OSCAR Human, Sf9
Description
Immune Regulation
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Antigen Presentation: OSCAR binds collagen motifs and facilitates antigen uptake in dendritic cells, promoting T-cell activation .
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Inflammatory Signaling: Activation of OSCAR triggers TNF-α release from CCR2+ monocytes, exacerbating conditions like rheumatoid arthritis (RA) and atherosclerosis .
Bone Homeostasis
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Osteoclastogenesis: OSCAR-FcRγ complexes provide co-stimulatory signals for osteoclast differentiation, mediated via integrin β3 subunits .
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Disease Link: Elevated OSCAR levels in RA patients correlate with increased osteoclast differentiation and bone resorption .
Therapeutic Targeting
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Inhibition Strategies: OSCAR-Fc fusion proteins block ligand interactions, reducing osteoclastogenesis in RA models .
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Disease Models: Used to study atherosclerosis, chronic obstructive pulmonary disease (COPD), and inflammatory bone loss .
Functional Assays
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ELISA Data: OSCAR-Fc binding to SP-D shows a dissociation constant (Kd) in the nanomolar range .
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Cytokine Release: OSCAR activation in monocytes increases TNF-α secretion by 3- to 5-fold compared to controls .
Disease Relevance
Production and Quality Control
Product Specs
OSCAR, PIgR-3, PIGR3, hOSCAR, PIgR-3, Poly-Ig Receptor 3.
DITPSVPPAS YHPKPWLGAQ PATVVTPGVN VTLRCRAPQP AWRFGLFKPG EIAPLLFRDV SSELAEFFLE EVTPAQGGSY RCCYRRPDWG PGVWSQPSDV LELLVTEELP RPSLVALPGP VVGPGANVSL RCAGRLRNMS FVLYREGVAA PLQYRHSAQP WADFTLLGAR APGTYSCYYH TPSAPYVLSQ RSEVLVISWE GEGPEARPAS SAPGMQAPGP PPSDPGAQAP SLSSFRPRGL VLQPLLPQTQ DSWDPAPPPS DPGVHHHHHH.
Q&A
What experimental approaches validate OSCAR-SP-D interactions in pulmonary studies?
OSCAR binds Surfactant Protein D (SP-D) through a conserved collagenous domain motif (GXPGPXGFXGXP) . Key validation steps include:
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Motif Identification:
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Binding Assays:
Table 1: Key Parameters for OSCAR-SP-D Binding Experiments
| Variable | Optimal Condition | Control Consideration |
|---|---|---|
| SP-D immobilization | 4°C overnight in acetic acid | BSA-coated wells |
| OSCAR-Fc concentration | 0.25 μg/mL | LAIR-1-Fc fusion proteins |
| Blocking buffer | 5% BSA in TBS | Validate with anti-OSCAR mAbs |
How do Sf9 cells enhance OSCAR-Fc fusion protein production?
Sf9 cells (derived from Spodoptera frugiperda) enable high-yield post-translational modification of OSCAR-Fc via baculovirus vectors:
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Vector Design: Clone OSCAR’s extracellular domain into pFastBac vectors with an IgG1 Fc tag using XhoI/XbaI restriction sites .
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Transfection: Use Cellfectin II reagent with 2 μg plasmid DNA per 1×10^6 cells. Harvest supernatants at 72 hr post-infection.
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Quality Control:
How to resolve contradictions in OSCAR binding affinity measurements across studies?
Discrepancies in reported Kd values (e.g., 10 nM vs. 150 nM) often stem from:
Technical Factors:
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Collagen Preparation: Rat tail collagen I batches vary in telopeptide content, altering OSCAR binding. Use mass spectrometry to verify collagen integrity.
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SP-D Isoforms: Trimeric vs. dodecameric SP-D assemblies show 8-fold differences in OSCAR avidity . Characterize oligomerization state via gel filtration chromatography.
Biological Variables:
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Monocyte Subtypes: Inflammatory CCR2+ monocytes exhibit 3× higher OSCAR surface density than resident subsets . Use FACS with CD14/CD16/CD192 markers to stratify populations.
Statistical Approach:
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Apply mixed-effects models to account for donor-to-donor variability in primary cell assays.
What methodologies optimize Sf9 cell systems for large-scale OSCAR mutagenesis studies?
High-throughput OSCAR mutagenesis requires:
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Bacmid Engineering:
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Use Tn7 transposition with >90% efficiency checks via blue/white screening.
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Implement site-saturation mutagenesis at collagen-binding residues (positions 42–55).
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Expression Troubleshooting:
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For insoluble proteins: Add 2 mM glutathione redox buffer during infection.
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For low yields: Test multiplicity of infection (MOI) from 0.1–5 and harvest at 48–96 hr intervals.
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Table 2: Sf9 Culture Optimization Matrix
| Parameter | Low Yield Scenario | High Purity Scenario |
|---|---|---|
| Cell density at infection | 2×10^6 cells/mL | 1.5×10^6 cells/mL |
| Serum concentration | 1% FBS (cost-effective) | 0% SFM (protein-free) |
| Harvest timepoint | 72 hr (balance) | 96 hr (mature glycosylation) |
How to model OSCAR-SP-D interactions in atherosclerotic microenvironments?
Advanced co-culture systems address this:
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3D Plaque Model:
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Seed human aortic endothelial cells (HAECs) on collagen I scaffolds (2 mg/mL).
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Add THP-1 monocytes pre-treated with 100 ng/mL SP-D for 24 hr.
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Readout Optimization:
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Quantify TNF-α via MSD U-PLEX (1 pg/mL sensitivity) to detect low-grade inflammation.
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Image OSCAR/SP-D co-localization using Airyscan confocal microscopy (63×/1.4 NA oil lens).
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What computational tools predict novel OSCAR-binding collagen motifs?
Beyond BLAST, machine learning pipelines improve prediction accuracy:
Product Science Overview
The Osteoclast Associated, Immunoglobulin-Like Receptor (OSCAR) is a protein encoded by the OSCAR gene. This receptor is a member of the leukocyte receptor complex protein family and plays a crucial role in the regulation of both innate and adaptive immune responses . OSCAR is specifically expressed in preosteoclasts and mature osteoclasts, making it an important regulator of osteoclast differentiation .
OSCAR is an IgG-like receptor that acts as a costimulatory receptor for osteoclast differentiation through the activation of NFATc1 . Osteoclasts are multinucleated cells responsible for bone resorption, a process essential for bone homeostasis . The receptor’s expression is highly conserved across different species, highlighting its significance in bone biology .
OSCAR has been identified as a key mediator in osteoimmunology, the study of the interface between the skeletal and immune systems . It plays a role in oxidative stress-mediated atherogenesis and monocyte adhesion . The receptor’s involvement in these processes underscores its importance in both bone health and immune regulation.
The human recombinant form of OSCAR, produced in Sf9 insect cells, is used in various research applications. This recombinant protein allows scientists to study the receptor’s function and its role in osteoclastogenesis and immune responses in a controlled environment.
