CLYBL Antibody

What is CLYBL and what are its primary enzymatic functions?

CLYBL is a mitochondrial enzyme with multiple enzymatic activities. It functions primarily as a citramalyl-CoA lyase, converting citramalyl-CoA into acetyl-CoA and pyruvate in the C5-dicarboxylate catabolism pathway. Additionally, CLYBL exhibits malate synthase activity (converting glyoxylate and acetyl-CoA to malate), malyl-CoA thioesterase activity, and β-methylmalate synthase activity (mediating conversion of glyoxylate and propionyl-CoA to β-methylmalate). The enzyme is part of detoxification pathways that prevent vitamin B12 poisoning by metabolites such as itaconate .

What applications are CLYBL antibodies validated for?

CLYBL antibodies are validated for several standard laboratory applications, including Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). Commercially available antibodies typically specify validation for particular applications. For instance, rabbit polyclonal antibodies are often validated for WB, ELISA, and IHC applications with human and mouse samples . When selecting an antibody, researchers should verify the specific applications for which each product has been validated through empirical testing.

What species reactivity should be considered when selecting a CLYBL antibody?

Most commercial CLYBL antibodies demonstrate reactivity with human samples, while some also cross-react with mouse samples. The high sequence conservation of CLYBL across mammals enables this cross-reactivity. Researchers should carefully evaluate the documented species reactivity in product specifications, particularly when studying CLYBL in non-human models. When available, species-specific validation data should be reviewed before application in experimental systems .

How should CLYBL antibodies be validated for specificity in experimental systems?

Comprehensive validation should include multiple approaches: (1) Comparing wild-type expression with CLYBL knockout cells/tissues, which should show absence of signal in knockout samples; (2) Using recombinant CLYBL protein as a positive control; (3) Performing peptide competition assays where pre-incubation with immunizing peptide should abolish signal; (4) Verifying detection of a band at the appropriate molecular weight (~40-42 kDa for human CLYBL); and (5) Comparing results from multiple antibodies targeting different CLYBL epitopes. For genetic models, CRISPR-engineered CLYBL knockout lines serve as essential negative controls, as demonstrated in studies with 3T3-L1 preadipocytes .

What are the optimal fixation and sample preparation methods for CLYBL detection in cellular studies?

For immunohistochemistry, paraformaldehyde fixation (4%) for 15-20 minutes at room temperature preserves mitochondrial morphology while maintaining CLYBL antigenicity. For subcellular localization studies, cell permeabilization with 0.3% Triton X-100 enables antibody access to mitochondrial compartments where CLYBL resides. When performing Western blotting, mitochondrial enrichment protocols significantly improve detection sensitivity. This involves cellular fractionation using sucrose gradient centrifugation followed by mitochondrial isolation before sample preparation for electrophoresis .

How can researchers distinguish between different CLYBL enzymatic activities in experimental systems?

To distinguish between CLYBL's multiple enzymatic activities, researchers should implement activity-specific biochemical assays. For citramalyl-CoA lyase activity, HPLC detection of acetyl-CoA and pyruvate formation from citramalyl-CoA substrate is recommended. For malate synthase activity, monitoring malate formation from glyoxylate and acetyl-CoA using coupled enzyme assays with malate dehydrogenase provides quantitative measurements. The malyl-CoA thioesterase activity can be assessed using Ellman's reagent (DTNB) to monitor CoA-SH production. These distinct assays, combined with site-directed mutagenesis of catalytic residues (e.g., D320A in human CLYBL), enable precise characterization of CLYBL's enzymatic versatility .

How does CLYBL loss-of-function impact cellular metabolism and vitamin B12 homeostasis?

CLYBL loss-of-function creates a complex metabolic phenotype characterized by accumulation of specific metabolites and altered vitamin B12 metabolism. CLYBL knockout cells show significant accumulation of both malyl-CoA (approximately 0.1 μM) and citramalyl-CoA (approximately 0.2 μM), representing 14-fold and 10-fold increases respectively compared to control cells. This metabolite accumulation leads to inhibition of methylmalonyl-CoA mutase (MCM), a key B12-dependent enzyme, resulting in decreased adenosylcobalamin levels and disrupted propionate metabolism. Isotope labeling experiments demonstrate that malate is a direct precursor of malyl-CoA, with 13C4-malate supplementation leading to 8% molar enrichment in 13C4-malyl-CoA in CLYBL knockout cells. Researchers investigating CLYBL function should incorporate metabolomic analyses focused on acyl-CoA species and vitamin B12 metabolites to fully characterize phenotypic consequences .

What is the molecular mechanism linking CLYBL to vitamin B12 metabolism?

The primary mechanism connecting CLYBL to vitamin B12 metabolism involves preventing inhibition of the B12-dependent enzyme methylmalonyl-CoA mutase (MCM). CLYBL deficiency leads to accumulation of malyl-CoA and citramalyl-CoA, metabolites that can inhibit MCM activity. Crystallography and spectroscopy studies have demonstrated that structurally similar metabolites like itaconyl-CoA can react with the 5'-deoxyadenosyl moiety of the B12 coenzyme, forming a biradical adduct that cannot be repaired by the B12 salvage pathway. This mechanism explains why individuals with CLYBL deficiency exhibit reduced circulating vitamin B12 levels despite the enzyme not directly participating in B12 metabolism. Researchers examining this relationship should employ enzymatic assays, metabolite supplementation experiments, and structural biology approaches to fully elucidate these molecular interactions .

How can CLYBL be used as a genomic safe-harbor for transgene expression?

CLYBL has been identified as a superior genomic safe-harbor locus for transgene expression in human stem cells. TALEN-mediated targeting of CLYBL intron 2 enables efficient transgene integration (38-58%) without disrupting critical cellular functions. Compared to conventional safe-harbor loci like PPP1R12C/AAVS1, CLYBL targeting results in up to 10-fold higher transgene expression and less perturbation of local gene expression. For optimal results, researchers should: (1) Design TALEN pairs targeting intron 2 of CLYBL; (2) Include 5' and 3' homology arms (~800bp each) flanking the transgene; (3) Use reporter systems with sensitive quantitative readouts; and (4) Verify genomic integration through Southern blot analysis using a probe specific for the integration site. This approach is particularly valuable for engineering human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) for sustained transgene expression during differentiation .

What role does CLYBL acetylation play in inflammatory responses?

CLYBL acetylation at lysine 154 (K154) serves as a crucial regulator of macrophage inflammatory responses. This post-translational modification participates in a positive feedback loop with AMPK (AMP-activated protein kinase), where TLR (toll-like receptor) activation triggers AMPK hypophosphorylation and CLYBL hyperacetylation. The deacetylase enzyme SIRT2 bridges AMPK phosphorylation and CLYBL acetylation states. Experimentally, blocking CLYBL-K154 acetylation restricts pro-inflammatory factor release, while CLYBL hypoacetylation decreases monocyte infiltration and alleviates cardiac remodeling in disease models. Researchers investigating inflammatory mechanisms should consider examining CLYBL acetylation status using acetylation-specific antibodies and implementing site-directed mutagenesis (K154R) to prevent acetylation at this critical residue .

How do genetic polymorphisms in CLYBL affect human metabolism?

CLYBL harbors polymorphic loss-of-function (LOF) mutations that are tolerated in seemingly healthy individuals, making it part of a group of approximately 150 LOF-tolerant genes in the human genome. These polymorphisms impact vitamin B12 metabolism, with affected individuals showing reduced circulating B12 levels. Unlike other LOF-tolerant genes (such as ALDH2 associated with alcohol flushing or G6PD deficiency conferring malaria protection), the physiological consequences of CLYBL deficiency remain less characterized. Researchers investigating these polymorphisms should incorporate genomic sequencing to identify variants, metabolomic profiling to assess downstream metabolic effects, and consider environmental interactions that may influence phenotypic expression in different populations .

What are the most effective strategies for monitoring CLYBL enzymatic activities in complex biological samples?

For accurate measurement of CLYBL enzymatic activities in complex biological samples, researchers should implement a multi-faceted approach: (1) Develop targeted metabolomic methods using liquid chromatography-high resolution mass spectrometry (LC-HRMS/MS) optimized for acyl-CoA detection; (2) Employ solid phase extraction techniques for acyl-CoA enrichment from cellular extracts; (3) Utilize isotope-labeled substrates (e.g., 13C4-malate) to trace metabolic flux through CLYBL-dependent pathways; (4) Implement enzymatic assays with Ellman's reagent (DTNB) to monitor CoA-SH production for thioesterase activity; and (5) Design specific HPLC methods to detect reaction products such as malate, β-methylmalate, and citramalate. These approaches provide complementary data on CLYBL function in native biological contexts .

How can researchers overcome challenges in detecting low-abundance CLYBL in certain tissues?

Detecting low-abundance CLYBL presents technical challenges that can be addressed through several methodological optimizations: (1) Implement subcellular fractionation to isolate mitochondria, where CLYBL is localized, significantly enriching signal-to-noise ratio; (2) Utilize signal amplification techniques such as tyramide signal amplification for immunohistochemistry or highly sensitive chemiluminescence substrates for Western blotting; (3) Consider tissues with high CLYBL expression (brown fat and kidney exhibit the highest expression levels) as positive controls; (4) Employ recombinant CLYBL protein for standard curves in quantitative assays; and (5) Use genetic overexpression systems to validate antibody specificity and sensitivity. These approaches collectively enhance detection capabilities for tissues where CLYBL expression is naturally low .

What considerations are important when using CLYBL antibodies for co-localization studies with other mitochondrial proteins?

When conducting co-localization studies involving CLYBL and other mitochondrial proteins, researchers should address several technical considerations: (1) Select fixation protocols that preserve mitochondrial morphology (4% paraformaldehyde is preferred over methanol fixation); (2) Verify antibody compatibility when performing multiplexed immunofluorescence, particularly when antibodies originate from the same host species; (3) Implement super-resolution microscopy techniques (STED, STORM, or SIM) to resolve mitochondrial subcompartments beyond the diffraction limit; (4) Include appropriate controls for spectral bleed-through and non-specific binding; and (5) Quantify co-localization using established coefficients (Pearson's, Manders') rather than relying solely on visual assessment. These methodological considerations enable precise determination of CLYBL's submitochondrial localization relative to other mitochondrial proteins .

How should researchers interpret contradictory results between different CLYBL antibodies?

When faced with contradictory results between different CLYBL antibodies, researchers should implement a systematic analysis protocol: (1) Compare antibody specifications including epitope locations, clonality, and validation parameters; (2) Verify antibody specificity using CLYBL knockout controls and recombinant protein standards; (3) Assess technical variables such as fixation methods, blocking conditions, and detection systems; (4) Consider isoform-specific detection capabilities of each antibody; and (5) Evaluate tissue-specific post-translational modifications that might affect epitope accessibility. Contradictory results often reveal important biological insights about protein conformation, processing, or interactions rather than simply representing technical failures. Resolution may require complementary non-antibody detection methods such as mass spectrometry or activity-based protein profiling .

What quantitative approaches provide the most reliable assessment of CLYBL expression across different tissues?

For reliable quantitative assessment of CLYBL expression across tissues, researchers should implement a multi-modal approach combining: (1) RT-qPCR with carefully validated reference genes specific to each tissue type; (2) Western blotting with recombinant protein standards for absolute quantification; (3) Mass spectrometry-based proteomics using stable isotope-labeled peptide standards; (4) Immunohistochemistry with automated image analysis for tissue distribution patterns; and (5) Single-cell RNA sequencing to resolve cell type-specific expression profiles. Published data indicate highest CLYBL expression in brown fat and kidney, providing important reference points. Integration of these complementary approaches provides a comprehensive assessment of CLYBL expression patterns, addressing limitations inherent to any single methodology .