G6PC2 Antibody
Description
Introduction to G6PC2 Antibody
The G6PC2 antibody is a polyclonal immunoglobulin (IgG) developed to detect the human glucose-6-phosphatase catalytic subunit 2 (G6PC2) protein. This antibody is critical for studying G6PC2’s role in pancreatic islet function, particularly in diabetes-related autoimmunity and glucose metabolism. Below is a detailed analysis of its specifications, applications, and research findings.
Detection in Pancreatic Islets
The antibody is pivotal for identifying G6PC2 expression in β-cells and α-cells, where it modulates glucose-stimulated insulin secretion (GSIS) and glucagon suppression . For example:
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Immunohistochemistry (IHC): Maps G6PC2 localization in human and murine pancreatic sections .
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Immunofluorescence (IF): Co-localizes G6PC2 with insulin (β-cells) or glucagon (α-cells) to study cell-specific roles .
Role in Autoimmunity and Diabetes
G6PC2 is a major autoantigen in type 1 diabetes (T1D), with the antibody aiding in:
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Autoantibody Profiling: Identifying G6PC2-specific autoantibodies in T1D patients .
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Therapeutic Target Validation: Assessing G6PC2 inhibitors’ impact on islet function .
G6PC2’s Dual Role in Glucose Regulation
Key Discoveries:
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Structural Motifs: Critical residues (e.g., R36, D252) in the substrate cavity modulate enzyme activity .
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Therapeutic Potential: Inhibiting G6PC2 may lower FBG and improve insulin secretion while reducing glucagon .
Genetic and Functional Studies
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GWAS Links: Variants near G6PC2 (e.g., rs560887) correlate with FBG levels, validated via G6pc2 knockout mice .
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α-cell Specificity: G6pc2 ablation in α-cells alters glucagon suppression without affecting insulin, confirming cell-specific roles .
Challenges in G6PC2 Antibody Use
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Cross-reactivity: Limited data on non-human reactivity (e.g., murine G6pc2) .
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Functional Studies: Antibody-based assays may not capture G6PC2’s enzymatic activity due to its lack of phosphohydrolase function .
Emerging Opportunities
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
This polyclonal anti-G6PC2 antibody is generated by immunizing rabbits with the recombinant human G6PC2 protein (78-115AA) and subsequently purifying the resulting antiserum using protein G. The antibody is unconjugated IgG with a purity of 95%+. It exhibits specific reactivity with human G6PC2 protein, which is localized to pancreatic islets. While G6PC2 lacks phosphohydrolase activity, it serves as a significant target for cell-mediated autoimmunity in diabetes. This antibody can be employed in various applications including ELISA, IHC, and IF for detecting the G6PC2 protein.
Target Background
G6PC2 is an enzyme that catalyzes the hydrolysis of glucose-6-phosphate to glucose within the endoplasmic reticulum. It plays a crucial role in glucose production through glycogenolysis and gluconeogenesis.
- Studies suggest that the GCKR and G6PC2 genes contribute to the risk of type 2 diabetes independently and/or in an interactive manner within the Han Chinese population. PMID: 30055620
- While the variant in TCF7L2 that increases fasting glucose levels leads to an increase in between-subject variance, variants in GCK and G6PC2 associated with elevated fasting glucose levels demonstrate a decrease in between-subject variance. PMID: 28783164
- All three allele variants of G6PC2 (rs560887, rs16856187, and rs573225) are associated with elevated fasting glucose. Notably, two of these variants (rs560887 in the Caucasian subgroup and rs16856187 under the allele and dominant model) are also linked to T2 diabetes. [meta-analysis] PMID: 28704540
- These investigations identify multiple G6PC2 variants that have the potential to influence altered FBG in humans. PMID: 27611587
- Evidence suggests that islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD4(+) helper T (Th) cells play a unique pathogenic role in adult-onset T1D (AT1D). PMID: 26341315
- The rs560887 polymorphism is associated with increased fasting glucose levels. PMID: 25078492
- This study identified coding variants at several GWAS loci, shedding light on the genes underlying these association signals. Notably, multiple coding variants in G6PC2 lead to a loss of protein function and lower fasting glucose levels. PMID: 25625282
- Three novel G6PC2 variants were discovered that are specifically present in islets. These variants result in protein truncations, frame shifts, and neo-sequences that are prone to immunogenicity. PMID: 24030068
- The GCKR rs780094 variant confers a high cross-ethnicity risk for developing T2DM. However, significant associations between GCK, MTNR1B, and G6PC2 variants and T2DM risk are primarily observed in Caucasians. PMID: 23840762
- Polymorphisms in the G6PC2 gene contribute to the association with higher fasting plasma glucose levels. PMID: 23508304
- A single nucleotide polymorphism (SNP) in the beta cell gene G6PC2 may be correlated with preserved insulin secretion in type 1 diabetes. PMID: 22438186
- One of the newly identified spontaneously reactive epitopes (P8 [IGRP(55-72)]) is highly conserved between mice and humans, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects. PMID: 22983906
- Variation at the rs560887 locus of G6PC2 is associated with worse glycated hemoglobin levels in individuals with GCK mutations; GG homozygotes are more likely to meet diagnostic criteria for diabetes based on HbA(1c) level. PMID: 22486180
- Children and adolescents carrying glucose-raising alleles of G6PC2, MTNR1B, GCK, and GLIS3 also exhibit reduced beta-cell function, as indicated by homeostasis model assessment of beta-cell function. PMID: 21515849
- The effects of hyperglycemia on insulin secretion supersede the more subtle effects of genetic variation in the G6PC2 locus on insulin secretion. PMID: 20826583
- Common variants of MTNR1B, G6PC2, and GCK are associated with elevated FPG and impaired insulin secretion, both individually and jointly. This suggests that these risk alleles may contribute to or exacerbate hyperglycemia in susceptible individuals. PMID: 20628598
- This study demonstrated that SNPs from GCK, G6PC2, and MTNR1B modulated fasting glucose levels in the normoglycaemic population, while SNPs from G6PC2 and GCKR were associated with type 2 diabetes. PMID: 20668700
- This research highlights the potential role linking single nucleotide polymorphisms in G6PC2 to variations in fasting blood glucose. PMID: 20622168
- The fasting glucose association at G6PC2 is reproducible across ethnic groups, although ethnic diversity in the pattern and strength of linkage disequilibrium exists. PMID: 19937311
- These results independently confirm the robust association of glucose-6-phosphatase catalytic 2 (G6PC2)/rs560887 with fasting plasma glucose levels in non-diabetic non-Hispanic white Americans. PMID: 20029179
- IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors for this molecule can be developed. PMID: 14722102
- Data demonstrate that islet-specific glucose-6-phosphatase-related protein is an endoplasmic reticulum membrane glycoprotein, and it is degraded through the proteasome pathway, generating major histocompatibility complex class I-presented peptides. PMID: 15044018
- Alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, preventing targeting by pathogenic CD8+ T cells. This implies that IGRP levels in beta cells could influence susceptibility to diabetes. PMID: 16012821
- This study demonstrates the prevalence of CD4+ IGRP-specific T cells not only in individuals with type I diabetes but also in healthy individuals carrying the DR0301 or DR0401 haplotypes. PMID: 16493034
- Differential tissue expression may aid in designing synthetic peptides for identifying IGRP-specific autoreactive T cells in patients with type 1 diabetes. PMID: 16520917
- A human CD8 T cell clone against this epitope has been identified, confirming that this IGRP epitope is shared across species. PMID: 17376840
- A missense mutation in exon 4, L173P, has been found in glycogen storage disease type Ia. PMID: 17607665
- The SNP rs560887 was associated with fasting plasma glucose (FPG). It is hypothesized that G6PC2 regulates FPG by modulating the set point for glucose-stimulated insulin secretion in pancreatic beta cells. PMID: 18451265
- Genetic polymorphisms of the G6PC2 gene may underlie variation in fasting blood glucose levels, and genetic polymorphisms of the ABCB11 gene may also contribute to such variation. PMID: 18521185
- Data suggest that a group of transcription factors, including MafA and Foxa2, regulate the expression of two major autoantigens in type 1 diabetes, including islet-specific glucose-6-phosphatase catalytic subunit-related protein. PMID: 18753309
- The rs573225 is a functional cis-regulatory (epi)-single-nucleotide polymorphism (SNP) of G6PC2 associated with glucose-insulin homeostasis in obese children. This SNP is likely to explain the results of recent genome-wide association studies in nondiabetic adults. PMID: 18984742
- Mutations and single nucleotide polymorphisms in this protein do not contribute to neonatal or early infant type 1 diabetes. PMID: 19238352
- Variations and single-nucleotide polymorphisms are associated with variations in fasting plasma glucose and an increased risk of type 2 diabetes. PMID: 19533084
- The common rs560887 G allele in the G6PC2/ABCB11 locus is associated with increased fasting glycaemia and an increased risk of IFG, associations that may be partly related to an increased basal hepatic glucose production rate. PMID: 19669124
- Data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion. PMID: 19741163
- The SNP rs16856187 was associated with type 2 diabetes and fasting plasma glucose in the Chinese population; carriers of the A allele displayed a higher risk for type 2 diabetes and a lower fasting plasma glucose level in the controls. PMID: 19082990
Q&A
What is G6PC2 and what is its primary function in pancreatic islet cells?
G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit that is primarily expressed in pancreatic islet β cells. Its main function is to modulate the sensitivity of insulin secretion to glucose, thereby regulating fasting blood glucose (FBG) levels . In β cells, G6PC2 opposes the action of glucokinase, creating a futile substrate cycle that determines the rate of glycolytic flux and consequently influences the sensitivity of glucose-stimulated insulin secretion (GSIS) to glucose . This substrate cycling activity has been confirmed through experiments showing reduced G6Pase activity, decreased glucose cycling, and elevated glycolysis in G6pc2 knockout islets compared to wild-type islets .
How does G6PC2 differ structurally and functionally from G6PC1?
While G6PC2 retains an identical structural fold to G6PC1, there are critical differences in their substrate cavities that contribute to functional variations. AlphaFold2 (AF2) predicted structures reveal that both G6PC1 and G6PC2 have conserved residues R40/R36 and D254/D252, respectively, but G6PC2 has lower catalytic activity .
A significant structural difference is that G6PC1 contains an aspartic acid at position 310, which can form an ionic interaction with R40, while G6PC2 has a histidine at the corresponding position (H308). Experimental evidence shows that substituting H308 with aspartic acid or glutamic acid in G6PC2 increases its G6Pase activity, supporting the hypothesis that this structural difference contributes to the markedly higher activity of G6PC1 .
What cell types express G6PC2 and how does this impact glucose homeostasis?
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In β cells: G6PC2 modulates insulin secretion sensitivity to glucose
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In α cells: G6PC2 controls glucose suppression of amino acid-stimulated glucagon secretion
What methods are recommended for validating G6PC2 antibody specificity?
When validating G6PC2 antibodies, a multi-faceted approach is essential:
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Genetic validation: Compare antibody staining in wild-type versus G6pc2 knockout tissues or cells. This provides the strongest evidence for specificity, as demonstrated in studies using G6pc2-/- mice .
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Western blot analysis: Verify a single band of appropriate molecular weight (~40 kDa for human G6PC2). The search results show successful detection of G6PC2 in transfected 832/13 cell lines, which can serve as a positive control for antibody validation .
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Epitope competition: Pre-incubate the antibody with recombinant G6PC2 protein or peptide before staining to confirm specific binding.
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Cross-reactivity testing: Assess potential cross-reactivity with G6PC1 and G6PC3, which share homology with G6PC2. This is particularly important given the structural similarities revealed by AlphaFold2 modeling .
How can researchers effectively solubilize and purify G6PC2 for antibody production?
Based on the search results, a novel method for solubilizing and purifying human G6PC2 has been developed . The protocol includes:
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Detergent selection: Lauryl maltose neopentyl glycol (LMNG) has proven effective for solubilizing G6PC2 while maintaining enzymatic activity .
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Expression system: Heterologous expression in islet-derived cell lines (such as 832/13) provides a suitable system for producing G6PC2 protein .
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Purification considerations: After solubilization with LMNG, G6PC2 actually showed increased G6Pase activity compared to microsomal preparations, suggesting this detergent preserves the enzyme's native conformation .
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Structural stability: Consider the importance of the intramolecular disulfide bond between cysteine residues 105 and 243, which is critical for protein stability. Disruption of this bond through mutation abolishes protein expression .
How is G6PC2 implicated in type 1 diabetes pathogenesis?
G6PC2 (previously known as IGRP) has been identified as a major target of autoreactive T cells implicated in type 1 diabetes pathogenesis in both mice and humans . Specifically:
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Autoantigen role: G6PC2 was identified as the molecular target of a CD8+ T-cell population that infiltrates the islets of NOD/ShiLtJ mice, a model of type 1 diabetes .
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Impact on disease progression: Interestingly, studies with G6pc2-/- mice on the NOD/ShiLtJ background demonstrated that while G6PC2 is an important driver for the selection and expansion of islet-reactive CD8+ T cells, autoreactivity to G6PC2 is not essential for autoimmune diabetes development .
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Therapeutic implications: The administration of G6PC2-derived peptides has been shown to abrogate or delay the disease process in NOD/ShiLtJ mice, suggesting potential tolerogenic therapeutic strategies .
What is the relationship between G6PC2 variants and metabolic phenotypes?
G6PC2 genetic variants have significant associations with metabolic traits:
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Fasting blood glucose: Genome-wide association studies have linked polymorphic variants in G6PC2 to variations in fasting blood glucose levels in humans .
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SNP rs492594 effect: This common non-synonymous SNP results in either valine or leucine at position 219, which is located within the cholesterol recognition amino acid consensus (CRAC) motif. The L219 variant has greater activity than the V219 variant in microsomal membrane preparations, though this difference disappears when G6PC2 is purified in detergent micelles .
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Cholesterol interaction: The V219 and L219 variants respond differently to cholesterol addition, with cholesteryl hemi-succinate decreasing the Vmax of V219 and L219 variants approximately 8-fold and 3-fold, respectively .
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Hemoglobin A1c: G6PC2 locus has been reproducibly associated with both fasting blood glucose and hemoglobin A1c levels .
How do the critical structural motifs in G6PC2 affect its function and antibody recognition?
Several structural motifs in G6PC2 are crucial for its function and may impact antibody recognition:
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Intramolecular disulfide bond: The bond between cysteine residues 105 and 243 is essential for G6PC2 expression. Mutation of these residues to alanine or serine abolishes protein expression . Antibodies targeting epitopes that depend on this disulfide bond may fail to recognize denatured protein.
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PAP2 motif: G6PC2 contains a modified type 2 phosphatidic acid phosphatase (PAP2) domain. Mutations in six regions of this motif significantly decrease enzyme activity . Researchers should consider developing antibodies that can distinguish between native and mutated PAP2 motifs.
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Substrate cavity: Residues R36 and D252 form part of the substrate cavity, and mutations affect both substrate affinity and maximum velocity. The R36A, D252A, and H308D variants increase the Km for G6P approximately 2.5- to 7-fold .
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CRAC motif: The cholesterol recognition amino acid consensus motif spans residues 219-226 and may interact with cholesterol. Mutation of residues 222 or 226 to alanine decreases expression .
What approaches are recommended for studying G6PC2's role in α cells?
Recent discoveries have expanded our understanding of G6PC2's role beyond β cells to include α cells, necessitating specialized approaches:
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Cell-specific knockout models: α cell-specific gene ablation of G6pc2 in mice has revealed that G6PC2 plays a critical role in controlling glucose suppression of amino acid-stimulated glucagon secretion, independent of alterations in insulin output, islet hormone content, or islet morphology .
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Human α cell validation: Findings from mouse models should be confirmed in primary human α cells to ensure translational relevance .
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Promoter analysis: Trait-associated variants in the G6PC2 promoter are located in open chromatin not just in β cells but also in α cells, suggesting cell-type-specific regulatory mechanisms .
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Allele-specific expression: Researchers should consider assessing allele-specific G6PC2 expression of linked variants in human α cells .
How can researchers effectively study the functional impact of G6PC2 inhibition?
Understanding the effects of G6PC2 inhibition is valuable for potential therapeutic applications:
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Bihormonal assessment: Since G6PC2 affects both insulin and glucagon secretion, researchers should simultaneously monitor both hormones when studying G6PC2 inhibition .
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Glucose homeostasis parameters: G6pc2-/- mice exhibit a 15% reduction in fasting blood glucose compared to wild-type littermates, providing a clear metabolic endpoint for inhibition studies .
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Experimental readouts: Key measurements should include:
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Potential therapeutic implications: Data suggest that G6PC2 inhibitors might help control blood glucose through a bihormonal mechanism, making this a promising area for drug discovery research .
What are the prospects for developing G6PC2 inhibitors as therapeutic agents?
G6PC2 inhibitors represent a promising therapeutic avenue for several reasons:
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Multiple therapeutic benefits: Because elevated fasting blood glucose is associated with increased risk for Type 2 diabetes, cardiovascular associated mortality, adverse pregnancy outcomes, heart disease, brain atrophy, and several types of cancer, G6PC2 inhibitors that lower FBG could have multiple therapeutic benefits .
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Rational drug design: The validated AlphaFold2 structure of G6PC2 and identification of critical functional motifs support the rational development of inhibitors designed to lower fasting blood glucose .
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Bihormonal mechanism: The discovery that G6PC2 affects glycemic control via actions in both α and β cells suggests inhibitors could control blood glucose through multiple mechanisms .
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Target validation: G6pc2 knockout models demonstrate reduced fasting blood glucose with minimal side effects, providing strong support for G6PC2 as a therapeutic target .
How can researchers utilize antibodies to investigate G6PC2 post-translational modifications?
G6PC2 function may be regulated through various post-translational modifications that can be studied using specialized antibody approaches:
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Disulfide bond formation: The critical disulfide bond between C105 and C243 suggests that oxidative folding is essential for G6PC2 stability. Researchers can develop redox-sensitive antibodies that specifically recognize properly folded G6PC2 .
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Phosphorylation states: Given G6PC2's role in metabolic signaling, phosphorylation may regulate its activity. Phospho-specific antibodies targeting potential regulatory sites would be valuable tools.
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Membrane association: G6PC2 is an ER membrane protein, and antibodies that distinguish between membrane-associated and solubilized forms could provide insights into its trafficking and localization.
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Cholesterol interaction: The CRAC motif suggests cholesterol binding may regulate G6PC2. Antibodies specifically designed to detect conformational changes induced by cholesterol binding could help investigate this mechanism .
