RABGGTA Antibody

What is RABGGTA and what is its function in cellular biology?

RABGGTA is the alpha subunit of Rab geranylgeranyltransferase (also known as GGTase II), an enzyme crucial for post-translational modification of Rab proteins. In humans, the canonical protein consists of 567 amino acid residues with a molecular mass of 65.1 kDa and is widely expressed across many tissue types . As a member of the Protein prenyltransferase subunit alpha family, RABGGTA catalyzes the transfer of geranylgeranyl moieties from geranylgeranyl diphosphate to cysteine residues in Rab proteins with C-terminal sequences -XXCC, -XCXC, and -CCXX .

This prenylation process is essential for:

• Proper membrane association of Rab proteins

• Regulation of intracellular vesicle trafficking

• Organelle identity and function

• Multiple signaling cascades

RABGGTA functions as part of a heterodimeric enzyme complex with RABGGTB (the beta subunit) and requires Rab escort proteins (REPs) for activity. Together, this machinery is critical for the proper functioning of over 60 Rab GTPases that orchestrate membrane trafficking events.

What are the common applications for RABGGTA antibodies in research protocols?

RABGGTA antibodies are employed in multiple experimental applications:

When selecting antibodies, researchers should verify validation data for their specific application and cell/tissue type of interest, as performance can vary significantly between manufacturers and antibody clones.

What species reactivity is typically observed with RABGGTA antibodies?

Commercial RABGGTA antibodies demonstrate varying degrees of cross-reactivity:

What is the observed molecular weight of RABGGTA in Western blot experiments?

When detected by Western blotting, RABGGTA typically appears at the following molecular weights:

• Theoretical molecular weight: 65.1 kDa based on amino acid sequence

• Observed molecular weight: 60-65 kDa in most experimental systems

The slight discrepancy between theoretical and observed molecular weights can be attributed to:

• Post-translational modifications

• Protein folding effects on electrophoretic mobility

• Variations in SDS-PAGE conditions

• Potential proteolytic processing

In the gunmetal mouse model, which has a RABGGTA mutation, Western blots show approximately 70% reduction in the 60 kDa RABGGTA protein compared to wild-type mice .

What are the alternative names and synonyms for RABGGTA in scientific literature?

Researchers should be aware of these alternative nomenclatures when searching literature:

• Geranylgeranyl transferase type-2 subunit alpha

• Rab GG transferase alpha

• Rab GGTase alpha

• Rab geranylgeranyltransferase alpha subunit

• PTAR3

Using multiple search terms is recommended when conducting comprehensive literature reviews on this protein.

How does the RABGGTA-mediated prenylation pathway impact Rab protein function and membrane trafficking?

RABGGTA functions within a complex prenylation machinery that regulates Rab GTPase activity through these mechanistic steps:

• Complex Formation: RABGGTA and RABGGTB form a heterodimer that constitutes the catalytic core of Rab geranylgeranyltransferase (RGGT)

• Substrate Recognition: Rab escort proteins (REP1 or REP2) bind newly synthesized Rab proteins and present them to RGGT

• Catalytic Transfer: RGGT catalyzes the transfer of one or two 20-carbon geranylgeranyl groups to C-terminal cysteine residues of Rab proteins

• Membrane Targeting: Prenylated Rabs are delivered to their target membranes by REPs

The prenylation status directly impacts:

• Membrane association capability of Rab proteins

• GTPase activity cycles

• Interaction with Rab effectors

• Vesicle budding, transport, and fusion events

Experimental evidence from the gunmetal mouse model demonstrates that reduced RABGGTA function (approximately 70% decrease) leads to significant reductions in Rab protein prenylation and subsequent phenotypes including macrothrombocytopenia and hypopigmentation .

In pancreatic β-cells, RABGGTA plays a critical role in glucose-stimulated insulin secretion (GSIS), as siRNA-mediated knockdown of RGGT subunits markedly attenuates insulin release . This links RABGGTA function directly to hormone secretion mechanisms that depend on coordinated vesicle trafficking.

What is known about RABGGTA mutations and their association with pathological conditions?

The most well-characterized RABGGTA mutation occurs in the gunmetal (gm) mouse model, providing valuable insights into RABGGTA function in disease:

Gunmetal Mouse Molecular Defect:

• Single G→A substitution at the terminal nucleotide of intron α

• Disrupts normal mRNA splicing, leading to exon skipping and usage of cryptic splice sites

• Results in ~70% reduction in RABGGTA protein levels

• Correspondingly reduces Rab GGTase enzymatic activity by ~70%

Phenotypic Consequences:

• Macrothrombocytopenia (reduced platelet count with enlarged platelets)

• Hypopigmentation

• These phenotypes are correctable by bone marrow transplantation

Relevance to Human Disease:
While the search results don't directly link RABGGTA mutations to human diseases, related research suggests potential implications in:

• Hematological disorders: Given the platelet defects in gunmetal mice

• Pigmentation disorders: Due to hypopigmentation phenotype

• Neurodegenerative diseases: RABGGTB (the partner of RABGGTA) shows differential expression in Amyotrophic Lateral Sclerosis (ALS) patients compared to healthy controls

• Metabolic disorders: RABGGTA plays a role in glucose-stimulated insulin secretion

How can researchers effectively validate RABGGTA knockdown experiments?

A multi-faceted validation approach is recommended:

Transcript Level Validation:

• RT-PCR using gene-specific primers spanning multiple exons

• Quantitative RT-PCR with normalization to housekeeping genes

• Analysis of splicing patterns (particularly important given the splicing defects in the gunmetal mouse)

Protein Level Validation:

• Western blotting with validated anti-RABGGTA antibodies

• Expected molecular weight: ~60-65 kDa

• Normalization to loading controls (actin, GAPDH, etc.)

• Quantitative densitometry to determine knockdown efficiency

Functional Validation:

• Rab GGTase enzymatic activity assays (detailed in question 2.2)

• Analysis of membrane association of Rab proteins

• Phenotypic assays relevant to the cell type being studied:

  • Insulin secretion in β-cells

  • Platelet production in megakaryocytes

  • Pigmentation in melanocytes

Controls and Specificity Assessment:

• Include non-targeting siRNA/shRNA controls

• Verify that RABGGTB, REP1, and REP2 levels remain unchanged, confirming specificity of RABGGTA targeting

• Rescue experiments with siRNA/shRNA-resistant RABGGTA constructs

What is the significance of RABGGTA in glucose-stimulated insulin secretion?

Research has revealed a critical role for RABGGTA in pancreatic β-cell function:

Expression Profile:

• RABGGTA is expressed in:

  • Clonal INS 832/13 β-cells

  • Normal rat islets

  • Human islets

• The relative abundance of RABGGTA is 30% higher in rat islets compared to INS 832/13 cells

Functional Evidence:

• siRNA-mediated knockdown of either α- or β-subunits of RGGT markedly attenuates glucose-stimulated insulin secretion (GSIS) in INS 832/13 cells

• Similarly, knockdown of REP1 (which works with RABGGTA) also impairs GSIS

Mechanistic Basis:

• Rab proteins, particularly Rab3A and Rab27A, are key regulators of insulin granule trafficking

• These Rabs require geranylgeranylation by RABGGTA-containing RGGT for proper membrane association

• Impaired prenylation disrupts the coordinated vesicle trafficking required for insulin secretion

This research identifies RABGGTA as a potential therapeutic target in diabetes research and highlights the importance of protein prenylation in hormone secretion pathways.

What are the critical factors for successful Western blot detection of RABGGTA?

To achieve optimal Western blot results with RABGGTA antibodies:

Sample Preparation:

• Use fresh tissues or cells with protease inhibitor cocktails

• For subcellular fractionation studies, sonicate fresh samples and separate membrane from soluble fractions by centrifugation

• Protein quantification using Bradford or BCA assays is essential for equal loading

Electrophoresis Conditions:

• 10-12% polyacrylamide gels are typically used for optimal resolution

• Load 20-50 μg of total protein per lane

Antibody Selection and Dilution:

• Primary antibody dilutions typically range from 1:1000-1:4000

• Secondary antibodies: peroxidase-labeled anti-rabbit IgG (for rabbit polyclonal primaries)

• Multiple exposures should be taken to ensure detection is within linear range

Controls:

• Include positive control samples (e.g., HeLa, Jurkat cells)

• Re-probe blots with antibodies to housekeeping proteins (e.g., actin) for normalization

• When studying knockdown/knockout samples, wild-type samples are essential controls

How should immunohistochemistry and immunofluorescence protocols be optimized for RABGGTA detection?

For optimal cellular and tissue detection of RABGGTA:

Tissue Processing (IHC):

• Formalin-fixed paraffin-embedded (FFPE) sections are commonly used

• Antigen retrieval is critical: TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Blocking with serum matching the host of the secondary antibody reduces background

Cell Preparation (ICC/IF):

• Fixation: 4% paraformaldehyde (10-15 minutes) is typically effective

• Permeabilization: 0.1-0.5% Triton X-100 or similar detergents

• Blocking with BSA or serum in PBS reduces non-specific binding

Antibody Parameters:

• Primary antibody dilutions: 1:50-1:500 for IF/ICC

• RABGGTA antibodies successfully detect the protein in various cell lines including HepG2, L929, and others

• Counterstain nuclei with DAPI for proper cellular orientation

Visualization:

• Fluorescent secondary antibodies or HRP-based detection systems

• Include controls for autofluorescence (unlabeled samples) and secondary antibody binding (primary antibody omitted)

• Confocal microscopy may be necessary to resolve subcellular localization

As shown in the research data, immunofluorescence analysis of L929 cells using RABGGTA antibody at a dilution of 1:100 successfully detects the protein with DAPI counterstaining for nuclear visualization .

What considerations are important when designing experiments to study RABGGTA interactions with other proteins?

When investigating RABGGTA's interactions with binding partners:

Experimental Approaches:

• Co-immunoprecipitation (Co-IP): Use antibodies against RABGGTA to pull down interacting proteins

• Proximity ligation assays: For detecting in situ protein-protein interactions

• Yeast two-hybrid screening: For identifying novel interaction partners

• In vitro binding assays with purified components

Key Interaction Partners to Consider:

• RABGGTB (β-subunit): Forms the functional heterodimeric enzyme

• REP1/REP2 (Rab escort proteins): Essential for presenting Rab substrates

• Rab proteins: The substrates for prenylation (RAB1A, RAB3A, RAB5A, RAB7A, etc.)

Technical Considerations:

• Lysis conditions must preserve protein-protein interactions (avoid harsh detergents)

• Crosslinking may be necessary for capturing transient interactions

• Controls should include IgG from the same species as the RABGGTA antibody

• Validate interactions using multiple approaches (Co-IP, IF colocalization, etc.)

Minimal Reconstitution Approaches:
Although not specifically described for RABGGTA, techniques like the GUV (giant unilamellar vesicle) assay described for Rab5 studies could be adapted to study RABGGTA interactions with Rab proteins and membranes.

How is RABGGTA expression altered in disease states?

RABGGTA expression and function can be altered in various conditions:

Genetic Disorders:

• In the gunmetal mouse model, a single G→A substitution causes abnormal splicing of RABGGTA mRNA, resulting in ~70% reduction in protein levels and activity

Neurodegenerative Diseases:

• While not directly examining RABGGTA, related research shows RABGGTB is significantly upregulated in monocytes and monocyte-derived macrophages from patients with Amyotrophic Lateral Sclerosis (ALS) compared to controls

• This differential expression appears specific to ALS, as it wasn't observed in Parkinson's disease or acute cerebrovascular disease patients

Other Conditions:

• Research indicates that RABGGTB (RABGGTA's partner) is downregulated in peripheral blood from multiple sclerosis patients compared to healthy controls

• Conversely, high RABGGTB expression has been reported in tumor-associated diseases

These variations suggest that the prenylation machinery, including RABGGTA, may be dysregulated in multiple pathological conditions, potentially contributing to disease mechanisms.

What experimental systems are available to study RABGGTA function in vivo?

Several experimental systems are available for in vivo RABGGTA research:

Mouse Models:

• Gunmetal (gm) mouse: Contains a naturally occurring mutation in the Rabggta gene causing ~70% reduction in protein levels and enzymatic activity

• The phenotype includes macrothrombocytopenia and hypopigmentation

• This model is valuable for studying RABGGTA function in hematopoiesis and pigmentation

Conditional Knockouts:

• While not specifically mentioned in the search results, conditional knockout approaches would allow tissue-specific deletion of RABGGTA

Cell-Based Systems:

• INS 832/13 β-cells: Used to study RABGGTA's role in insulin secretion

• Primary cell cultures from various tissues expressing RABGGTA

• siRNA/shRNA-mediated knockdown systems for acute reduction of RABGGTA levels

Emerging Technologies:

• CRISPR/Cas9 gene editing could be employed to create specific RABGGTA mutations or tagged versions of the endogenous protein

• Patient-derived iPSCs differentiated into relevant cell types could provide human-specific insights

How can antibody-based approaches be used to develop therapeutic strategies targeting the RABGGTA pathway?

While the search results don't directly address therapeutic applications, several antibody-based approaches could potentially target the RABGGTA pathway:

Diagnostic Applications:

• RABGGTA antibodies could serve as biomarkers for diseases involving prenylation defects

• The differential expression of RABGGTB in ALS suggests potential diagnostic applications for prenylation machinery components

Therapeutic Approaches:

• Intrabodies: Engineered antibody fragments expressed intracellularly could modulate RABGGTA function

• Blocking peptides: Designed based on antibody epitope mapping to interfere with specific RABGGTA interactions

• Antibody-drug conjugates: Could potentially deliver payloads to cells with aberrant RABGGTA expression

Antibody Engineering Considerations:

• The computational antibody design framework described in search result could potentially be applied to develop antibodies against RABGGTA or its interaction partners

• RosettaAntibodyDesign (RAbD) allows for the optimization of antibody-antigen interactions

Research Tools for Therapeutic Development:

• RABGGTA antibodies are essential tools for validating target engagement in drug discovery efforts

• Immunohistochemistry applications help identify tissues where RABGGTA-targeting therapeutics might act

What emerging technologies might advance our understanding of RABGGTA biology?

Several cutting-edge approaches could enhance RABGGTA research:

Structural Biology Advances:

• Cryo-electron microscopy of the complete RGGT complex (RABGGTA-RABGGTB-REP-Rab)

• Single-particle tracking of RABGGTA dynamics in live cells

• Super-resolution microscopy for detailed subcellular localization studies

Single-Cell Technologies:

• Single-cell RNA-seq to identify cell-specific expression patterns of RABGGTA

• Single-cell proteomics to assess protein levels and modifications across cell populations

Organoid Systems:

• Patient-derived organoids to study RABGGTA function in three-dimensional tissue contexts

• Organoid-based disease modeling for conditions with altered prenylation

In Vitro Reconstitution:

• Minimal reconstitution systems using giant unilamellar vesicles (GUVs), similar to the approach described for Rab5 studies

• Cell-free systems to study RABGGTA-mediated prenylation kinetics

Computational Approaches:

• Antibody design frameworks like RosettaAntibodyDesign could generate novel reagents for RABGGTA research

• Molecular dynamics simulations of RABGGTA-substrate interactions

What are the most significant knowledge gaps in RABGGTA research?

Despite significant progress, several crucial knowledge gaps remain:

Regulatory Mechanisms:

• How is RABGGTA expression regulated at transcriptional, post-transcriptional, and post-translational levels?

• What factors determine tissue-specific variations in RABGGTA abundance?

Substrate Specificity:

• What determines the specificity of RABGGTA for different Rab protein substrates?

• Are there non-Rab proteins that undergo RABGGTA-mediated prenylation?

Pathological Relevance:

• Are RABGGTA mutations or expression changes associated with human diseases beyond what's been modeled in the gunmetal mouse?

• What is the role of RABGGTA in neurodegenerative conditions where Rab dysfunction has been implicated?

Therapeutic Targeting:

• Can RABGGTA activity be selectively modulated for therapeutic benefit?

• Would targeting RABGGTA have advantages over directly targeting individual Rab proteins?

System Integration:

• How does RABGGTA function integrate with other post-translational modification systems?

• What is the interplay between prenylation and other Rab regulatory mechanisms?

Addressing these questions will require interdisciplinary approaches combining cell biology, biochemistry, genetics, and clinical research.