CD72 Human, Sf9

Why is CD72 produced in Sf9 cells for research applications?

CD72 Human is produced in Sf9 Baculovirus cells because this expression system offers significant advantages for complex mammalian proteins:

• The baculovirus-insect cell system allows for proper folding of the protein's complex domains

• Sf9 cells can perform post-translational modifications including glycosylation

• This system typically yields higher amounts of recombinant protein compared to mammalian systems

• The resulting protein is functional for most research applications while maintaining cost-effectiveness

The Sf9-expressed CD72 typically contains a single, glycosylated polypeptide chain with the extracellular domain necessary for ligand binding, making it suitable for structural and functional studies .

What are the structural characteristics of CD72 Human Recombinant protein?

CD72 Human Recombinant produced in Sf9 Baculovirus cells has the following structural characteristics:

• It is a single, glycosylated polypeptide chain containing 485 amino acids (residues 117-359 of the native sequence)

• It has a calculated molecular mass of 55.3 kDa, though on SDS-PAGE it appears at approximately 50-70 kDa due to glycosylation

• It is expressed with a 242 amino acid hIgG-His tag at the C-Terminus, facilitating purification and detection

• The protein is purified by proprietary chromatographic techniques

• The standard formulation contains 50mM Tris-HCl buffer (pH6.8), 0.2M NaCl, 2mM DTT and 50% glycerol

How does CD72 expression differ in healthy individuals versus autoimmune disease patients?

CD72 expression patterns show significant disease-specific variations:

• In primary Sjögren's Syndrome (pSS) patients, the percentage of CD72+ B cells is significantly increased compared to healthy controls (85.31 ± 8.37% vs 76.91 ± 8.50%, p < 0.001)

• The mean fluorescence intensity (MFI) of CD72 on CD19+ B cells is slightly, but not statistically, higher in pSS patients compared to healthy controls

• Serum levels of soluble CD72 (sCD72) are significantly elevated in pSS patients compared to healthy controls (0.41 ng/mL vs 0.07 ng/mL, p < 0.001)

• In contrast to pSS, studies have shown that CD72 expression on B cells is decreased in SLE patients and in patients with multiple sclerosis

• These differential expression patterns suggest disease-specific roles of CD72 in immune regulation and B cell function across different autoimmune conditions

How does the CD72-mediated signaling pathway regulate B cell function?

CD72 signaling involves complex regulatory mechanisms that can both inhibit and enhance B cell activation:

• Negative regulation: When B cells are stimulated by antigens, CD72 molecules are recruited to the BCR complex and phosphorylated. The phosphorylated CD72 recruits SHP-1 through its immunoreceptor tyrosine-based inhibition motifs (ITIMs), and the CD72-associated SHP-1 negatively regulates BCR signals .

• Positive regulation: CD72-bound Grb2 can reduce the strength of negative signals transmitted by CD72-associated SHP-1. Additionally, ligation of CD72 by CD100 (Semaphorin 4D) can dissociate SHP-1 from CD72 .

• BCR-independent activation: CD72 can also act as a positive regulator through BCR-independent pathways, activating signals such as Btk or mitogen-activated protein kinases (MAPK) by associating with CD19 .

In pSS patients, the upregulation of CD72 expression on B cells correlates with increased serum IgG levels, suggesting CD72 positively regulates B cell functions in this disease context .

What experimental approaches are recommended for studying CD72-CD100 interactions?

To investigate CD72-CD100 (Semaphorin 4D) interactions effectively, several complementary approaches are recommended:

• Binding assays:

  • ELISA-based binding assays using immobilized CD72 Human, Sf9 and recombinant CD100

  • Surface Plasmon Resonance (SPR) to determine binding kinetics and affinity constants

  • Flow cytometry to measure CD100 binding to cell surface CD72

• Functional consequence experiments:

  • Analysis of SHP-1 dissociation from CD72 following CD100 ligation

  • Assessment of BCR signaling changes upon CD100-mediated CD72 engagement

  • Measurement of downstream B cell activation markers, proliferation, and antibody production

• Structural studies:

  • Domain mapping using truncated or mutated versions of CD72 Human, Sf9

  • Crystallography or cryo-EM of CD72-CD100 complexes

• Validation controls:

  • CD100 mutants with altered binding to CD72

  • Blocking antibodies against CD72 or CD100

  • CD72-deficient cells with reconstituted CD72 expression

How can researchers interpret the dual positive and negative regulatory roles of CD72?

The apparently contradictory roles of CD72 in B cell regulation can be interpreted through several conceptual frameworks:

• Context-dependent signaling: CD72's regulatory effect may depend on the activation state of B cells, with predominance of negative regulation in resting B cells and positive regulation in activated B cells.

• Temporal regulation: Initial negative regulation through SHP-1 recruitment may be followed by positive regulation after CD100 ligation and SHP-1 dissociation.

• Threshold modulation: CD72 may help establish activation thresholds that prevent inappropriate B cell activation while still allowing robust responses to strong stimuli.

• Disease-specific mechanisms: In pSS, the correlation between CD72+ B cells and serum IgG levels suggests a predominant positive regulatory role, whereas other autoimmune conditions may show different balances .

• Isoform-specific functions: Different CD72 isoforms or post-translational modifications may preferentially activate either negative or positive regulatory pathways.

Researchers should design experiments that can distinguish between these possibilities, using time-course studies and multiple readouts of B cell activation.

What is the significance of increased soluble CD72 (sCD72) in autoimmune diseases?

The elevation of serum soluble CD72 (sCD72) in autoimmune diseases carries several important implications:

• Biomarker potential: Studies have shown significantly higher sCD72 levels in pSS patients (0.41 ng/mL) compared to healthy controls (0.07 ng/mL), suggesting sCD72 could serve as a biomarker for disease activity or B cell dysregulation .

• Mechanism of generation: Increased sCD72 levels likely result from enhanced shedding of membrane CD72 from activated B cells, potentially through the action of specific proteases upregulated in inflammatory conditions .

• Functional consequences: sCD72 may act as a decoy receptor, competing with membrane-bound CD72 for ligand binding and potentially interfering with normal CD72-mediated signaling.

• Disease correlation: While sCD72 is elevated in pSS patients, studies did not find significant correlations between sCD72 levels and anti-SSA antibodies or IgG levels, suggesting complex regulatory mechanisms .

• Therapeutic implications: Understanding sCD72 regulation may reveal potential therapeutic targets for modulating B cell hyperactivity in autoimmune diseases.

The precise biological function of sCD72 remains to be fully elucidated, but its consistent elevation in autoimmune conditions suggests it plays a role in disease pathogenesis.

What are the optimal conditions for functional studies using CD72 Human, Sf9?

For optimal functional studies with CD72 Human, Sf9, researchers should consider the following conditions:

• Storage and handling:

  • Store the protein at 4°C if using within 2-4 weeks

  • For long-term storage, maintain at recommended temperatures with 50% glycerol

  • Avoid repeated freeze-thaw cycles that may compromise protein integrity

  • Verify protein quality by SDS-PAGE before experiments

• Buffer conditions:

  • Standard working buffer: 50mM Tris-HCl (pH 7.2-7.5), 150mM NaCl

  • Include 1-2mM calcium for C-type lectin domain functionality

  • Add 0.1% BSA to prevent non-specific adhesion

  • Consider including 1mM DTT to maintain reduced state when necessary

• Experimental parameters:

  • Protein concentration: Typically 1-10 μg/mL for binding studies

  • Temperature: Most assays perform optimally at 37°C for cellular studies or room temperature for binding assays

  • Incubation time: 30 minutes to 2 hours depending on the specific application

  • Include proper positive controls (such as anti-IgM for B cell activation)

• Cell-based considerations:

  • Use freshly isolated B cells when possible

  • Standardize cell density (typically 1-2 × 10^6 cells/mL)

  • Pre-warm media and reagents to avoid temperature shock

  • Include appropriate vehicle controls

How should researchers validate the biological activity of CD72 Human, Sf9 preparations?

To ensure CD72 Human, Sf9 preparations maintain their biological activity, researchers should implement a multi-faceted validation approach:

• Biochemical characterization:

  • SDS-PAGE and Western blotting to confirm expected molecular weight (50-70 kDa) and purity

  • Size-exclusion chromatography to detect potential aggregation

  • Verification of glycosylation status using glycosidase treatments

• Functional binding assays:

  • ELISA or SPR-based binding assays with known CD72 ligands (e.g., CD100/Semaphorin 4D)

  • Cell-based binding assays using flow cytometry with B cells

  • Competition assays with anti-CD72 antibodies of known epitope specificity

• Signaling activity verification:

  • Co-immunoprecipitation assays to confirm interaction with SHP-1

  • Phosphorylation studies examining CD72 ITIM motif phosphorylation

  • Effects on calcium flux or other activation markers in B cells

• Batch consistency testing:

  • Implement lot-to-lot comparison using standardized assays

  • Maintain reference standards from validated lots

  • Document critical quality attributes for each preparation

What controls are essential when studying CD72's role in B cell signaling?

When investigating CD72's role in B cell signaling, the following controls are essential:

• Positive controls for B cell activation:

  • Anti-IgM antibodies (to directly stimulate the BCR)

  • CD40L (to provide co-stimulatory signals)

  • Combination of PMA and ionomycin (to bypass receptor signaling)

• Negative controls:

  • Untreated B cells

  • Isotype control antibodies

  • Irrelevant recombinant proteins produced in the same Sf9 system

• Specificity controls:

  • Blocking anti-CD72 antibodies to inhibit the effect of CD72 Human, Sf9

  • CD72-depleted B cells (using siRNA or CRISPR)

  • Dose-response experiments to establish concentration-dependent effects

• System controls:

  • Heat-inactivated CD72 Human, Sf9 (to control for non-specific protein effects)

  • Endotoxin testing (to rule out LPS contamination)

  • Experiments with different B cell subsets (naive, memory) as CD72 effects may vary

• Signaling pathway controls:

  • Inhibitors of specific kinases or phosphatases to determine pathway specificity

  • Phosphatase-dead SHP-1 expression to study the importance of phosphatase activity

  • Analysis of multiple timepoints to capture both early and late signaling events

What methods are recommended for analyzing CD72 phosphorylation status?

Analyzing CD72 phosphorylation status requires careful methodological considerations:

• Sample preparation:

  • Rapid cellular lysis to preserve phosphorylation status

  • Inclusion of phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers

  • Maintenance of cold temperature throughout processing

  • Standardized stimulation protocols (e.g., anti-IgM for specific timepoints)

• Detection methods:

  • Western blotting using phospho-specific antibodies against CD72 ITIM motifs

  • Immunoprecipitation of CD72 followed by blotting with general phospho-tyrosine antibodies

  • Flow cytometry with phospho-specific antibodies for single-cell analysis

  • Mass spectrometry approaches for detailed phosphorylation site mapping

• Validation approaches:

  • Use of phosphatase treatments as negative controls

  • Mutation of ITIM tyrosine residues as specificity controls

  • Correlation with functional readouts (SHP-1 recruitment, downstream signaling)

  • Time-course experiments to capture phosphorylation dynamics

• Data analysis:

  • Normalization to total CD72 protein levels

  • Quantification of the proportion of phosphorylated to non-phosphorylated CD72

  • Correlation with downstream signaling events

  • Integration with other signaling pathway analyses

How can researchers address inconsistent results in CD72 functional studies?

Inconsistencies in CD72 functional studies may arise from several sources. Here are strategies to address common issues:

• Protein quality and stability:

  • Verify CD72 Human, Sf9 integrity by SDS-PAGE before experiments

  • Confirm activity using binding assays with known ligands

  • Avoid repeated freeze-thaw cycles that can compromise protein function

  • Ensure consistent protein lot usage throughout related experiments

• Experimental conditions:

  • Standardize buffer compositions, pH, and temperature across experiments

  • Optimize protein concentration based on dose-response curves

  • Ensure consistent cell densities and activation states in cellular assays

  • Consider the impact of serum components on CD72 function

• Biological variability:

  • Document donor characteristics when using primary B cells

  • Consider genetic background effects on CD72 signaling

  • Verify expression levels of CD72 binding partners (e.g., CD100)

  • Account for the heterogeneity of B cell subpopulations

• Methodological approaches:

  • Implement rigorous positive and negative controls in each experiment

  • Use multiple readouts to assess CD72 function

  • Blind sample analysis to minimize bias

  • Standardize protocols for cell isolation and culture

How should researchers interpret correlations between CD72 expression and B cell hyperactivity?

The interpretation of correlations between CD72 expression and B cell hyperactivity requires careful consideration:

• Evidence supporting positive regulation:

  • Studies in pSS patients show that CD72+ B cell percentage positively correlates with serum IgG levels [β = 0.018(0.001–0.036), p = 0.034]

  • This correlation persists after adjusting for age and gender with linear regression analyses

  • These findings suggest CD72 may positively regulate B cell functions in pSS patients

• Mechanistic considerations:

  • CD72 can activate signals such as Btk or MAPK by associating with CD19 in a BCR-independent manner

  • CD100 ligation of CD72 can dissociate SHP-1, potentially converting CD72 from a negative to a positive regulator

  • The balance between these opposing functions may determine the net effect on B cell activity

• Disease-specific context:

  • Different autoimmune diseases show distinct patterns of CD72 expression

  • The local cytokine environment may influence CD72 function

  • Genetic variants or post-translational modifications might alter CD72 function in disease states

• Correlation versus causation:

  • Direct experimental manipulation of CD72 levels is needed to establish causal relationships

  • Longitudinal studies could help determine whether CD72 upregulation precedes or follows B cell hyperactivity

What explains the discrepancy between CD72 membrane expression and soluble CD72 levels in different diseases?

Several mechanisms may explain the discrepancies between CD72 membrane expression and soluble CD72 (sCD72) levels observed in different autoimmune diseases:

• Disease-specific shedding mechanisms:

  • In pSS, both membrane CD72 expression and sCD72 levels are increased, suggesting enhanced expression and moderate shedding

  • In SLE, decreased membrane CD72 with increased sCD72 suggests predominant shedding mechanisms

  • Different proteases may be activated in different disease contexts

• Regulation of expression versus shedding:

  • Transcriptional upregulation of CD72 in certain diseases

  • Post-translational regulation of membrane shedding in others

  • Differential stability of membrane-bound versus soluble forms

• Source heterogeneity:

  • sCD72 may derive from multiple cell types, not just B cells

  • Different B cell subsets may contribute differently to the sCD72 pool

  • Tissue-resident B cells might have different CD72 expression/shedding patterns than circulating cells

• Functional implications:

  • sCD72 might serve as a decoy receptor in some contexts

  • In other situations, it might have independent signaling functions

  • The balance between membrane-bound and soluble forms may determine net regulatory effects

What are the key considerations when designing experiments to study CD72's role in autoimmune diseases?

When designing experiments to investigate CD72's role in autoimmune diseases, researchers should consider:

• Patient selection and stratification:

  • Clearly define disease criteria (e.g., American-European Consensus Group criteria for pSS)

  • Match control and patient groups for age and gender

  • Consider disease duration, treatment history, and activity scores

  • Stratify patients based on autoantibody profiles or other disease features

• Comprehensive CD72 assessment:

  • Measure both membrane CD72 expression (percentage and MFI) and soluble CD72 levels

  • Analyze CD72 expression on different B cell subsets

  • Consider functional assays alongside expression analysis

  • Correlate findings with clinical parameters and B cell activation markers

• Mechanistic investigations:

  • Examine CD72 phosphorylation status and SHP-1 recruitment

  • Assess the impact of CD72 ligation on B cell function

  • Investigate the effects of blocking CD72-ligand interactions

  • Consider genetic approaches (siRNA, CRISPR) to modulate CD72 expression

• Translational considerations:

  • Evaluate CD72 as a potential biomarker for disease activity or progression

  • Assess whether targeting CD72 might have therapeutic potential

  • Consider how findings in one autoimmune disease might apply to others

  • Design longitudinal studies to track CD72 expression during disease course