ART4 Human

What is ART4 and what are its primary structural features?

ART4 (ADP-Ribosyltransferase 4) is a glycosylphosphatidylinositol (GPI)-linked membrane protein that carries Dombrock blood group antigens. It belongs to the Ribosyltransferase family and contains a mono-ADP-ribosylation (ART) motif, although its enzymatic activity has not been conclusively demonstrated experimentally .

The mature human ART4 protein consists of 239 amino acids (positions 47-285) with a molecular mass of approximately 28.8 kDa, though it typically appears as 28-40 kDa on SDS-PAGE due to glycosylation . The protein is anchored to the erythrocyte membrane via a GPI anchor and has several sites for post-translational modifications, including phosphorylation at positions Ser307, Ser308, Tyr362, and Tyr398 .

When working with ART4, researchers should consider:

• The presence of the GPI anchor affects membrane localization and protein behavior

• Glycosylation patterns may influence protein function and antibody recognition

• The mature protein (aa 47-285) rather than the full-length sequence should be used for recombinant protein expression

How is ART4 expression regulated during development and cell differentiation?

ART4 shows developmental regulation with highest expression levels in the fetal liver . Its expression appears to be tightly controlled during erythroid differentiation, suggesting a specific role in red blood cell development .

For researchers investigating ART4 expression patterns:

• Compare expression across developmental stages and cell lineages

• Employ erythroid differentiation models to track temporal expression changes

• Use specific antibodies validated for detecting ART4 in different applications (WB, IHC)

• Consider tissue-specific expression patterns when designing experiments

The relationship between ART4 expression and erythroid development makes it an interesting target for hematological research, particularly in understanding the molecular basis of erythropoiesis.

What is the relationship between ART4 and the Dombrock blood group system?

ART4 serves as the molecular carrier of the Dombrock blood group antigens. Several antigens have been identified in this system, including DOA, DOJO1, DOA-WL, DODOYA, DOB, DOB-WL, DOB-SH-Q149K, DOB-(WL)-I175N, DOHY1, DOHY2, and DO*DOMR .

These antigens result from allelic variations in the ART4 gene. Some of these variations lead to adverse transfusion reactions, making them clinically significant . For blood typing research, understanding the molecular basis of these variations is critical for:

• Developing improved blood typing methodologies

• Investigating immunological responses to transfusions

• Studying the structure-function relationships of ART4 variants

What are the optimal antibodies and recombinant proteins for studying human ART4?

Based on the available research tools, the following options have been validated for ART4 research:

Antibodies:

• Polyclonal antibodies targeting specific regions (aa 61-160, 181-230, 1-314)

• Phospho-specific antibodies for post-translational modifications (Ser307, Ser308, Tyr362, Tyr398)

• Monoclonal antibody 2F8 targeting amino acids 61-160

Antibody Selection Table:

Recombinant Proteins:

• Human ART4 recombinant protein expressed in Sf9 insect cells (aa 47-285)

• His-tagged versions for purification and detection

For optimal experimental results, researchers should:

• Validate antibodies in their specific experimental systems

• Consider the specific application requirements (Western blot, IHC, etc.)

• Select appropriate species reactivity for comparative studies

• Use recombinant proteins as positive controls and standards

How should researchers design experiments to investigate potential ART4 enzymatic activity?

Despite belonging to the ADP-ribosyltransferase family, ART4's enzymatic activity remains to be conclusively demonstrated. When designing experiments to investigate this activity:

• In vitro enzymatic assays:

  • Use purified recombinant ART4 protein (amino acids 47-285)

  • Include appropriate positive controls (known active ART family members)

  • Test various potential protein substrates

  • Employ multiple detection methods (radiometric, antibody-based, mass spectrometry)

• Structural considerations:

  • Analyze the conservation of catalytic residues compared to enzymatically active ART family members

  • Design mutations of predicted catalytic sites to test structure-function relationships

  • Consider the impact of post-translational modifications on potential activity

• Experimental controls:

  • Generate catalytically inactive mutants as negative controls

  • Include assays with and without potential cofactors

  • Consider the effect of membrane association via the GPI anchor on activity

What principles should guide the experimental design for ART4 knockout or knockdown studies?

For researchers planning gene modification approaches to study ART4 function:

• Model system selection:

  • Cell lines: CRISPR/Cas9 can be used for complete knockout

  • Mouse models: A conditional knockout mouse line (Art4 tm1a(KOMP)Wtsi) is available

• Verification strategies:

  • Genomic: PCR and sequencing to confirm modification

  • Transcript: RT-PCR and RNA-seq

  • Protein: Western blotting with validated antibodies

  • Functional: Blood typing for Dombrock antigens

• Experimental design considerations:

  • Include heterozygous models to study gene dosage effects

  • Implement proper controls (age and sex-matched, isogenic)

  • Analyze both cellular and systemic phenotypes

  • Consider temporal aspects for developmental studies

• Specific phenotypes to analyze:

  • Erythroid differentiation and maturation

  • Membrane protein composition

  • Blood group antigen expression

  • Cellular response to physiological stressors

How can researchers resolve contradictory findings regarding ART4 function in the literature?

To address conflicting reports about ART4 function, especially regarding its enzymatic activity:

• Systematically evaluate methodological differences:

  • Compare protein preparations (recombinant vs. native, different expression systems)

  • Assess assay conditions (buffers, cofactors, temperature, pH)

  • Examine detection method sensitivity and specificity

• Consider protein modifications:

  • Analyze the effect of post-translational modifications (phosphorylation sites Ser307, Ser308, Tyr362, Tyr398)

  • Evaluate the impact of the GPI anchor on protein function

  • Assess glycosylation patterns across experimental systems

• Design definitive experiments:

  • Use complementary approaches (biochemical, cellular, genetic)

  • Include appropriate positive and negative controls

  • Employ both in vitro and cellular systems

  • Consider structural biology approaches

• Apply the principles of experimental design for art conservation research:

  • Define clear research questions

  • Control variables systematically

  • Use appropriate statistical approaches for data analysis

  • Consider sample size and statistical power

What advanced techniques are recommended for studying ART4 protein interactions?

To elucidate the interactome of ART4, researchers can employ several cutting-edge methodologies:

• Affinity-based approaches:

  • Immunoprecipitation using validated antibodies

  • Tandem affinity purification with tagged ART4 constructs

  • Crosslinking followed by mass spectrometry (XL-MS)

• Proximity-based labeling:

  • BioID or TurboID fusion proteins to identify proximal proteins

  • APEX2-based approaches for temporal resolution

  • Controls should include catalytically inactive biotin ligase fusions

• Structural and biophysical methods:

  • Surface plasmon resonance (SPR) for direct binding studies

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational changes

• Validation strategies:

  • Confirm key interactions using orthogonal methods

  • Perform domain mapping to identify interaction interfaces

  • Employ functional assays to assess biological relevance

How does polymorphism in ART4 impact experimental design and interpretation?

ART4 exhibits significant polymorphism, particularly in regions encoding Dombrock blood group antigens. These variations affect experimental approaches:

• Sample selection and characterization:

  • Genotype samples for known ART4 polymorphisms

  • Include diverse genotypes to represent population variation

  • Document ethnic/ancestry information due to population differences in allele frequencies

• Antibody selection considerations:

  • Verify antibody recognition across different ART4 variants

  • Use antibodies targeting conserved regions for universal detection

  • Employ variant-specific antibodies for distinguishing polymorphisms

• Functional studies:

  • Compare wild-type and variant proteins in parallel

  • Assess whether functional differences correlate with specific polymorphisms

  • Consider the evolutionary conservation of polymorphic sites

• Technical adjustments:

  • Design primers that account for known variations

  • Use sequencing to confirm genotypes

  • Consider allele-specific expression analyses

What methodological approaches are recommended for studying rare ART4 variants in blood group research?

Researching rare Dombrock blood group phenotypes presents unique challenges:

• Sample acquisition strategies:

  • Collaborate with blood banks and transfusion centers

  • Develop screening protocols for rare phenotypes

  • Consider biobanking of rare samples for sustained research

• Low-input methodologies:

  • Optimize protocols for limited sample amounts

  • Employ single-cell approaches when possible

  • Develop targeted sequencing approaches for ART4 variants

• Validation approaches:

  • Use multiple methodologies to confirm rare variants

  • Combine serological and molecular typing methods

  • Generate recombinant proteins expressing rare variants for functional studies

• Alternative approaches when samples are unavailable:

  • Use CRISPR/Cas9 to introduce rare variants into cell lines

  • Develop computational models based on protein structure prediction

  • Create synthetic biology systems to mimic rare phenotypes

How can ART4 research inform blood typing methodologies?

Advanced understanding of ART4 has implications for improving blood typing approaches:

• Molecular vs. serological typing:

  • DNA-based Dombrock typing offers advantages in specificity

  • Molecular methods can detect silent alleles missed by serology

  • Integration into comprehensive blood group genotyping panels increases efficiency

• Novel methodological approaches:

  • Mass spectrometry-based blood group protein profiling

  • Next-generation sequencing for comprehensive variant detection

  • Recombinant protein-based assays for antibody identification

• Validation and standardization:

  • Reference materials for rare phenotypes

  • Proficiency testing across methodologies

  • Correlation studies between genotype and phenotype

• Implementation considerations:

  • Cost-effectiveness analyses for new methodologies

  • Adaptation for resource-limited settings

  • Training requirements for specialized techniques

By focusing on these methodological approaches, researchers can advance our understanding of ART4 biology while contributing to improvements in transfusion medicine and hematological research.