LMBRD1 Antibody

What is LMBRD1 and why is it important to study?

LMBRD1 encodes the LMBD1 protein, a predicted lysosomal cobalamin transport protein with 9 putative transmembrane domains. It plays critical roles in multiple cellular functions. Mutations in the LMBRD1 gene cause the rare inborn cblF defect of cobalamin metabolism, characterized by free cobalamin accumulation in lysosomes and loss of mitochondrial succinyl-CoA synthesis and cytosolic methionine synthesis . Children with these mutations suffer from heart defects, developmental delay, and megaloblastic anemia. Additionally, LMBRD1 functions as an insulin receptor-specific adaptor for endocytosis, regulating insulin signaling . Complete loss of Lmbrd1 function causes early embryonic lethality in mice, highlighting its essential role in development .

What experimental techniques are commonly used to detect LMBRD1 protein expression?

Multiple techniques can effectively detect LMBRD1 expression in experimental settings:

• Immunofluorescence microscopy: Fixed embryonic tissues or cells can be labeled using anti-LMBRD1 antibodies (such as rabbit anti-LMBRD1 antibody at 1:500 dilution), followed by fluorophore-conjugated secondary antibodies (e.g., anti-rabbit Cy3 at 1:5000) .

• Western blot analysis: Particularly useful for detecting LMBRD1 in fractionated cell lysates and determining its association with vesicular structures.

• Whole mount in situ hybridization: For analyzing expression patterns in intact embryos, as demonstrated in studies of C57BL/6 wild type embryos where Lmbrd1 was found ubiquitously expressed with strong signals in the primitive streak and extraembryonic tissues at E7.5 .

• Real-time PCR: For quantitative analysis of Lmbrd1 gene expression, allowing comparison between wild-type and heterozygous models .

• ELISA: Commercial ELISA kits are available for quantitative measurement of LMBRD1 protein levels .

What is the expression pattern of LMBRD1 during embryonic development?

LMBRD1 displays a dynamic expression pattern during embryonic development. Whole mount in situ hybridization analysis in C57BL/6 wild type embryos shows that Lmbrd1 is ubiquitously expressed with strong signals in the primitive streak and in extraembryonic tissues at E7.5 . During further development, Lmbrd1 expression becomes strongest in the neuronal fold at E8.5 . This expression pattern correlates with its critical function in embryonic development, as Lmbrd1 deficient mice exhibit early embryonic lethality. While the initial formation of the proximal-distal axis remains unaffected in early embryonic stages of these mice, the initiation of gastrulation is disturbed, as evidenced by altered expression patterns of even skipped homeotic gene 1 and fibroblast growth factor 8 .

How should LMBRD1 antibodies be validated for research applications?

Proper validation of LMBRD1 antibodies is critical for reliable research results:

• Specificity testing: Compare staining patterns between wild-type and knockout/knockdown models. In Lmbrd1-deficient embryos, antibody staining should be absent or significantly reduced compared to wild-type counterparts .

• Multiple detection methods: Validate antibody performance across different applications (Western blot, immunofluorescence, immunoprecipitation) to ensure consistent results.

• Immunoprecipitation validation: Confirm that the antibody can successfully pull down LMBRD1 and its known interacting partners such as adaptor protein-2 (AP-2) and clathrin heavy chain (CHC) .

• Cross-reactivity assessment: Test for potential cross-reactivity with related proteins, particularly other members of the limb region 1 domain-containing family.

• Comparison of commercial sources: When possible, compare results obtained with antibodies from different vendors or different clones to confirm consistency of findings.

How does LMBRD1 participate in clathrin-mediated endocytosis of the insulin receptor?

LMBRD1 functions as a specific adaptor in the clathrin-mediated endocytosis of the insulin receptor (IR) through several molecular mechanisms:

• LMBRD1 contains two highly conserved AP-2 binding motifs in its cytosolic loop between transmembrane domains 5 and 6: the μ2 subunit binding motif YXXΦ and the α-appendage binding motif WXX(F/W) .

• LMBRD1 co-immunoprecipitates with both AP-2 and clathrin heavy chain (CHC), confirming its direct interaction with the endocytic machinery .

• Mutation studies have demonstrated that altering either the AP-2 μ2 subunit binding motif (Y233A) or the α-appendage binding motif (W295A) substantially decreases LMBRD1's binding ability to AP-2 .

• lmbrd1 knockdown hinders the internalization of the IR, allowing it and its downstream signaling molecules (particularly Akt) to remain activated for longer periods .

• Rescue experiments with wild-type LMBRD1 effectively reduce IR phosphorylation status compared to the AP-2 binding motif mutants, indicating these domains are critical for LMBRD1's function in IR internalization .

The specificity of LMBRD1 for IR endocytosis is notable, as it does not appear to influence the internalization of other receptors such as the transferrin receptor .

What methodologies are most effective for analyzing LMBRD1 knockout phenotypes?

Based on published research, the following methodologies have proven effective for characterizing LMBRD1 knockout phenotypes:

• Cre/LoxP system for targeted gene disruption: The generation of conditional knockouts by flanking critical exons (such as exon 3) with LoxP sites allows for tissue-specific or temporal control of gene deletion .

• Timed pregnancy analysis: Essential for identifying the developmental stage at which embryonic lethality occurs in complete knockouts (around E8.0 for Lmbrd1-/- embryos) .

• Whole mount in situ hybridization: Using markers like bone morphogenetic protein 4, Nodal, even skipped homeotic gene 1, and fibroblast growth factor 8 to assess developmental processes such as axis formation and gastrulation .

• Immunofluorescence microscopy: For confirming protein absence in knockout tissues and examining effects on cellular organization .

• PCR-based genotyping: Crucial for identifying homozygous, heterozygous, and wild-type embryos, particularly at early developmental stages .

• Real-time PCR quantification: For confirming reduced mRNA expression levels in heterozygous models and complete absence in homozygous knockouts .

• Micro-positron emission tomography: For functional analysis, as demonstrated by studies showing increased 18F-fluorodeoxyglucose uptake in murine hearts with single-allele knockout of lmbrd1 .

What are the most common technical challenges when working with LMBRD1 antibodies?

Researchers working with LMBRD1 antibodies frequently encounter several technical challenges:

• Membrane protein detection issues: As LMBRD1 contains 9 transmembrane domains, proper sample preparation is crucial to maintain protein integrity while ensuring membrane solubilization.

• Epitope accessibility: The complex topology of LMBRD1 means certain epitopes may be inaccessible in native conformations, requiring careful selection of antibodies targeting accessible regions.

• Cross-reactivity with NESI: The lmbrd1 gene encodes two major proteins - LMBD1 (540 amino acids) and NESI (467 amino acids), with NESI being identical to LMBD1 except for lacking 73 N-terminal amino acids . Antibodies must be carefully selected to differentiate between these two proteins if studying specific isoform functions.

• Fixation sensitivity: Certain fixation methods may alter LMBRD1 epitopes, requiring optimization of fixation protocols for immunohistochemistry and immunofluorescence applications.

• Validation in knockout models: Given the embryonic lethality of complete Lmbrd1 knockout, validation of antibody specificity requires careful timing of embryo collection or the use of conditional/inducible knockout systems .

• Subcellular localization complexity: As LMBRD1 functions in both lysosomes and the plasma membrane, proper fractionation techniques are necessary to accurately assess its distribution between these compartments .

How can researchers distinguish between LMBRD1's roles in cobalamin transport versus insulin receptor endocytosis?

Distinguishing between LMBRD1's dual functions requires specialized experimental approaches:

• Compartment-specific markers: Co-localization studies using lysosomal markers (LAMP1, LAMP2) versus plasma membrane and endocytic vesicle markers (clathrin, AP-2) can help determine which pool of LMBRD1 is being studied .

• Domain-specific mutations: The AP-2 binding motifs (Y233 and W295) are critical for endocytosis functions but may not affect lysosomal cobalamin transport . Creating point mutations that selectively disrupt one function while preserving the other can help delineate these roles.

• Functional readouts:

  • For cobalamin transport: Measure intracellular cobalamin accumulation, succinyl-CoA synthesis, or methionine synthesis

  • For insulin receptor endocytosis: Assess insulin receptor phosphorylation status, downstream Akt activation, and glucose uptake

• Temporal manipulation: Using inducible knockdown or knockout systems may reveal different phenotypes depending on developmental timing, as cobalamin transport may be critical during embryogenesis while insulin receptor regulation becomes more important postnatally.

• Cell-type specific analysis: Different cell types may predominantly utilize one function over the other, allowing for comparative studies between, for example, hepatocytes (high cobalamin processing) versus adipocytes (high insulin sensitivity).

What is the significance of the LMBRD1 gene in developmental disorders?

The LMBRD1 gene plays a critical role in early development, with significant implications for developmental disorders:

• Embryonic lethality: Complete loss of Lmbrd1 function causes early embryonic death in mice around E8.0, with abnormally developed embryos that are smaller in size and poorly developed compared to wild-type or heterozygous littermates .

• Gastrulation defects: While initial formation of the proximal-distal axis remains unaffected in Lmbrd1-deficient embryos, the initiation of gastrulation is disturbed, as evidenced by altered expression patterns of developmental markers .

• Clinical manifestations: In humans, mutations in LMBRD1 cause the cblF defect of cobalamin metabolism, which manifests as heart defects, developmental delay, and megaloblastic anemia .

• Metabolic pathway disruption: The developmental abnormalities likely stem from disrupted cobalamin-dependent metabolic pathways, affecting both mitochondrial energy metabolism (via succinyl-CoA synthesis) and cytosolic one-carbon metabolism (via methionine synthesis) .

• Insulin signaling implications: Given LMBRD1's role in insulin receptor endocytosis, alterations in insulin signaling during development may contribute to developmental phenotypes, as insulin and insulin-like growth factors are known to influence embryonic growth and differentiation .

This multifaceted role of LMBRD1 in development highlights its potential importance in understanding various congenital disorders and emphasizes the value of continued research using LMBRD1 antibodies for developmental studies.

What are the optimal conditions for using LMBRD1 antibodies in immunofluorescence studies?

Based on published protocols, the following conditions have been successfully employed for LMBRD1 immunofluorescence studies:

• Fixation protocol: 4% paraformaldehyde in 1× PBS for 30 minutes has proven effective for embryonic tissues while preserving LMBRD1 epitopes .

• Permeabilization: PBS containing 0.1% Triton X-100 (PBTx) allows antibody access to intracellular epitopes without excessive damage to membrane structures .

• Blocking conditions: 1× PBTx with 1% BSA effectively reduces non-specific binding .

• Primary antibody: Rabbit anti-LMBRD1 antibody at 1:500 dilution (HPA019547; Sigma-Aldrich) incubated overnight at 4°C has shown good specificity and signal strength .

• Secondary antibody: Anti-rabbit Cy3 conjugated secondary antibody at 1:5000 dilution (111-166-045; Jackson ImmunoResearch) provides strong signal with low background .

• Mounting medium: Standard fluorescence mounting medium (such as Dako) preserves signal and allows for extended imaging sessions .

• Imaging parameters: For optimal visualization of subcellular distribution, confocal microscopy with appropriate z-stack acquisition is recommended, as demonstrated with the Zeiss Apotome Axiovert 200 system .

• Controls: Include Lmbrd1-deficient samples as negative controls to confirm antibody specificity .

How can researchers effectively design knockdown experiments to study LMBRD1 function?

Effective knockdown studies of LMBRD1 should incorporate the following design elements:

• Target sequence selection: When designing shRNA or siRNA, target unique regions of lmbrd1 that don't overlap with other genes or alternative transcripts. Researchers have successfully generated lmbrd1 knockdown constructs that effectively reduce protein expression .

• Rescue experiments: Include shRNA-resistant LMBRD1 constructs to confirm phenotype specificity. This approach has been used to validate the role of LMBRD1 in insulin receptor endocytosis . The shRNA-resistant constructs can be generated by introducing silent mutations at the wobble positions in the target sequence, as demonstrated in the following table:

  Construct	Description	Function
  pLMBD1-shRNA lmbrd1R-myc-His	Wild-type shRNA-resistant LMBRD1	Complete rescue of function
  pLMBD1-shRNA lmbrd1R(Y233A)-myc-His	Y233A mutant (disrupted μ2 binding motif)	Partial rescue
  pLMBD1-shRNA lmbrd1R(W295A)-myc-His	W295A mutant (disrupted α-appendage binding)	Partial rescue

• Functional readouts: Include appropriate assays to measure the specific function being studied:

  • For insulin signaling: Measure IR phosphorylation status and Akt activation

  • For cobalamin transport: Assess intracellular cobalamin levels and dependent metabolic pathways

  • For endocytosis: Use fluorescently labeled receptors to track internalization kinetics

• Temporal considerations: Due to LMBRD1's role in development, inducible knockdown systems may be preferable to study functions at specific developmental stages without interfering with early embryogenesis .

• Partial versus complete knockdown: While complete knockout causes embryonic lethality, partial knockdown may allow study of hypomorphic phenotypes that better model certain disease states .

What are the best practices for co-immunoprecipitation studies involving LMBRD1?

For effective co-immunoprecipitation (co-IP) studies of LMBRD1 and its interaction partners:

• Lysate preparation: Given LMBRD1's multiple transmembrane domains, use lysis buffers containing appropriate detergents (such as 1% Triton X-100 or 0.5% NP-40) that solubilize membranes while preserving protein-protein interactions.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding to the beads themselves.

• Antibody selection: For immunoprecipitation of endogenous LMBRD1, antibodies that recognize epitopes in cytoplasmic domains generally perform better than those targeting transmembrane regions.

• Validated interactions: Previous studies have successfully demonstrated co-immunoprecipitation of LMBRD1 with both adaptor protein-2 (AP-2) and clathrin heavy chain (CHC) , providing positive controls for protocol optimization.

• Negative controls: Include isotype-matched control antibodies and, when available, samples from knockdown or knockout models to confirm specificity of detected interactions.

• Sequential immunoprecipitation: For multi-protein complexes, consider sequential immunoprecipitation (first pulling down LMBRD1, then a suspected interacting partner from the initial precipitate) to confirm direct vs. indirect interactions.

• Analysis techniques: LC-MS/MS analysis of LMBRD1 co-immunoprecipitates has successfully identified interacting partners such as clathrin heavy chain , providing an unbiased approach to discovering novel interactions.

What are the potential therapeutic implications of targeting LMBRD1 pathways?

Understanding LMBRD1 function opens several potential therapeutic avenues:

• Cobalamin metabolism disorders: As mutations in LMBRD1 cause the cblF defect characterized by lysosomal cobalamin accumulation, therapeutic approaches that bypass or enhance alternative cobalamin transport pathways could benefit affected patients .

• Insulin signaling modulation: Given LMBRD1's role as an insulin receptor-specific adaptor for endocytosis, modulating its function could potentially enhance insulin receptor signaling in insulin-resistant states . Single-allele knockout of lmbrd1 increased glucose uptake in murine hearts, suggesting that partial inhibition of LMBRD1 might enhance insulin sensitivity .

• Developmental disorders: Understanding LMBRD1's critical role in gastrulation and early embryonic development may inform therapies for congenital disorders with similar developmental disruptions .

• Receptor-specific endocytosis pathways: The specificity of LMBRD1 for insulin receptor endocytosis (but not transferrin receptor) suggests it may be possible to selectively modulate specific receptor populations without broadly affecting endocytosis .

• Precision medicine approaches: Genetic screening for LMBRD1 variants could help identify individuals who might benefit from targeted nutritional interventions or metabolism-modifying therapies.

How might single-cell analysis techniques advance our understanding of LMBRD1 function?

Single-cell technologies offer powerful new approaches to understanding LMBRD1 biology:

• Cell-specific expression patterns: Single-cell RNA sequencing could reveal previously unrecognized cell populations with high Lmbrd1 expression, potentially identifying new functional roles.

• Dynamic regulation: Single-cell temporal analysis during development could map the precise timing of Lmbrd1 expression changes relative to developmental milestones, particularly during gastrulation when LMBRD1 function is critical .

• Pathway interactions: Single-cell multi-omics approaches combining transcriptomics, proteomics, and metabolomics could reveal how LMBRD1-dependent pathways interact with other cellular processes in different cell types.

• Heterogeneous responses: In models with Lmbrd1 mutations or knockdown, single-cell analysis could identify compensatory mechanisms that might explain why some cells or tissues are more affected than others.

• Spatial context: Emerging spatial transcriptomics techniques could map Lmbrd1 expression within complex tissues, potentially revealing gradient patterns that correlate with developmental processes or metabolic activities.

• Rare cell populations: Given LMBRD1's role in both cobalamin transport and insulin signaling, single-cell approaches might identify specialized cell populations that disproportionately rely on one function over the other.