CD9 Human, Sf9

What is CD9 and what are its key biological functions?

CD9 belongs to the tetraspanin family of four membrane-spanning proteins that function in a wide range of physiological processes in higher organisms. Its major functions include:

• Regulation of cell migration and proliferation

• Mediation of cell fusion processes

• Essential role in fertilization (CD9-knockout female mice exhibit infertility due to failure of sperm-egg fusion)

• Involvement in virus infection pathways

• Participation in numerous signaling pathways

CD9 is expressed in various blood cell types, including precursor B-lymphocytes, and plays crucial roles in cell motility, cell-cell adhesion, and membrane reorganization. In pathological contexts, CD9 influences cancer cell motility, invasiveness and proliferation in multiple tumor types, including prostate carcinoma, melanoma, and glioblastoma .

Why are Sf9 cells preferred for expression of human CD9 protein?

Sf9 cells provide several methodological advantages for CD9 expression:

• Enable high-yield expression of complex membrane proteins like CD9

• Support proper post-translational modifications essential for protein functionality

• Compatible with the Bac-to-Bac baculovirus expression system for efficient protein production

• Allow for purification yields sufficient for structural studies

For optimal expression of human CD9, researchers have successfully used modified pFastBac1 expression vectors in Sf9 cells, including N-terminal His8 tags, GFP tags, and TEV protease cleavage sites. The CD9 gene can be PCR-amplified and inserted into expression vectors using KpnI and EcoRI restriction sites .

What expression construct modifications improve CD9 protein yield and quality?

Several modifications to CD9 expression constructs have proven beneficial:

• Deletion of five residues in the extracellular loop region

• Truncation of three C-terminal residues (Δ226-228)

• Introduction of specific cysteine mutations (e.g., at Ile20) for experimental purposes

• Integration of purification tags (His8) and reporter proteins (GFP)

The expression protocol typically involves transforming the CD9 expression plasmid into DH10Bac competent E. coli cells to generate recombinant bacmid. After isolation, the bacmid DNA can be transfected into Sf9 cells at a density of approximately 3 × 10^6 cells/ml using FuGENE HD, followed by incubation at 27°C for four days .

What methodologies are used to study CD9 function in cells?

Multiple experimental approaches have been developed to investigate CD9 function:

• Genetic manipulation techniques:

  • Lentiviral shRNA for CD9 knockdown (demonstrating decreased CD9 mRNA and protein expression)

  • Site-directed mutagenesis for structure-function studies

  • CD9-knockout models (particularly useful in fertility studies)

• Functional assays:

  • Cell proliferation analysis (CD9 knockdown suppresses proliferation)

  • Adhesion and migration assays (CD9 influences cell adhesion to ECM components)

  • Apoptosis quantification (CD9 knockdown promotes apoptosis via p53-dependent pathways)

  • Drug sensitivity testing (CD9 knockdown enhances chemotherapy efficacy)

• Protein detection methods:

  • Flow cytometry using PE-conjugated anti-CD9 antibodies (1:50 dilution)

  • Western blotting

  • Immunofluorescence microscopy

What are the optimal methods for crystallizing human CD9?

Successful crystallization of human CD9 requires specialized techniques due to its membrane protein nature:

• Protein preparation strategy:

  • Expression in Sf9 cells using the Bac-to-Bac baculovirus system

  • Purification via metal-affinity chromatography followed by size-exclusion chromatography

  • Critical modification: truncation of the flexible loop region in the large extracellular loop (LEL)

• Crystallization technique:

  • Reconstitution into lipidic cubic phase (LCP) by mixing with liquefied monoolein in a 2:3 (w:v) protein:lipid ratio

  • Sandwich-drop crystallization with 50 nl protein-LCP mixture overlaid with 800 nl precipitant solution

  • Initial screening using specialized kits (e.g., MemMeso)

• Optimization parameters:

  • Reservoir solution: 36-42% PEG 200, 10-50 mM Tris-HCl pH 7.5 or 10-50 mM MOPS pH 6.6

  • For phase determination: mercury derivatization through introduced cysteine mutations (e.g., I20C)

This approach significantly improved crystal quality compared to wild-type CD9, which typically produced crystals with dimensions of only 10 × 10 × 5 μm that diffracted X-rays to a maximum of 10 Å resolution .

How does CD9 structure correlate with its functional properties?

CD9's structure-function relationship reveals several key insights:

• Domain influence on cellular processes:

  • The C-terminal tail contributes significantly to CD9's function in regulating cell adhesion and spreading

  • Wild-type CD9 diminishes adhesion to fibronectin compared to mutant CD9 (lacking functional C-terminal domain)

  • CD9 inhibits spreading of both MOLT-4 and K562 cells on fibronectin through C-terminal domain interactions

• Morphological effects:

  • Wild-type CD9 expression correlates with formation of abundant filopodia and microvilli-like projections

  • CD9 localizes within these projections, co-localizing with F-actin

  • CD9 mutants (particularly C-terminal mutants) fail to induce these cytoskeletal projections

• Cell aggregation properties:

  • Wild-type CD9 promotes cell-cell aggregation when cells are plated on collagen I, laminin-5, or in FBS

  • Mutant CD9 shows minimal pro-aggregation effect

  • CD9's pro-aggregation activity appears consistent with a role for β1 integrins

What considerations are critical for successful CD9 knockdown experiments?

When designing CD9 knockdown studies, researchers should consider:

• Validation methodology:

  • Verify knockdown efficiency at both mRNA and protein levels

  • Assess membrane expression using flow cytometry with PE-conjugated anti-CD9 (1:50 dilution)

  • Use appropriate controls (e.g., PE-conjugated mouse IgG1 κ isotype control)

• Functional assessment:

  • Comprehensive panel of assays including:

    • Cell proliferation (CD9 knockdown suppresses proliferation)

    • Adhesion, migration and invasion (CD9 knockdown reduces these behaviors)

    • Apoptosis (CD9 knockdown promotes apoptosis)

    • Drug sensitivity (CD9 knockdown enhances efficacy of chemotherapeutics and tyrosine kinase inhibitors)

• Mechanistic investigation:

  • CD9 knockdown suppresses cell proliferation via p53-dependent pathways

  • For apoptosis studies, consider preincubation with caspase inhibitors (e.g., 0.6 μmol/l Z-DEVD-FMK)

  • Analyze downstream effects on RAC1 activation and chemokine receptor signaling

How can proximity labeling be used to study the CD9 interactome during infection?

CD9 proximity labeling offers powerful insights into dynamic protein interactions:

• Experimental design:

  • Creation of CD9:TurboID fusion proteins for proximity-dependent biotinylation

  • Collection of samples at multiple timepoints during bacterial infection (30, 60, 240 minutes)

  • Comparison of infected versus uninfected conditions to identify infection-specific interactions

• Key findings:

  • Thirteen proteins proximal to CD9 are enriched during meningococcal infection

  • Different temporal patterns emerge: 30 mins (4 proteins), 60 mins (2 proteins), 240 mins (7 proteins)

  • YTHDF3 is the most enriched protein at 30 minutes post-infection

  • Several metalloproteases are observed throughout all timepoints

• Pathway insights:

  • CD9 associates with both canonical and non-canonical interaction partners

  • Proteins involved in bacterial adherence pathways are enriched during infection

  • Interference with CD9 can diminish bacterial adherence

How do post-translational modifications influence CD9 function?

CD9 undergoes several important post-translational modifications that affect its function:

• Disulfide bonding:

  • Four cysteine residues in the large extracellular loop form critical disulfide bonds

  • These bonds stabilize the tertiary structure of the extracellular domain

  • Proper disulfide formation is essential for CD9's role in sperm-egg fusion

• Palmitoylation:

  • Six cysteine residues at the intracellular ends of the transmembrane helices undergo heterogeneous palmitoylation

  • This lipid modification influences CD9's membrane microdomain localization

  • Palmitoylation affects CD9's ability to interact with partner proteins

• Experimental considerations:

  • For uniform labeling in crystallography, researchers have introduced additional cysteine residues (e.g., at Ile20)

  • Protein expression in Sf9 cells preserves most post-translational modifications

  • When designing CD9 mutants, consider the potential disruption of modification sites

What is the molecular mechanism by which CD9 regulates cell adhesion and migration?

CD9 influences cell adhesion and migration through several molecular pathways:

• Integrin modulation:

  • CD9 regulates integrin-dependent cell adhesion and spreading

  • Wild-type CD9 expression in K562 cells diminishes adhesion to fibronectin

  • This effect is observable at both early (30 minutes) and later (2 hours) timepoints after plating

• Cytoskeletal reorganization:

  • CD9 promotes formation of filopodia and microvilli-like projections

  • CD9 co-localizes with F-actin within these projections

  • C-terminal mutations disrupt this cytoskeletal reorganization capacity

• Cell-cell interactions:

  • CD9 promotes cell aggregation on various substrates (collagen I, laminin-5, serum)

  • This aggregation appears to involve β1 integrin-dependent mechanisms

  • CD9 enhances C-X-C motif chemokine receptor 4-mediated migration

• Signaling pathway activation:

  • CD9 promotes RAC1 activation in B-ALL cells

  • CD9 knockdown in Ph+ ALL cells suppresses cell proliferation and promotes apoptosis via p53-dependent pathways

What methods are most effective for purifying CD9 for structural studies?

Successful purification of CD9 for structural studies involves several critical steps:

• Expression optimization:

  Parameter	Optimal Condition
  Expression system	Sf9 cells with Bac-to-Bac baculovirus
  Cell density	3 × 10^6 cells/ml
  Incubation	27°C for 4 days post-infection
  Construct design	N-terminal His8 tag, GFP tag, TEV cleavage site
  Domain modification	Truncation of LEL and C-terminal residues

• Purification protocol:

  • Disruption of cells in buffer containing 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM PMSF, 5 μg/ml leupeptin

  • Membrane fraction isolation via ultracentrifugation

  • Solubilization using appropriate detergents

  • Metal-affinity chromatography using the His8 tag

  • TEV protease cleavage to remove tags

  • Size-exclusion chromatography for final purification

• Quality control:

  • SDS-PAGE analysis to verify purity

  • Western blotting to confirm identity

  • Assessment of monodispersity via size-exclusion chromatography

  • Functional verification when possible

How does CD9 contribute to therapeutic resistance in malignancies?

CD9 plays significant roles in therapeutic resistance through several mechanisms:

• Drug resistance pathways:

  • CD9+ B-ALL cells exhibit drug resistance properties

  • CD9 knockdown enhances sensitivity to chemotherapeutic drugs (vincristine, daunorubicin, cyclophosphamide, dexamethasone)

  • CD9 knockdown also increases sensitivity to tyrosine kinase inhibitors like imatinib

• Cell survival mechanisms:

  • CD9 expression influences apoptotic pathways through p53-dependent mechanisms

  • CD9+ cells show enhanced leukemogenic potential with asymmetric cell division-like proliferation

  • Anti-CD9 monoclonal antibodies have shown anti-proliferative effects on B-ALL cells

• Clinical correlations:

  • Patients with CD9+ ALL exhibit higher positive rates of the BCR-ABL fusion gene

  • CD9 expression correlates with poor prognosis in ALL patients

  • CD9 enhances C-X-C motif chemokine receptor 4-mediated B-ALL cell migration and bone marrow engraftment

• Therapeutic implications:

  • CD9-targeted therapies may represent a novel approach for B-ALL treatment

  • Combining CD9 inhibition with conventional chemotherapy shows promise

  • RNA interference targeting CD9 demonstrates in vitro anti-leukemia activity in Ph+ ALL cells