function, several important questions remain:

• Tissue-specific effects: Why do ATP6V0A2 mutations predominantly affect certain tissues (skin, skeletal) despite the protein's ubiquitous expression? Research into tissue-specific interacting partners or compensatory mechanisms could provide insights.

• Therapeutic approaches: Can pharmacological agents that modify vesicular pH or enhance protein secretion ameliorate cellular defects in ATP6V0A2 deficiency? Drug screening studies using patient-derived cells might identify potential therapeutic candidates.

• Functional domains: Which domains of ATP6V0A2 are critical for different aspects of cellular function? Structure-function studies correlating specific mutations with cellular phenotypes could elucidate this question.

• Developmental impact: How does ATP6V0A2 deficiency affect developmental processes, particularly during critical periods of elastogenesis? Developmental models might help address this question.

• Interaction with other V-ATPase subunits: How do mutations in ATP6V0A2 affect assembly and function of the complete V-ATPase complex? Co-immunoprecipitation and structural studies could provide insights into these molecular interactions.

How can ATP6V0A2 antibodies be used to advance research in related connective tissue disorders?

ATP6V0A2 antibodies can advance research in connective tissue disorders through several approaches:

• Comparative studies: Immunostaining of tissues from patients with various connective tissue disorders can identify whether ATP6V0A2 dysregulation is a common feature across multiple conditions, potentially revealing shared pathogenic mechanisms.

• Biomarker development: ATP6V0A2 expression or localization patterns might serve as diagnostic or prognostic biomarkers for certain connective tissue disorders. Antibody-based assays could enable such applications.

• Therapeutic response monitoring: In experimental treatments targeting vesicular trafficking or elastin secretion, ATP6V0A2 antibodies could help monitor cellular responses through analysis of protein localization and Golgi morphology.

• Genetic modifier studies: In connective tissue disorders with variable expressivity, ATP6V0A2 antibodies could help determine whether differences in protein expression or localization contribute to phenotypic variability.

• Drug screening: High-content screening approaches using ATP6V0A2 antibodies to monitor protein localization or Golgi morphology could identify compounds that normalize trafficking defects in models of connective tissue disorders.

What are common issues when using ATP6V0A2 antibodies and how can they be resolved?

When working with ATP6V0A2 antibodies, researchers may encounter several technical challenges:

• High background in immunostaining:

  • Increase blocking time and concentration (try 5-10% normal serum)

  • Optimize antibody dilution (typically starting at 1:500)

  • Include 0.1-0.3% Triton X-100 in wash buffers

  • Reduce primary antibody incubation time or temperature

• Multiple bands in Western blotting:

  • Ensure complete protein denaturation (heat samples at 95°C for 5 minutes)

  • Include protease inhibitors in sample preparation

  • Use freshly prepared samples

  • Consider that additional bands might represent isoforms, post-translational modifications, or degradation products

• Inconsistent immunostaining patterns:

  • Standardize fixation protocols (4% paraformaldehyde for 10-15 minutes is often optimal)

  • Optimize antigen retrieval conditions

  • Ensure consistent blocking and washing steps

  • Use positive control tissues with known ATP6V0A2 expression

• Weak or absent signal:

  • Increase antibody concentration

  • Extend incubation time (overnight at 4°C)

  • Ensure sample contains adequate ATP6V0A2 expression

  • Check secondary antibody compatibility and functionality

• Non-specific nuclear staining:

  • Increase blocking time and serum concentration

  • Pre-absorb antibody with nuclear extract

  • Use more stringent washing conditions

How should researchers quantify and interpret ATP6V0A2 expression data?

Accurate quantification and interpretation of ATP6V0A2 expression data requires attention to several methodological considerations:

For Western blot quantification:

• Always normalize ATP6V0A2 signal to appropriate loading controls (β-actin, GAPDH, or total protein staining)

• Use technical replicates (minimum of three) and biological replicates (different samples/independent experiments)

• Employ densitometry software with linear dynamic range

• Present data as fold-change relative to control conditions

• Consider that the 98 kDa molecular weight corresponds to the full-length protein

For immunohistochemistry/immunofluorescence quantification:

• Define objective scoring criteria (intensity scale, percentage of positive cells)

• Use automated image analysis when possible to reduce subjective bias

• Include multiple fields per sample (minimum 5-10)

• Account for background staining in quantification

• Consider both expression level and subcellular localization in interpretation

For mRNA expression analysis:

• Choose appropriate reference genes for normalization

• Be aware that mutations may affect mRNA stability through nonsense-mediated decay

• Use appropriate primers that detect all relevant transcripts

• Consider validating mRNA expression changes with protein-level analyses

When interpreting ATP6V0A2 expression data, particularly in disease contexts, remember that different mutations can affect expression through distinct mechanisms. While PTC mutations typically reduce mRNA levels through NMD, missense mutations may show normal mRNA expression despite altered protein function .