Recombinant Human CD82 antigen (CD82)

What is CD82 and what are its basic structural characteristics?

CD82 (also known as KAI-1) is a tetraspanin family membrane protein that plays crucial roles in cell adhesion, migration, and signaling. The mature human CD82 protein consists of 267 amino acids with a predicted molecular mass of approximately 15 kDa, though glycosylation increases its apparent molecular mass to 25-33 kDa in SDS-PAGE analysis . The protein contains four transmembrane domains, forming a characteristic tetraspanin structure with intracellular N- and C-termini, and two extracellular loops. The large extracellular loop (LEL) mediates most protein-protein interactions and contains most of the glycosylation sites.

When working with recombinant forms, researchers commonly use fragments containing the functionally important regions, such as the Gly111-Leu228 segment that includes portions of the LEL .

How does CD82 expression vary across different cell types?

CD82 is expressed on various cell types, including immune cells (particularly T lymphocytes), epithelial cells, and certain stem/progenitor populations. Expression levels are dynamically regulated in different physiological and pathological contexts:

• Immune cells: CD82 is constitutively expressed on resting T cells but significantly upregulated (up to 5-fold in CD8+ T cells and 9-fold in CD4+ T cells) following TCR activation and IL-2 stimulation .

• Cancer cells: CD82 expression is frequently downregulated in metastatic cancer tissues compared to primary tumors or normal tissues, consistent with its role as a metastasis suppressor .

• Developmental contexts: CD82 expression marks specific progenitor populations, such as pancreatic β-cell precursors, where it can serve as a selection marker for cell isolation .

When designing experiments, researchers should consider these tissue-specific and context-dependent expression patterns to accurately interpret results.

What are the primary biological functions of CD82?

CD82 functions as a molecular organizer within the plasma membrane, influencing multiple cellular processes:

• Immune cell regulation: CD82 enhances T-cell activation, cytokine production (particularly IFN-γ, TNF-α, and IL-2), and memory subset accumulation .

• Metastasis suppression: CD82 inhibits cell migration and invasion by regulating integrin-dependent adhesion, membrane organization, and cytoskeletal dynamics .

• Cell differentiation: CD82 contributes to the maturation of certain progenitor populations, such as pancreatic β-cell precursors into insulin-producing cells .

• Membrane organization: CD82 modulates membrane mechanics, affects caveolae integrity, and influences stress fiber formation, focal adhesions, and mechanotransduction pathways .

These diverse functions make CD82 relevant to multiple research fields, including immunology, oncology, and developmental biology.

How does CD82 influence T-cell activation and function?

CD82 plays multifaceted roles in T-cell biology, functioning as both a marker and modulator of activation:

• Activation marker: CD82 expression increases substantially following TCR stimulation, with up to 9-fold increases in CD4+ T cells and 5-fold increases in CD8+ T cells, making it a reliable activation indicator .

• Co-stimulatory function: CD82 can function as a co-stimulatory molecule analogous to CD28. When T cells are stimulated with anti-CD3 in combination with anti-CD82 antibodies, they exhibit enhanced activation compared to anti-CD3 alone .

• Cytokine production: T cells with high CD82 expression (CD82high) produce significantly higher levels of cytotoxic cytokines compared to CD82low cells:

  • IFN-γ: ~900 pg/mL in CD82high vs. minimal in CD82low cells

  • TNF-α: ~2000 pg/mL in CD82high vs. ~150 pg/mL in CD82low cells

• IL-2 regulation: CD82 co-stimulation promotes sustained IL-2 production in CD4+ T cells over 72 hours, with a steeper production curve than even CD28 co-stimulation .

• Memory cell differentiation: CD82high T cells more efficiently differentiate from naive to central memory phenotypes. In CD82high populations, the naive subset (CCR7+/CD45RA+) decreases to 7-10% while the central memory subset (CCR7+/CD45RA-) increases to 70-80% .

For immunological research, these findings suggest CD82 could be targeted to enhance T-cell responses in immunotherapy approaches.

What methodologies are recommended for studying CD82's role in cytotoxic T-cell function?

When investigating CD82's impact on cytotoxic T-cell function, consider these methodological approaches:

• Cell sorting strategies: Sort T cells into CD82high and CD82low populations using flow cytometry, then compare their functional properties. For optimal results, sort cells after 2 days of IL-2R stimulation and anti-CD3/28 activation .

• Cytotoxicity assays: Co-culture CD82-expressing T cells with target cells and measure cytolysis using real-time imaging or flow cytometry-based assays. CD8+ T cells overexpressing CD82 can achieve up to 80% target cell cytolysis by 36 hours .

• Cytokine profiling: Measure cytokine secretion using ELISA following TCR stimulation. Compare supernatants from CD82high and CD82low T cells to assess differences in IFN-γ, TNF-α, and other effector molecules .

• Co-stimulation experiments: Compare T-cell responses to anti-CD3 alone versus anti-CD3 plus anti-CD82 antibodies (5 μg/mL) to evaluate CD82's co-stimulatory potential. Include anti-CD28 as a positive control .

• Memory subset analysis: Use flow cytometry with CCR7 and CD45RA markers to track changes in naive (CCR7+/CD45RA+), central memory (CCR7+/CD45RA-), and effector memory (CCR7-/CD45RA-) subsets over time (7-day culture recommended) .

When designing these experiments, standardize stimulation conditions (anti-CD3 concentration: 0.5 μg/mL; IL-2 supplementation) to ensure reproducibility.

How does CD82 function as a metastasis suppressor?

CD82 (KAI-1) has been extensively characterized as a metastasis suppressor, with multiple mechanisms contributing to this function:

• Cell migration inhibition: CD82 overexpression significantly reduces cell motility and migratory capacity, as demonstrated in trophoblast and various cancer cell models . This occurs through:

  • Regulation of integrin-dependent adhesion

  • Modulation of cell membrane organization

  • Alteration of cytoskeletal dynamics

• ECM remodeling regulation: CD82 influences the gelatinolytic activities of matrix metalloproteinases (MMPs), enzymes crucial for degrading extracellular matrix components during invasion .

• Membrane mechanics modulation: CD82 affects fundamental membrane properties by:

  • Regulating stress fiber formation

  • Controlling focal adhesion dynamics

  • Influencing caveolae integrity

  • Modulating membrane tension

  • Affecting YAP nuclear translocation in a caveolin-1-dependent manner

• Signaling pathway interference: CD82 interacts with and modulates multiple signaling pathways implicated in metastasis, including:

  • EGFR-mediated signaling

  • Integrin-dependent pathways

  • Mechanotransduction cascades involving YAP/TAZ

Loss of CD82 expression is frequently observed in advanced stages of multiple cancer types, including breast cancer, correlating with increased metastatic potential .

What experimental models are most appropriate for studying CD82's anti-metastatic properties?

When investigating CD82's anti-metastatic functions, consider these experimental models and approaches:

• 2D migration assays: Track single-cell trajectories using time-lapse microscopy in CD82-overexpressing versus control cells. EGF stimulation can be used to trigger migration .

• 3D invasion models: Use transwell chambers coated with Matrigel or collagen to assess invasive capacity. Compare invasion rates between CD82-manipulated and control cells, quantifying the number of cells crossing the matrix barrier .

• Cell line models: Several well-characterized cell lines are suitable for CD82 metastasis studies:

  • HTR8/SVneo (trophoblast cells) - well-established for studying CD82's effects on migration and invasion

  • Breast cancer cell lines (MCF-7, MDA-MB-231) - frequently used due to CD82's relevance in breast cancer progression

  • Prostate cancer cell lines - where CD82 was initially identified as a metastasis suppressor

• Mechanistic studies: To understand the molecular basis of CD82's effects:

  • Visualize focal adhesions using Talin-GFP labeling in CD82-expressing cells

  • Track YAP nuclear localization through immunofluorescence

  • Monitor actin cytoskeleton organization with phalloidin staining

  • Assess caveolae integrity using electron microscopy or caveolin-1 immunostaining

• In vivo metastasis models: For translational relevance, use mouse models with CD82-manipulated cancer cells to assess:

  • Primary tumor growth

  • Metastatic spread

  • Target organ colonization

These complementary approaches provide a comprehensive assessment of CD82's role in the metastatic cascade.

What role does CD82 play in progenitor cell differentiation?

CD82 has emerged as an important regulator of stem and progenitor cell differentiation, particularly in the context of pancreatic β-cell development:

• Progenitor cell marker: CD82 serves as a surface marker for isolating specific progenitor populations. In pancreatic differentiation, CD82 expression identifies late-stage pancreatic progenitor cells with enhanced potential to differentiate into endocrine cells .

• Functional contribution: Beyond its utility as a marker, CD82 actively contributes to differentiation processes:

  • CD82+ progenitor cells show superior insulin secretion capacity compared to CD82- cells

  • Knockdown of CD82 impairs β-cell function, indicating its functional role in maturation

  • CD82 likely influences membrane organization and signaling pathways critical for differentiation

• Developmental dynamics: CD82 expression changes dynamically during differentiation, with stage-specific patterns that reflect developmental progression. Flow cytometric analysis reveals distinct CD82 expression profiles at different stages (day 7, day 12, and day 22) of pancreatic differentiation .

These findings have significant implications for regenerative medicine approaches, particularly for generating functional β-cells for diabetes treatment.

How can CD82 be utilized to improve progenitor cell isolation and differentiation protocols?

Researchers can leverage CD82 to enhance stem cell differentiation protocols through these approaches:

• Positive selection strategy: Use fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) with anti-CD82 antibodies to enrich for progenitor populations with enhanced differentiation potential. This approach has been successfully applied to isolate β-cell precursors from mixed populations .

• Quality control marker: Monitor CD82 expression during differentiation protocols as an indicator of proper progression. Flow cytometric analysis at key timepoints can help identify successful versus suboptimal differentiation.

• Differentiation enhancement: Consider manipulating CD82 expression or function to improve differentiation outcomes:

  • CD82 overexpression might enhance differentiation efficiency

  • CD82-activating antibodies could potentially stimulate differentiation pathways

  • Targeting CD82-associated signaling could optimize progenitor maturation

• Purity assessment: Use immunofluorescence staining of clusters derived from CD82+ versus CD82- sorted cells to compare differentiation outcomes. Look for co-expression of CD82 with lineage-specific markers .

• Functional readouts: Evaluate the functional superiority of CD82-selected cells through:

  • Glucose-stimulated insulin secretion assays

  • Calcium imaging to assess stimulus-secretion coupling

  • Transcriptional profiling of maturation markers

These strategies can help address a major challenge in regenerative medicine: generating homogeneous, functional cell populations from pluripotent stem cells.

What are the recommended protocols for manipulating CD82 expression in research models?

Effective manipulation of CD82 expression is crucial for investigating its functions. Consider these methodological approaches:

CD82 Overexpression

• Vector selection: For membrane protein expression, vectors with CMV or EF1α promoters provide robust expression. Include epitope tags (FLAG, HA) at the C-terminus to avoid interfering with membrane insertion .

• Verification methods:

  • Immunocytochemistry using anti-FLAG/tag antibodies to confirm proper membrane localization

  • Western blotting to detect the expected molecular weight patterns (typically 25-33 kDa due to glycosylation)

  • Flow cytometry to quantify surface expression levels

• Expression considerations: After CD82 overexpression, an additional band at ~55 kDa may appear in Western blots, likely representing a different glycosylation form .

CD82 Knockdown/Knockout

• siRNA approach: For transient knockdown, use specific siRNAs targeting CD82 mRNA. Validate knockdown efficiency by Western blot and flow cytometry .

• shRNA approach: For stable knockdown, lentiviral delivery of shRNAs provides longer-term suppression.

• CRISPR/Cas9: For complete knockout, design gRNAs targeting early exons of CD82. Confirm knockout by sequencing and absence of protein expression.

Cell Sorting Strategy

To study cells with different CD82 expression levels:

• Stimulate T cells with IL-2 and anti-CD3/28 for 2 days

• Sort into CD82highCD81high and CD82lowCD81low populations

• Culture sorted cells with continued IL-2 supplementation

• Validate sustained expression differences after sorting

These approaches provide complementary strategies to investigate CD82 function through gain-of-function and loss-of-function experiments.

What are the critical considerations when working with recombinant CD82 protein?

When utilizing recombinant CD82 protein in research applications, consider these important factors:

Protein Characteristics

• Domain selection: Most commercially available recombinant CD82 proteins contain partial sequences focusing on the functionally important large extracellular loop (LEL), such as Gly111-Leu228 .

• Expression system: Human cell lines (e.g., HEK293) are preferred for producing properly folded and glycosylated CD82, as glycosylation affects protein function .

• Tags and fusion partners: C-terminal His-tags are commonly used and generally don't interfere with function. Consider the impact of tag position and size on structural integrity and activity .

• Molecular weight considerations: While the predicted molecular mass of the core protein may be 15 kDa, expect 25-33 kDa bands in SDS-PAGE due to glycosylation .

Storage and Handling

• Reconstitution: Reconstitute lyophilized protein in sterile water to a stock concentration of 0.2 μg/μl. Centrifuge the vial at 4°C before opening to recover all content .

• Storage conditions: Store at -20°C to -80°C under sterile conditions. Aliquot to avoid repeated freeze-thaw cycles that can compromise protein integrity .

• Stability: Properly stored samples typically remain stable for twelve months from the date of receipt .

Quality Control Parameters

• Purity: Verify >90% purity by SDS-PAGE before experimental use .

• Endotoxin levels: Ensure <1.0 EU per μg of protein as determined by the LAL method to prevent experimental artifacts from endotoxin contamination .

• Functional validation: Confirm biological activity through binding assays or cell-based functional tests appropriate to your experimental context.

These considerations help ensure experimental reproducibility and reliable results when working with recombinant CD82 protein.

How might CD82 be leveraged for immunotherapeutic approaches?

CD82's multiple functions in T-cell biology suggest several promising immunotherapeutic applications:

• Enhanced T-cell activation: CD82's co-stimulatory properties could be exploited to boost T-cell responses in cancer immunotherapy:

  • Anti-CD82 agonistic antibodies could provide co-stimulation alongside TCR engagement

  • CD82 overexpression in CAR-T cells might enhance their cytotoxic potential

  • CD82-targeting approaches could sustain IL-2 production, supporting T-cell persistence

• Memory T-cell generation: CD82high T cells show enhanced differentiation toward central memory phenotypes, which are associated with improved long-term anti-tumor responses:

  • Selecting or engineering CD82high T cells might improve the persistence of adoptive cell therapies

  • Modulators of CD82 expression could potentially enhance memory formation during vaccination

• Cytotoxic function enhancement: CD82 overexpression significantly impacts cytolytic activity against target cells:

  • CD8+ T cells overexpressing CD82 achieve up to 80% target cell cytolysis at 36 hours

  • CD4+ T cells with CD82 overexpression demonstrate approximately 40% target cell cytolysis

• Combination approaches: CD82-targeting could complement existing immunotherapies:

  • Checkpoint inhibitors might synergize with CD82 agonists

  • Cytokine therapies could be optimized by CD82 modulation

These approaches would require careful validation in preclinical models before clinical translation, but the significant effects of CD82 on T-cell function suggest therapeutic potential.

What are the unresolved questions and contradictions in CD82 research?

Despite significant advances, several important questions about CD82 remain unresolved:

• Mechanistic uncertainties:

  • The precise molecular mechanisms by which CD82 organizes membrane microdomains and influences receptor clustering remain incompletely understood

  • How CD82 coordinates its diverse functions across different cell types (immune cells, epithelial cells, progenitors) is unclear

  • The relationship between CD82's anti-metastatic and immunomodulatory functions has not been fully explored

• Functional contradictions:

  • While CD82 generally suppresses cell migration, its role in enhancing T-cell activation seems paradoxical since activated T cells must migrate to sites of inflammation

  • CD82 appears to have context-dependent effects on signaling pathways, sometimes enhancing and sometimes suppressing the same pathway in different cell types

  • The relative importance of CD82's scaffolding function versus direct signaling capabilities remains debated

• Regulatory gaps:

  • Factors controlling CD82 expression in different contexts are not completely mapped

  • Post-translational modifications affecting CD82 function, particularly different glycosylation patterns, need further characterization

  • The significance of CD82 internalization and recycling dynamics to its function requires additional investigation

• Therapeutic translation challenges:

  • Whether CD82-targeting approaches can selectively affect specific cell populations without unwanted effects on other CD82-expressing cells

  • The potential for CD82-mediated immunomodulation to overcome immunosuppressive tumor microenvironments

• Interaction complexity:

  • CD82 interacts with multiple partners including other tetraspanins, integrins, and signaling receptors, forming a complex "tetraspanin web" whose composition and function vary by context

Addressing these questions will require integrated approaches combining structural biology, advanced imaging, proteomics, and in vivo models to fully unravel CD82's multifaceted biology.

What advanced techniques are emerging for studying CD82's membrane organization functions?

Cutting-edge technologies are enabling deeper insights into CD82's membrane organizing functions:

• Super-resolution microscopy techniques:

  • Single-molecule localization microscopy (PALM/STORM) can visualize CD82 nanoclusters with 10-20 nm resolution

  • Stimulated emission depletion (STED) microscopy allows visualization of dynamic CD82 interactions with partner proteins

  • These approaches overcome the diffraction limit of conventional microscopy to reveal CD82's true membrane organization

• Proximity labeling proteomics:

  • BioID or APEX2 fusion proteins can identify proteins in close proximity to CD82 in living cells

  • This reveals the dynamic CD82 "interactome" in different cellular contexts and activation states

  • Quantitative comparative analysis can show how CD82's interaction network changes during processes like T-cell activation

• Membrane biophysics approaches:

  • Fluorescence recovery after photobleaching (FRAP) measures CD82's effects on membrane fluidity

  • Förster resonance energy transfer (FRET) detects direct molecular interactions between CD82 and partner proteins

  • Atomic force microscopy can measure CD82's effects on membrane mechanical properties

• Cryo-electron tomography:

  • This technique enables visualization of membrane protein organization in near-native states

  • Can reveal how CD82 influences the three-dimensional architecture of membrane domains

• Organoid and tissue imaging:

  • Light-sheet microscopy combined with clearing techniques allows visualization of CD82 in complex 3D tissues

  • This bridges the gap between cellular studies and in vivo relevance

These technologies, often used in combination, are revealing unprecedented details about how CD82 organizes the plasma membrane to coordinate diverse cellular functions.

CD82 Expression and T-Cell Function Data

Recombinant Human CD82 Protein Specifications

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CD82 in Different Research Applications