GPC4 511 aa Human

What is the molecular structure of GPC4 511 aa Human protein?

GPC4 511 aa Human is a recombinant protein produced in E.Coli as a single, non-glycosylated polypeptide chain spanning from Ala19 to Ser529, containing 521 amino acids with an additional 10 amino acid histidine tag at the N-terminus . The total calculated molecular mass of this recombinant protein is 58.7kDa. Unlike native GPC4, this recombinant version lacks glycosylation modifications and is designed for research applications requiring purified protein. The absence of glycosylation should be considered when interpreting experimental results, as native GPC4 contains functionally important N-linked glycosylation at Asn514 .

How should GPC4 511 aa Human be stored for optimal stability?

For optimal stability of GPC4 511 aa Human, multiple storage recommendations apply depending on intended usage timeframe. For short-term use (2-4 weeks), storage at 4°C is sufficient if the entire vial will be used within this period . For longer storage periods, the protein should be kept frozen at -20°C. To enhance stability during long-term storage, it is recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) . Multiple freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of biological activity. For researchers conducting long-term studies, aliquoting the protein before freezing is advisable to minimize freeze-thaw cycles.

What are the key biological functions of GPC4 protein?

GPC4 belongs to the glypican family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during embryonic and tissue development . Based on research findings, GPC4 functions as:

• A regulator of morphogenesis during developmental processes

• An insulin-sensitizing adipokine that serves as a marker for body mass index and insulin resistance in humans

• A potential marker for kidney function, with high serum GPC4 being associated with decreased prevalent and future kidney function

• A developmental regulator whose loss of function is associated with Keipert syndrome, characterized by a prominent forehead, flat midface, hypertelorism, broad nose, and digital abnormalities

The protein plays critical roles in cell signaling pathways and is expressed during development in a variety of tissue types, making it relevant to multiple developmental and metabolic processes .

What experimental approaches are optimal for studying GPC4 protein interactions?

Studying GPC4 protein interactions requires multiple complementary approaches to fully characterize its binding partners and signaling mechanisms:

• Recombinant Protein Expression Systems: Utilize V5-tagged wild-type and mutant (e.g., p.Glu496* and p.Gln506*) GPC4 in HEK293 cells for comparative binding studies .

• Protein Stability Assays: Assess stability through proteasome inhibition experiments using compounds like MG-132 (10μM) to inhibit the ubiquitin proteasome system (UPS), followed by immunoblot analysis to quantify steady-state protein levels .

• Flow Cytometry Analysis: For cell surface expression studies, flow cytometry using anti-GPC4 antibodies can detect GPC4 on cell surfaces, as demonstrated in TF-1 human erythroleukemic cells and BG01V human embryonic stem cells .

• Immunocytochemistry: Visualization of cellular localization patterns of wild-type versus mutant GPC4 can reveal trafficking defects or altered membrane insertion .

• Co-immunoprecipitation: To identify binding partners, particularly growth factors whose signaling is regulated by GPC4.

When analyzing results, researchers should account for the differences between recombinant non-glycosylated GPC4 and the native glycosylated form, as glycosylation at Asn514 and the GPI anchor at Ser529 are functionally important .

How can researchers effectively measure GPC4 levels in clinical samples?

Accurately measuring GPC4 levels in clinical samples requires careful consideration of methodology:

• ELISA Development: Paired antibody systems, such as the Mouse Anti-Human Glypican 4 Monoclonal Antibody (Clone #961609) as a capture antibody, can be used to develop sensitive ELISA assays . This approach is particularly valuable for serum samples where GPC4 has been linked to kidney function and metabolic parameters.

• Sample Collection Standardization: For serum GPC4 analysis, standardized collection methods are crucial as time-dependent degradation may occur. Samples should be processed within 2 hours of collection and stored at -80°C until analysis.

• Clinical Correlation Studies: When measuring GPC4 in patient samples, researchers should collect comprehensive clinical data including:

  • Estimated glomerular filtration rate (eGFR)

  • Albuminuria status

  • Body mass index

  • Insulin resistance markers

• Longitudinal Sampling: For predictive studies of kidney function, a longitudinal approach with baseline and follow-up measurements (e.g., 3.4 years apart) is recommended, as employed in coronary angiography patient cohorts .

• Statistical Analysis Considerations: Odds ratios adjusted for relevant confounders should be calculated per standard deviation of GPC4 levels to standardize reporting and enable inter-study comparisons .

What are the current challenges in interpreting GPC4 mutation analysis in clinical genetics?

Interpreting GPC4 mutation analysis presents several challenges that researchers must address:

• Phenotypic Heterogeneity: The clinical presentation of GPC4 mutations shows significant variability. For example, while Keipert syndrome was initially characterized to include sensorineural hearing loss, subsequent cases with confirmed GPC4 pathogenic variants did not exhibit this feature, suggesting it might be relatively infrequent .

• Variant Classification Complexity: The presence of hemizygous loss-of-function variants in population databases like gnomAD (albeit at very low frequency: 1.1 × 10^-5) complicates the classification of novel variants . Researchers must consider:

  • Segregation analysis in families

  • X-inactivation studies in carrier females

  • Functional validation of variants

• Overlapping Phenotypes: Distinguishing Keipert syndrome from similar conditions is challenging. A hemizygous missense variant (c.1235G>A [p.Arg412Lys]) was reported in an individual with Robinow-syndrome-like phenotype sharing features with Keipert syndrome, yet presenting additional findings not observed in classic Keipert syndrome .

• Genotype-Phenotype Correlations: The relationship between specific mutation types and clinical features remains unclear, particularly regarding intellectual disability (observed in 8/10 cases with variable severity) and hearing loss (found in only 3/9 cases) .

These challenges highlight the importance of combining genetic findings with functional studies and detailed phenotyping for accurate diagnosis and counseling.

How can GPC4 511 aa Human be used in functional studies of development?

GPC4 511 aa Human can be employed in multiple developmental biology research paradigms:

• In vitro Developmental Models: The recombinant protein can be used to supplement culture media in embryoid body or organoid systems to assess the impact on morphogenesis and tissue patterning. Researchers should consider concentration-dependent effects, typically testing ranges from 10-500 ng/mL.

• Synaptogenesis Research: GPC4 has been utilized in studying the formation of active synapses . Recombinant GPC4 can be applied to neuronal cultures to examine its effects on synapse number, morphology, and function through electrophysiological recordings and immunocytochemistry.

• Competitive Binding Assays: GPC4 511 aa Human can be used to compete with endogenous GPC4 for binding partners, helping elucidate signaling pathways regulated by this protein during development.

• Rescue Experiments: In cellular models with GPC4 knockdown or knockout, adding recombinant GPC4 can determine whether developmental phenotypes can be rescued, providing insight into protein domain functionality.

• Heparan Sulfate Interaction Studies: Though non-glycosylated, the recombinant protein can still be used in comparative studies with native GPC4 to understand the specific contributions of the protein backbone versus its glycosaminoglycan chains in developmental signaling.

When designing these experiments, researchers should include appropriate controls and consider that the recombinant protein lacks glycosylation and GPI anchor present in the native protein .

What are the methodological considerations for studying GPC4 in relation to kidney function?

Research into GPC4's relationship with kidney function requires careful methodological planning:

• Cohort Selection: Studies should include subjects with varying degrees of kidney function, as demonstrated in research with 456 Caucasian coronary angiography patients that revealed associations between serum GPC4 and kidney function .

• Kidney Function Assessment:

  • eGFR calculation using standardized formulas (CKD-EPI or MDRD)

  • Albuminuria measurement (spot urine albumin-to-creatinine ratio)

  • Definition of chronic kidney disease (CKD) as eGFR < 60 mL/min/1.73 m² or presence of albuminuria

• Statistical Analysis Approach:

  • Adjustment for confounding variables in multivariate models

  • Calculation of odds ratios per standard deviation of GPC4 levels

  • ROC analysis to assess additional predictive value beyond existing markers

• Longitudinal Study Design: Prospective studies with median follow-up periods of at least 3-4 years allow assessment of GPC4's predictive value for future kidney function .

• Experimental Models: In vitro studies using kidney cell lines or ex vivo kidney tissue can explore the mechanistic relationships between GPC4 and kidney function.

Data from a prospective cohort study with 456 Caucasian coronary angiography patients

How should researchers design experiments to compare wild-type and mutant GPC4 proteins?

Designing experiments to compare wild-type and mutant GPC4 proteins requires systematic approaches:

• Expression System Selection: Stable transfection of HEK293 cells with expression constructs encoding V5-tagged wild-type or truncated (e.g., p.Glu496* and p.Gln506*) GPC4 provides a consistent cellular context for comparisons . Cells should be cultured for approximately 3 weeks in media with 400 μg/mL geneticin to establish stable populations.

• Protein Stability Assessment:

  • Baseline protein quantification via immunoblotting

  • Proteasome inhibition experiments using 10μM MG-132 for 14 hours

  • Calculation of fold-change in protein levels before and after proteasome inhibition

• Subcellular Localization Studies:

  • Immunocytochemistry with antibodies against the V5 tag

  • Co-localization studies with markers for endoplasmic reticulum, Golgi apparatus, and plasma membrane

  • Quantitative image analysis of cellular distribution patterns

• Functional Assays:

  • Growth factor binding capacity comparisons

  • Cell signaling pathway activation assessment

  • Developmental effects in relevant cell models

• Data Analysis Framework:

  • Normalization to housekeeping proteins/genes

  • Statistical comparison using paired t-tests or ANOVA

  • Fold-change calculations relative to wild-type protein

As demonstrated in previous research, wild-type GPC4 shows stable expression levels, while mutants like p.Gln506* and p.Glu496* exhibit approximately 2.5-fold and 6-fold decreases in steady-state levels, respectively, when the ubiquitin proteasome system is active . This indicates reduced stability of the mutant proteins, providing insight into the pathogenic mechanism underlying Keipert syndrome.

What are the potential applications of GPC4 as a biomarker in metabolic and kidney diseases?

GPC4 shows considerable promise as a biomarker in several clinical contexts:

• Kidney Function Prediction: High serum GPC4 is associated with decreased prevalent and future kidney function, demonstrating potential as a predictive biomarker for declining renal function . ROC analysis indicates GPC4 provides additional predictive value beyond basic prediction models for newly diagnosed CKD and eGFR <60 mL/min/1.73 m² .

• Insulin Resistance Assessment: As an insulin-sensitizing adipokine, GPC4 serves as a marker for insulin resistance and may complement existing biomarkers in diabetes risk assessment and management .

• Cardiovascular Risk Stratification: In coronary angiography patients, GPC4 could potentially enhance risk prediction models for adverse outcomes, particularly when combined with kidney function parameters.

• Developmental Disorders: Circulating GPC4 levels might serve as biomarkers for certain developmental disorders, though more research is needed to establish reference ranges and clinical correlations.

• Therapeutic Monitoring: In future GPC4-targeted therapies, serum levels could potentially serve as pharmacodynamic markers.

Researchers exploring these applications should design studies with appropriate control groups, standardized sample collection and processing protocols, and rigorous statistical analyses that account for potential confounding factors.

How does GPC4 interact with other glypican family members in signaling pathways?

Understanding GPC4 interactions with other glypican family members remains an active area of research:

• GPC3-GPC4 Functional Relationships: GPC4 duplication has been implicated in Simpson-Golabi-Behmel syndrome (SGBS), an X-linked disorder typically caused by pathogenic variation in GPC3 . This suggests potential functional relationships or compensatory mechanisms between these two glypicans.

• Redundancy and Specificity: Researchers should design experiments to determine which functions are specific to GPC4 versus shared among glypican family members. Approaches may include:

  • Simultaneous knockdown/knockout of multiple glypicans

  • Domain-swapping experiments between glypican family members

  • Competitive binding assays for shared ligands

• Tissue-Specific Interaction Networks: Different glypicans may form tissue-specific interaction networks that regulate development and homeostasis. Single-cell transcriptomic approaches coupled with protein-protein interaction studies can help map these networks.

• Co-receptor Functions: Glypicans often function as co-receptors for growth factors and morphogens. Investigating how GPC4 cooperates with or antagonizes other glypican family members in these co-receptor functions is crucial for understanding developmental signaling.

• Evolutionary Conservation: Comparative studies across species can reveal conserved and divergent aspects of glypican family member functions and interactions, providing insight into their fundamental biological roles.

What are the current gaps in understanding GPC4 genetics and phenotypic expression?

Several significant knowledge gaps exist in GPC4 research:

• Incomplete Penetrance and Variable Expressivity: The mechanisms underlying the variability in Keipert syndrome phenotypes remain poorly understood. For instance, sensorineural deafness, initially described as a cardinal feature, was observed in only 3 of 9 patients with confirmed GPC4 pathogenic variants .

• Domain-Specific Functions: While C-terminal truncations affecting glycosylation and GPI anchor sites clearly cause disease, the functions of other GPC4 domains are less well characterized. This limits our understanding of how different variants might affect protein function.

• Tissue-Specific Effects: The expression pattern of GPC4 "in all tissues except ovary" suggests broad biological roles, but the tissue-specific consequences of GPC4 dysfunction remain unclear .

• Genotype-Phenotype Correlations: Current data does not fully explain why some GPC4 variants cause more severe phenotypes than others. A systematic analysis of mutation type, protein domain affected, and clinical features is needed.

• X-Inactivation Patterns: While extreme skewing of X-inactivation (>90%) has been observed in female carriers from multiple families, the mechanisms driving this skewing and its potential protective effects require further investigation .

• Modifier Genes: The influence of genetic background and modifier genes on GPC4-associated phenotypes remains largely unexplored but may explain some of the phenotypic variability observed in affected individuals.

Addressing these gaps will require comprehensive approaches combining clinical genetics, functional genomics, and developmental biology research methodologies.

What are the best practices for integrating GPC4 research across different disciplines?

Effective integration of GPC4 research across disciplines requires:

• Standardized Nomenclature and Reporting: Consistent reporting of GPC4 variants, protein domains, and phenotypic features enables cross-disciplinary comparison of findings.

• Multidisciplinary Collaboration Frameworks: Teams combining expertise in developmental biology, clinical genetics, nephrology, metabolism, and molecular biology can address complex questions about GPC4 function and dysfunction.

• Translational Research Pipelines: Establishing pathways to translate basic GPC4 findings into clinical applications for diagnosis, prognosis, and potential therapeutic development.

• Data Sharing Initiatives: Contributing to and utilizing resources like GeneMatcher to identify additional cases of GPC4-related disorders and expand our understanding of genotype-phenotype correlations .

• Methodological Triangulation: Employing multiple complementary approaches (genetic, biochemical, cellular, clinical) to address research questions about GPC4 biology and pathology.

By implementing these best practices, researchers can accelerate progress in understanding GPC4's complex roles in development, disease, and potential therapeutic applications.

How should researchers interpret conflicting data on GPC4 function across different model systems?

When faced with conflicting data on GPC4 function, researchers should:

• Systematically Evaluate Model System Differences:

  • Cell line-specific factors (expression of co-receptors, signaling pathways)

  • Species differences in GPC4 sequence, modification, and interacting partners

  • In vitro versus in vivo contexts that may affect protein localization and function

• Assess Protein Form and Modifications:

  • Consider whether studies used full-length versus truncated GPC4

  • Evaluate presence/absence of post-translational modifications like glycosylation

  • Determine if GPI anchor was present, as this affects membrane localization

• Examine Experimental Conditions:

  • Protein concentration differences that may affect signaling outcomes

  • Temporal aspects of experiments (acute vs. chronic exposure)

  • Environmental factors that might influence protein stability or function

• Statistical and Methodological Rigor Assessment:

  • Sample size and power calculations

  • Appropriate controls and normalizations

  • Validation across multiple experimental approaches

• Consider Biological Complexity:

  • GPC4 may have context-dependent functions in different tissues/developmental stages

  • Compensatory mechanisms might mask phenotypes in some model systems

  • Interaction with other glypican family members may vary across models