CD2 Human, GST

What is the structure and function of human CD2?

Human CD2 is a surface glycoprotein found on virtually all T cells, thymocytes, and natural killer (NK) cells. It binds to CD58 glycoprotein present on antigen-presenting cells (APCs) and promotes the initial stages of T cell contact with APCs even before T cell receptor (TCR) recognition of peptide-MHC complexes .

The extracellular segment of CD2 consists of two immunoglobulin superfamily domains:

• A nine-stranded N-terminal V set domain (lacking the first half of strand A)

• A seven-stranded membrane-proximal C2 set domain

The N-terminal domain mediates adhesion by binding to CD58, with the binding surface located on the highly charged GFCC′C″ face of the protein . The CD2 cytoplasmic tail contains multiple functional regions, including five proline-rich (PXXP) segments that are important for signaling .

What is the role of GST in cellular processes?

Glutathione S-transferases (GSTs) are a family of enzymes that play an essential role in cellular detoxification of toxic or carcinogenic compounds, including reactive oxygen species (ROS) . Their primary function is to catalyze the conjugation of glutathione (GSH) to various xenobiotic substrates, forming products that can be more easily excreted.

GSTs are particularly important in:

• Detoxification of environmental stressors and toxins

• Protection against oxidative stress

• Metabolism of certain drugs and chemicals

The activity of GSTs depends on GSH supply from γ-glutamylcysteine synthetase and glutathione synthetase, as well as transporters to remove GSH conjugates from cells . Compounds that induce GSTs or serve as substrates typically share a common chemical signal: a carbon-carbon double bond adjacent to an electron-withdrawing group .

How can researchers identify and characterize CD2-binding proteins?

For identifying proteins that interact with the CD2 cytoplasmic tail, researchers have successfully employed the yeast two-hybrid screening system. The methodology involves:

• Creating a bait construct with CD2 tail cDNA sequence fused to a DNA-binding domain (e.g., LexA)

• Screening against a prey library (e.g., T cell-derived cDNA library) fused to an activation domain

• Detecting positive interactions through reporter gene activation (leucine synthetase or lacZ)

Using this approach, researchers identified CD2BP1, which specifically associates with the CD2 cytoplasmic tail in T lymphocytes . Further characterization can be performed through:

• Biochemical mapping of interaction domains using GST fusion proteins

• In vitro binding assays with purified components

• Co-immunoprecipitation from cell lysates to verify interactions in a cellular context

• Mutational analysis to pinpoint critical binding residues

For example, the interaction between CD2 and CD2BP1 was mapped to the SH3 domain of CD2BP1 and the PPLP sequence (amino acids 302-305) in the C-terminal region of the CD2 tail .

What methods are most reliable for measuring GST activity across different experimental conditions?

The most widely used method for measuring GST activity is spectrophotometric monitoring of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione (GSH) at λmax=340nm at 37°C . The standard protocol involves:

• Preparing an enzyme assay mixture containing:

  • 0.5mM CDNB (in 2% ethanol)

  • 1.0mM GSH

  • 100mM Phosphate buffer (K₂HPO₄/KH₂PO₄; pH=6.5)

  • Distilled water

• Pre-incubating the Phosphate buffer-CDNB mixture for 10 minutes at 37°C

• Starting the reaction by adding GSH and measuring absorbance changes at 340nm

• Not all GST isoforms use CDNB as a substrate, potentially leading to underestimation of certain GST activities

• Activity is dependent on adequate GSH supply

• Hemoglobin concentration should be determined when measuring GST activity in erythrocytes to control for spectrophotometric interference

• Cell lysis methods (e.g., freezing/thawing) can affect enzyme activity measurements

For comprehensive GST activity assessment, using multiple substrates beyond CDNB is recommended.

How can GST be effectively used as an affinity tag for CD2 protein purification and characterization?

GST serves as an effective affinity tag for CD2 purification and characterization due to several advantages:

• The structure of GST has been fully determined, making it useful for finding the conformation of target molecules through phase information in crystallographic studies .

• GST fusion proteins can be purified using glutathione affinity chromatography, offering a straightforward single-step purification process.

• Heat treatment can be employed during purification as GST-tagged proteins often maintain their conformations during this process, providing an alternative method for producing small proteins and peptides .

• For structural studies, GST tags can be employed during NMR data acquisition without affecting the quality of NMR data of the fused partner protein .

When designing CD2-GST fusion constructs, researchers should consider:

• The linker between GST and CD2, which affects fusion protein flexibility

• Potential dimerization effects (GST naturally forms dimers)

• The need for protease cleavage sites if tag removal is required

The use of GST as an affinity tag allows for easier purification and characterization of CD2 protein while maintaining its native conformation, which is crucial for functional and structural studies.

What controls are essential when studying CD2-mediated T cell activation?

When investigating CD2-mediated T cell activation, several critical controls should be included:

• Domain-specific mutations: The CD2 cytoplasmic tail contains multiple functional regions with distinct roles. Controls should include mutations in:

  • The two N-terminal PPPGHR sequences (amino acids 260-265 and 274-279), which are crucial for IL-2 production and calcium flux

  • The PPLP sequence (amino acids 302-305), which interacts with CD2BP1

  • The highly conserved C-terminal region (amino acids 297-314)

• Divalent cation controls: The interaction between CD2 and binding partners like CD2BP1 is dependent on divalent cations. Experiments should include:

  • Conditions with various concentrations (2 μM to 2 mM) of Zn²⁺, Mg²⁺, or Ca²⁺

  • EDTA-treated controls to demonstrate cation dependency

• Cell type controls: Compare CD2 expression and function across:

  • Primary T cells from different sources

  • T cell lines with varying CD2 expression levels

  • Transgenic models with human CD2 expression

• Stimulation controls: Include appropriate positive and negative controls:

  • Anti-CD3 stimulation (TCR pathway)

  • PMA/ionomycin (bypass proximal signaling)

  • Isotype control antibodies

• Readout controls: Multiple T cell activation markers should be assessed:

  • IL-2 production

  • Calcium flux

  • Surface activation markers (CD69, CD25)

  • Proliferation

These controls help distinguish CD2-specific effects from other signaling pathways and ensure experimental validity.

How should researchers design experiments to study the effects of Cd²⁺ on GST activity and expression?

When investigating cadmium (Cd²⁺) effects on GST activity and expression, researchers should implement the following design considerations:

• Media and treatment conditions:

  • Use serum-free media for all Cd²⁺ treatments to prevent interactions between Cd²⁺ and serum proteins

  • Determine appropriate Cd²⁺ concentrations through preliminary dose-response studies (e.g., 3 μM Cd²⁺ for 24h induced <5% cell death in fibroblasts)

• Cell viability assessment:

  • Include trypan blue-exclusion assay or other viability tests to ensure observed effects aren't due to cytotoxicity

• Quantitative proteomic approach:

  • Employ SILAC-based metabolic labeling with both forward and reverse labeling schemes

  • Conduct multiple biological replicates (minimum three) to ensure reproducibility

• Pathway analysis:

  • Examine multiple cellular pathways potentially affected by Cd²⁺, including:

    • Glutathione metabolism

    • Glycolysis and gluconeogenesis

    • Pyruvate metabolism

    • Pentose phosphate pathway

    • Fatty acid metabolism

    • Citric acid cycle

• Validation experiments:

  • Confirm proteomic findings with Western blot analysis for key proteins

  • Measure functional outcomes (e.g., NO production using nitrate/nitrite assays)

  • Assess mRNA levels of relevant genes using RT-PCR

The table below shows examples of proteins significantly upregulated in human skin fibroblasts after Cd²⁺ treatment:

This comprehensive approach helps elucidate the mechanisms underlying Cd²⁺-induced changes in GST activity and expression .

What factors should be considered when using CD2-GST fusion proteins for structural studies?

When conducting structural studies with CD2-GST fusion proteins, researchers should consider several critical factors:

• Fusion protein design:

  • Linker selection: The linker between GST and CD2 significantly impacts flexibility and may cause signal loss in NMR studies due to decreased T2 relaxation rates upon dimerization

  • Fusion orientation: Consider whether N-terminal or C-terminal GST fusion is more appropriate for the specific structural study

  • Cleavage sites: Include protease recognition sequences if tag removal is necessary for certain analyses

• Dimerization effects:

  • GST naturally forms dimers, which can complicate structural analyses

  • Account for the potential impact of dimerization on CD2 conformation and function

  • Consider using monomeric GST variants for certain applications

• NMR considerations:

  • Despite signal loss for the GST portion, the target protein signals can remain intact in 1H-15N HSQC spectra

  • The method allows collecting structural information on the protein/peptide of interest while employing the GST-tagged target during NMR data acquisition

• Temperature stability:

  • GST fusion proteins often maintain their conformations during heat treatment

  • This property can be exploited for purification steps prior to structural studies

• Buffer optimization:

  • For CD2-specific considerations, include appropriate divalent cations (Zn²⁺, Mg²⁺, or Ca²⁺) as these are critical for CD2 interactions

  • Optimize pH and ionic strength based on the specific properties of the CD2 domain being studied

By carefully addressing these factors, researchers can maximize the quality and relevance of structural data obtained from CD2-GST fusion proteins.

How can researchers reconcile contradictory findings regarding GST polymorphisms and disease associations?

Contradictory findings regarding GST polymorphisms and disease associations are common in the literature. Researchers can address these discrepancies through several methodological approaches:

By implementing these approaches, researchers can develop more nuanced and reliable interpretations of the relationships between GST polymorphisms and disease risk.

What are common pitfalls in measuring GST activity across different experimental systems?

Several common pitfalls can affect GST activity measurements across experimental systems:

• Substrate limitations:

  • CDNB is the most common substrate used in GST activity assays

  • Not all GST isoforms can use CDNB efficiently, potentially leading to underestimation of certain GST activities

  • The Ile105Val polymorphism in GSTP results in decreased GST activity towards CDNB

• GSH dependency:

  • GST activity depends on adequate GSH supply

  • Variations in γ-glutamylcysteine synthetase, glutathione synthetase, or GSH transporters across experimental systems affect measurements

  • GSH levels should be measured alongside GST activity

• Genetic variation effects:

  • Polymorphisms in GSTs significantly alter enzyme activity

  • The GSTT1null and GSTM1null genotypes lead to loss of protein expression and detoxification activity

  • Genotyping should accompany activity measurements when possible

• Disease state interference:

  • Pathological conditions can alter GST activity independently of genetic factors

  • For example, erythrocytes infected with Plasmodium falciparum show significantly lower GST activity compared to healthy controls

• Cell-specific expression patterns:

  • Different GST classes are expressed in different organs and cell types

  • Activity comparisons across cell types must account for baseline expression differences

• Methodological variations:

  • Buffer composition, temperature, and pH affect enzyme kinetics

  • Standardization of assay conditions is essential for cross-study comparisons

By addressing these pitfalls through careful experimental design and appropriate controls, researchers can obtain more reliable and comparable GST activity measurements.

How should researchers interpret the divalent cation dependency of CD2-CD2BP1 interactions?

The divalent cation dependency of CD2-CD2BP1 interactions has significant implications for experimental design and data interpretation:

• Physiological relevance:

  • The requirement for divalent cations (Zn²⁺, Mg²⁺, or Ca²⁺) at concentrations from 2 μM to 2 mM suggests that these interactions are regulated by physiological fluctuations in cation availability

  • Previous sequence analysis of the CD2 tail raised the possibility that it might contain a cation-binding site

• Experimental design considerations:

  • All binding experiments must include appropriate divalent cations

  • Negative controls should include chelating agents (e.g., EDTA) to demonstrate cation dependency

  • Multiple cations should be tested to determine specificity and optimal conditions

• Structural implications:

  • Divalent cations may induce conformational changes in either CD2 or CD2BP1 that facilitate binding

  • The SH3 domain of CD2BP1 mediates this interaction, suggesting a non-typical SH3-binding mechanism that involves cations

  • Traditional SH3 domain interactions typically don't require divalent cations, making this interaction unique

• Signaling context:

  • Intracellular cation concentrations fluctuate during T cell activation

  • Changes in cation availability may represent a regulatory mechanism for CD2-CD2BP1 interactions during T cell signaling

  • The cation dependency links CD2 signaling to cellular ion homeostasis

• Binding site localization:

  • The interaction was mapped to the PPLP sequence (amino acids 302-305) in the CD2 tail

  • This region falls within a sequence of 18 amino acids that is the most highly conserved CD2 cytoplasmic tail region across all species studied

  • The conservation suggests functional importance of this cation-dependent interaction

Understanding this cation dependency is crucial for correctly interpreting CD2-CD2BP1 interaction studies and their physiological relevance to T cell function.

How are transgenic models enhancing our understanding of CD2 regulation and function?

Transgenic models expressing human CD2 have provided significant insights into CD2 regulation and function:

• Genomic organization and expression control:

  • A 28.5 kb segment of DNA containing the human CD2 gene (including 4.5 kb of 5' flanking sequences, 15 kb containing the gene's five exons, and 9 kb of 3' flanking sequences) directs CD2 expression specifically on thymocytes, circulating T cells, and megakaryocytes in transgenic mice

  • This demonstrates that this DNA segment contains all necessary regulatory elements for tissue-specific expression

• Copy number and integration site effects:

  • Expression of each copy of the human CD2 transgene appears equivalent to the endogenous mouse CD2 gene and human T lymphocyte CD2 expression

  • Expression is independent of the integration site but dependent on the copy number of integrated genes

  • This suggests robust regulatory mechanisms controlling CD2 expression

• Tissue-specific expression mechanisms:

  • The transgenic models confirm CD2 expression is tightly restricted to specific hematopoietic lineages

  • This provides a system to study the cis-regulatory elements controlling this specificity

• Cross-species conservation:

  • The successful expression of human CD2 in mice indicates conservation of the regulatory machinery controlling CD2 expression

  • This facilitates comparative studies of CD2 function across species

These transgenic approaches provide valuable tools for studying CD2's role in T cell development, immune responses, and potential therapeutic approaches targeting CD2 signaling pathways.

What recent proteomic approaches have advanced our understanding of GST function in oxidative stress?

Recent proteomic approaches have significantly enhanced our understanding of GST function in oxidative stress response:

• SILAC-based quantitative proteomics:

  • Stable Isotope Labeling with Amino acids in Cell culture (SILAC) combined with LC-MS/MS analysis has revealed comprehensive changes in protein expression following oxidative stress

  • This approach identified numerous proteins with altered expression in human skin fibroblasts after cadmium (Cd²⁺) treatment

• Pathway and network analysis:

  • Bioinformatic tools like DAVID and STRING have enabled the identification of perturbed cellular pathways

  • These analyses revealed Cd²⁺-induced changes in multiple interconnected pathways including glutathione metabolism, glycolysis, pyruvate metabolism, and the citric acid cycle

• Post-translational modification analysis:

  • Proteomic approaches can now identify oxidative modifications of GSTs and their substrates

  • This helps understand how oxidative stress directly affects GST function beyond expression changes

• Integration with transcriptomics:

  • Combined analysis of protein and mRNA levels provides insights into regulatory mechanisms

  • For example, RT-PCR analysis showed significant reductions in mRNA levels of cholesterol biosynthesis genes (HMGCR and FDPS) following Cd²⁺ exposure, complementing proteomic findings

The table below highlights key proteins in antioxidant and GST pathways upregulated after Cd²⁺ treatment:

These proteomic approaches provide comprehensive insights into how GSTs function within the broader cellular response to oxidative stress .