DRM3 Antibody

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Description

DRM3: A Plant-Specific DNA Methylation Regulator

DRM3 is a methyltransferase in Arabidopsis thaliana that plays a role in RNA-directed DNA methylation (RdDM). It interacts with RNA Polymerase V (Pol V) to maintain genome-wide DNA methylation patterns, particularly in CHH contexts (non-CG methylation) .

Key Functions of DRM3:

  • General RdDM Factor: DRM3 acts as a weak but widespread RdDM component, affecting DNA methylation at most RdDM target sites .

  • Pol V Interaction: DRM3 physically interacts with Pol V, stabilizing Pol V-dependent transcripts and regulating chromatin occupancy .

  • siRNA Regulation: DRM3 loss reduces 24-nt siRNA abundance at downstream RdDM clusters, indicating its role in siRNA stability .

Table 1: Comparative DNA Methylation Deficits in Arabidopsis Mutants

MutantCHH Methylation Loss (%)Pol V Occupancy ChangesKey Phenotype
drm3~15–20%Mixed (gain/loss)Weak RdDM defects
drm2~50–60%Loss at hypomethylated sitesSevere RdDM disruption
nrpe1 (Pol V mutant)~70–80%Global lossComplete RdDM failure
Data sourced from bisulfite sequencing and chromatin immunoprecipitation studies .

Antibody CDR-H3: Structure and Design

While unrelated to DRM3, the Complementarity-Determining Region H3 (CDR-H3) is a hypervariable loop in antibody heavy chains critical for antigen binding. Recent advances in computational modeling and AI-driven design have improved CDR-H3 engineering for therapeutic applications .

Key Advances in CDR-H3 Research:

  • Structural Diversity: CDR-H3 exhibits kinked or extended conformations depending on sequence and environmental factors .

  • AI-Driven Design: Tools like IgFold and PALM-H3 predict CDR-H3 structures with high accuracy (RMSD < 2 Å for short loops) and generate novel sequences targeting antigens like SARS-CoV-2 variants .

  • Therapeutic Applications: Engineered CDR-H3 loops enable antibodies to neutralize emerging viral strains (e.g., SARS-CoV-2 XBB variant) .

Table 2: Performance of AI Models in CDR-H3 Prediction

ModelTraining Data SizeCDR-H3 RMSD (Å)Key Application
IgFold558 million sequences1.64 (short loops)High-accuracy structure prediction
PALM-H31.2 million antigen-CDRH3 pairsN/ADe novo CDR-H3 generation
PIGS~17,260 CDR structures2.5–4.0Homology modeling
RMSD = Root Mean Square Deviation .

Potential Misinterpretation of "DRM3 Antibody"

No literature in the provided sources describes an antibody targeting DRM3. The confusion likely arises from:

  1. Terminology Overlap: "DRM3" refers to a plant methyltransferase, while "CDR-H3" pertains to antibody structure.

  2. Therapeutic Antibody Context: Recent studies focus on engineering antibodies (e.g., CDR-H3 optimization) rather than antibodies against DRM3 .

Future Directions

  • DRM3: Further studies on its role in Pol V transcript stability and cross-talk with other RdDM components (e.g., DRM2) .

  • CDR-H3: Refinement of generative AI models to improve conformational predictions for long loops (>15 residues) .

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
DRM3 antibody; At3g17310Probable inactive DNA antibody; cytosine-5)-methyltransferase DRM3 antibody; Protein DOMAINS REARRANGED METHYLTRANSFERASE 3 antibody
Target Names
DRM3
Uniprot No.

Target Background

Function
DRM3 Antibody is a catalytically inactive DNA methyltransferase that serves as a regulatory factor for DRM2-mediated DNA methylation. It is essential for maintaining non-CpG DNA methylation levels. Additionally, DRM3 plays a crucial role in the establishment and maintenance of RNA-directed DNA methylation (RdDM) and the accumulation of specific repeat-associated small interfering RNAs (siRNAs). It is also required for proper nucleolus organizer region (NOR) nuclear organization during interphase. DRM3 functions downstream of siRNA production, potentially promoting RNA polymerase V (Pol V) transcriptional elongation or assisting in the stabilization of Pol V transcripts.
Gene References Into Functions
  1. Both DRM3 and DRM2 are essential for NOR loci nuclear organization but do not influence overall peri and centromeric heterochromatin organization. PMID: 25316414
  2. Research indicates that despite its catalytic inactivity, DRM3 (At3g17310) is necessary for the normal maintenance of non-CG DNA methylation, the establishment of RNA-directed DNA methylation triggered by repeat sequences, and the accumulation of repeat-associated small RNAs. PMID: 21060858
Database Links

KEGG: ath:AT3G17310

STRING: 3702.AT3G17310.1

UniGene: At.5773

Protein Families
Class I-like SAM-binding methyltransferase superfamily, DRM-methyltransferase family
Subcellular Location
Nucleus.

Q&A

What structural features define antibody specificity, particularly in relation to the heavy chain complementarity-determining region 3 (CDRH3)?

The heavy chain complementarity-determining region 3 (CDRH3) plays a critical role in antibody specificity and diversity. This region exhibits high sequence variability compared to other framework regions, as visualized in sequence logo plots which display significantly smaller residue letters in the CDR loops, particularly CDR-H3 . The length of CDRH3 directly influences binding characteristics, with longer loops (>10 amino acids) demonstrating greater structural complexity and potentially more specific antigen recognition. When designing experiments to characterize antibody specificity, researchers should analyze both sequence and structural features, as CDRH3 loops ranging from 10-25 amino acids show distinct optimization potential compared to shorter or longer variants .

For experimental quantification, techniques such as cell-based assays rather than peptide-based ELISA should be utilized, as these methods better preserve conformational epitopes. Flow cytometry and modified On-Cell Western (OCW) assays have demonstrated higher detection rates (60-75%) for conformational epitope-binding antibodies compared to conventional ELISA .

How does the binding mechanism of antibodies to receptors differ from binding to protein antigens?

Receptor-targeting antibodies, such as anti-muscarinic type 3 receptor (anti-M3R) antibodies, interact with multi-pass transmembrane proteins that contain conformational epitopes spanning multiple extracellular domains. This differs from binding to soluble protein antigens which often present linear epitopes.

Methodologically, evaluating receptor-targeting antibodies requires specialized techniques that preserve native receptor conformation. Cell-based assays that express the target receptor in its native membrane environment show significantly higher detection sensitivity. For example, anti-M3R prevalence varies from 1.92% to 97% depending on the assay system, with cell-based methods providing more reliable detection . These antibodies can disrupt receptor function not just through physical blocking but also by altering receptor conformation, internalization, or downstream signaling pathways, requiring functional assays beyond simple binding assays.

What are the optimal methodologies for validating novel antibodies against multi-pass membrane receptors like muscarinic receptors?

Validation of antibodies targeting multi-pass membrane receptors requires a multi-faceted approach beyond simple binding assays:

  • Cell-based binding assays: Flow cytometry or On-Cell Western assays that maintain native receptor conformation are preferred over conventional ELISA. These methods have demonstrated 60-75% detection rates for anti-M3R antibodies compared to lower rates with peptide-based methods .

  • Functional assays: Receptor-targeting antibodies should be validated for their effects on:

    • Signal transduction (measuring secondary messengers)

    • Agonistic/antagonistic properties

    • Receptor internalization dynamics

  • Tissue cross-reactivity studies: Especially important for receptors with multiple subtypes (M1-M5 for muscarinic receptors) to confirm specificity.

  • Comparative validation: Receiver operating characteristic (ROC) curve analysis should be performed to establish optimal cut-off values for positive/negative determination. The anti-M3R antibody studies demonstrated high discriminatory power with AUC values of 0.95 (95% CI 0.92 to 0.98) when distinguishing Sjögren's syndrome from healthy controls .

  • Correlation with clinical parameters: For autoantibodies like anti-M3R, correlation with disease-specific parameters (e.g., ocular dryness scores, ESSDAI domains) provides functional validation beyond simple binding.

How can computational modeling approaches improve antibody design and characterization for difficult-to-crystallize targets?

Computational modeling offers powerful solutions for antibody structure prediction when crystallization is challenging:

For antibodies with long CDRH3 loops and limited templates:

  • RosettaCM multi-template comparative modeling outperforms single-template methods, particularly for antibodies with long CDRH3s and few available templates .

  • H3-OPT, which combines features of AlphaFold2 and Protein Language Models (PLMs), achieves lower average RMSD for CDR-H3 loops across varying difficulty subsets .

Methodological approach for structure prediction:

The accuracy of these methods correlates with CDRH3 loop length: loops <10 amino acids are predicted accurately by AlphaFold2, while loops 10-25 amino acids benefit from specialized refinement methods. Loops >25 amino acids remain challenging for all current methods .

How do pathogenic autoantibodies against cell surface proteins mediate tissue damage in autoimmune diseases?

Pathogenic autoantibodies targeting cell surface proteins employ multiple mechanisms to cause tissue damage:

Direct mechanisms:

  • Disruption of cellular adhesion: Pathogenic antibodies against desmoglein 3 (Dsg3) in pemphigus vulgaris directly disrupt keratinocyte adhesion by binding to Dsg3, resulting in intraepithelial blister formation .

  • Interference with receptor function: Anti-M3R antibodies in Sjögren's syndrome bind to muscarinic receptors on secretory cells, disrupting normal cholinergic neurotransmission and exocrine secretion, leading to glandular hypofunction .

Methodological considerations for investigation:

  • Utilize recombinant monoclonal antibodies like PVMAB786 (a Dsg3-specific IgG4 antibody) to study mechanism of action in controlled systems .

  • Employ both in vitro models (cell cultures) and in vivo models (skin/mucous membrane explants, neonatal mice) to verify pathogenic effects.

  • Test antibody pathogenicity through functional assays relevant to the target tissue (e.g., acantholysis assays for skin integrity, sialometry for salivary function).

Translational applications:

  • Correlation of autoantibody levels with clinical disease severity: Anti-M3R antibodies correlate with ocular dryness and glandular hypofunction in Sjögren's syndrome .

  • Development of diagnostic criteria: Anti-M3R antibodies enhance sensitivity and specificity for Sjögren's syndrome diagnosis, with 92% agreement with established criteria when substituted for lip biopsy (kappa value of 0.824) .

What is the significance of antibody isotype and subclass in determining pathogenicity and diagnostic utility?

Antibody isotype and subclass critically influence both pathogenic potential and diagnostic value:

Pathogenic implications:

  • IgG4 subclass antibodies, such as the Dsg3-specific PVMAB786 from pemphigus vulgaris patients, demonstrate direct pathogenic effects in tissue models, inducing acantholysis in normal human skin and mucous membranes .

  • Different isotypes possess distinct effector functions: IgG1/IgG3 effectively activate complement and Fc-receptor-bearing cells, while IgG4 typically does not fix complement but can still disrupt molecular interactions.

Diagnostic considerations:

  • When designing assays, targeting specific isotypes improves diagnostic accuracy. For example, IgG anti-M3R antibodies demonstrated superior discriminatory power (AUC 0.95, 95% CI 0.92 to 0.98) compared to other isotypes when differentiating Sjögren's syndrome from other conditions .

  • Combining different antibody markers enhances diagnostic performance: anti-M3R in combination with anti-Ro/SSA outperformed single analytes in discriminating Sjögren's syndrome patients from other groups .

Methodological approach:
For comprehensive antibody characterization, researchers should:

  • Determine isotype and subclass distribution using isotype-specific secondary antibodies

  • Evaluate pathogenic potential through relevant functional assays

  • Assess diagnostic utility through ROC curve analysis with calculation of AUC, sensitivity, specificity, and likelihood ratios

  • Consider combined biomarker panels rather than single antibody tests

How can artificial intelligence approaches facilitate de novo antibody generation for emerging pathogens?

AI-driven approaches now enable de novo antibody generation without requiring natural antibody isolation from serum, significantly accelerating antibody development:

Methodological framework:

  • Pre-training on large antibody datasets: Pre-trained Antibody generative Large Language Models (PALM-H3) can be trained on unpaired antibody sequences to learn fundamental antibody structure patterns .

  • Fine-tuning on paired data: Models are subsequently fine-tuned on smaller datasets of antigen-antibody pairs with known binding affinities .

  • Architecture optimization: Encoder-decoder architectures like those used in PALM-H3 combine ESM2-based antigen models as encoders with antibody Roformer as decoders, leveraging cross-attention mechanisms to generate antigen-specific CDRH3 sequences .

Performance metrics:
The PALM-H3 approach has successfully generated antibodies with binding ability to SARS-CoV-2 antigens, including emerging variants like XBB, as confirmed through both in-silico analysis and in-vitro assays .

Implementation considerations:

  • Training data composition significantly impacts model performance. The statistics of training data should be carefully documented (see example in supplementary tables from the original research) .

  • Model hyperparameters require optimization for specific antibody classes (supplementary note 1 and table S2 in original research) .

  • Binding prediction models (e.g., A2binder) should be incorporated to validate generated antibodies before experimental testing .

What are the key challenges in predicting antibody structure, particularly for CDRH3 regions with limited template availability?

Predicting antibody structures, particularly CDRH3 regions, presents specific challenges that must be addressed through specialized approaches:

CDRH3 length-dependent challenges:

  • Short loops (<10 amino acids): Generally predicted accurately by AlphaFold2, with PLM-based models offering minimal improvement .

  • Medium loops (10-25 amino acids): Benefit significantly from specialized refinement models like H3-OPT, which outperforms general methods .

  • Long loops (>25 amino acids): Remain challenging for all current methods due to high degrees of freedom and lack of homologous sequences .

Template-dependent challenges:

  • When modeling antibodies with long HCDR3s and few available templates, RosettaCM multi-template comparative modeling outperforms single-template methods .

  • Limited availability of good structural templates for some CDR conformations necessitates grafting on templates with low sequence identity, requiring specialized refinement approaches .

Methodological solutions:

  • Dataset construction: Create non-redundant datasets with high-resolution structures (<2.5 Å) from antibody databases like SAbDab .

  • Model selection strategy:

    CDRH3 LengthRecommended MethodExpected Accuracy
    <10 aaAlphaFold2RMSD 0-2 Å
    10-25 aaH3-OPT/RosettaCMRMSD 2-4 Å
    >25 aaCombined approachRMSD >4 Å
  • Validation protocols: Split test datasets into subgroups based on difficulty (e.g., RMSD ranges: 0-2 Å, 2-4 Å, >4 Å) to properly benchmark prediction methods .

How can antibody assays be optimized for clinical diagnostic applications in autoimmune diseases?

Optimizing antibody assays for clinical diagnostics requires careful consideration of methodological approaches:

Assay platform selection:
Cell-based assays that preserve conformational epitopes show superior performance over conventional ELISA for detecting antibodies against membrane proteins. Anti-M3R detection rates vary from 1.92% to 97% depending on assay methodology, with cell-based assays demonstrating higher sensitivity (60-75%) .

Diagnostic performance optimization:

  • Establish optimal cut-off values based on maximum positive likelihood ratios (+LR) obtained from ROC curve analysis comparing patient and control populations .

  • Calculate area under curve (AUC) with 95% confidence intervals to quantify discriminatory power. Anti-M3R demonstrated excellent discrimination with AUC 0.95 (95% CI 0.92 to 0.98) for Sjögren's syndrome vs. healthy controls .

  • Compare different diagnostic criteria combinations using Cohen's kappa to determine level of agreement. When substituting anti-M3R for lip biopsy in Sjögren's syndrome diagnosis, 92% agreement was achieved with a significant kappa of 0.824 .

Clinical correlation analysis:

  • Perform correlation analyses between antibody levels and clinically relevant parameters.

  • Group data into antibody-positive and negative categories for comparison with laboratory and clinical features using appropriate statistical tests (Pearson χ2 test or Fisher's exact test) .

  • Evaluate disease activity correlation using validated scoring systems (e.g., ESSDAI domains for Sjögren's syndrome) .

What are the best practices for validating monoclonal antibodies for research applications?

Comprehensive validation of monoclonal antibodies for research requires a systematic approach:

Essential validation parameters:

  • Specificity validation:

    • Against target protein vs. related family members

    • Using knockout/knockdown controls

    • Through cross-reactivity testing with recombinant proteins

  • Functional validation:

    • Verify expected biological effects (e.g., PVMAB786 induces acantholysis in normal human skin and mucous membranes)

    • Confirm reproducibility in relevant model systems (e.g., cell cultures, tissue explants, animal models)

  • Structural characterization:

    • Determine binding epitope through competition assays or structural biology techniques

    • Analyze antibody structure through computational modeling with specialized tools like H3-OPT or RosettaCM

    • Quantify binding affinity through surface plasmon resonance or bio-layer interferometry

Validation methodology framework:

Validation ParameterMethodsExpected Outcomes
SpecificityWestern blot, IP, flow cytometryBinding to expected molecular weight target; no binding in knockout samples
Epitope bindingELISA, SPR, epitope mappingConfirmed binding to target epitope with quantifiable affinity
Functional effectsCell-based assays, tissue explantsReproducible biological effect consistent with target function
Structure predictionH3-OPT, RosettaCMAccurate structural models for rational optimization

Documentation requirements:

  • Detailed methods including all validation steps performed

  • Raw data and analysis methods

  • Known limitations and optimal applications

  • Recommended positive and negative controls

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