SPPL2A Antibody
Description
SPPL2A Antibody Overview
SPPL2A antibodies target the SPPL2A protein, a member of the Peptidase A22B family. Key features include:
Target Protein Characteristics
Antibody Applications
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Western Blot (WB): Detects SPPL2A in lysates from mouse, rat, and human samples .
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Immunoprecipitation (IP): Isolates SPPL2A for functional studies .
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Immunofluorescence (IF): Visualizes SPPL2A localization in lysosomes/endosomes .
Role in Immune Cell Development
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B Cell Maturation: SPPL2A processes the CD74/p8 fragment, preventing its toxic accumulation. SPPL2A-deficient mice show arrested B cell development beyond the transitional T1 stage, rescued by CD74 co-ablation .
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Antigen Presentation: SPPL2A regulates MHC class II trafficking by degrading CD74-NTF, ensuring efficient antigen sampling in dendritic cells and B cells .
Therapeutic Implications
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Autoimmune Disease Targeting: Inhibiting SPPL2A (e.g., compound SPL-707) reduces mature B cells and myeloid dendritic cells, mimicking knockout phenotypes. This suggests potential for treating autoimmune disorders like rheumatoid arthritis .
Mechanistic Insights
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Fas Ligand Processing: SPPL2A cleaves Fas-L, modulating apoptosis and immune responses .
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Endosomal Traffic: SPPL2A maintains endosomal integrity by preventing CD74-NTF accumulation, which disrupts PI3K/Akt signaling and promotes apoptosis .
Technical Considerations
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Immunogen Sequences: Common immunogens include the peptide KFWKGNSYQMMDHLDCATNEE .
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Buffer Compatibility: Most antibodies are supplied in PBS with 0.09% sodium azide .
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Storage: Stable at 4°C for short-term; long-term storage at -20°C recommended .
Future Directions
SPPL2A antibodies remain pivotal in elucidating the protease's role in:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research suggests that elements within the transmembrane segment and the luminal juxtamembrane domain facilitate intramembrane proteolysis of CD74 by SPPL2a. PMID: 26987812
- In human B cells, SPPL2a is essential for the turnover of the CD74 N-terminal fragment. A human 15q21.2 microdeletion results in the loss of SPPL2a transcript and protein. PMID: 25035924
- Evidence indicates that endogenous SPPL2a, consistent with overexpression studies, is localized in the membranes of lysosomes/late endosomes. PMID: 21896273
- ADAM10 and SPPL2a have been identified as two proteases involved in FasL processing and the release of the FasL intracellular domain, which has been demonstrated to be critical for retrograde FasL signaling. PMID: 17557115
Q&A
What experimental strategies validate SPPL2A antibody specificity in Western blotting?
To confirm antibody specificity:
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Knockout validation: Compare wild-type vs. Sppl2a<sup>−/−</sup> B cell lysates. A valid antibody shows signal loss in knockout samples (Fig. 2B in ).
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Subcellular fractionation: SPPL2A localizes to endosomal/lysosomal membranes. Validate via fractionation followed by immunoblotting with organelle markers (e.g., LAMP1 for lysosomes ).
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Competition assays: Preincubate antibody with recombinant SPPL2A protein (aa 200-300 containing immunogenic epitopes) to test signal reduction .
Critical data: In , anti-SPPL2A antibodies detected a 50 kDa band in wild-type splenocytes absent in knockouts, confirming specificity.
How does SPPL2A deficiency alter B cell populations? Design a flow cytometry panel to assess this.
SPPL2A ablation causes a block at the transitional T1→T2 B cell stage (Table 2 ). Essential markers:
Method: Use a 10-color panel including B220, CD19, IgM, CD21, CD24, CD23, and viability dye. Gate sequentially: lymphocytes → singlets → live cells → B220<sup>+</sup> → subpopulations .
What controls are essential when measuring CD74/p8 accumulation as a SPPL2A activity readout?
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Time-course analysis: CD74/p8 peaks 16–24 hr post-BCR stimulation in WT cells but persists in SPPL2A-inhibited cells .
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Inhibitor specificity: Co-treat with γ-secretase inhibitor (e.g., DAPT) to exclude off-target effects. SPL-707 shows >1,000× selectivity over γ-secretase .
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Rescue experiment: Transfect SPPL2A cDNA into KO cells and verify p8 reduction by Western blot (Fig. 3B in ).
Data conflict resolution: If p8 accumulates in WT cells, check IFN-γ pretreatment (enhances CD74 synthesis ).
How to resolve contradictory findings about SPPL2A’s role in dendritic cells vs. B cells?
Some studies report DC numbers unchanged while others note mDC reductions . Resolution strategies:
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Strain-specific effects: Compare C57BL/6 (used in ) vs. BALB/c (prone to DC defects).
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Activation state: Assess DCs post-LPS/IFN-γ treatment. SPPL2A<sup>−/−</sup> DCs show 2.3× higher CD74 surface expression when activated (Fig. 3B in ).
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Temporal analysis: DC defects manifest after 14 days of SPPL2A inhibition, unlike acute B cell effects .
Experimental design: Use tamoxifen-inducible Sppl2a KO mice to separate developmental vs. maintenance roles.
What pharmacological tools differentiate SPPL2A inhibition from genetic knockout phenotypes?
Small-molecule inhibitors (e.g., SPL-707) enable reversible, dose-dependent effects unlike constitutive KO:
| Parameter | SPL-707 (10 mg/kg BID) | Sppl2a<sup>−/−</sup> |
|---|---|---|
| B cell depletion | 60–70% at day 11 | 85–90% |
| DC depletion | 50% reduction | Variable |
| CD74/p8 half-life | 25 min (washout) | Sustained accumulation |
Method: Treat WT mice with SPL-707 for 11 days and compare to KO via splenic B220<sup>+</sup> counts (FACS) and CD74 processing (Western blot) .
How to troubleshoot nonspecific binding of SPPL2A antibodies in IHC?
Artifacts arise from homologous epitopes in SPPL2B/SPPL2C. Solutions:
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Epitope mapping: Use antibodies targeting SPPL2A-specific regions (e.g., aa 252–270 containing chompB mutation site ).
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Multiplex RNAscope: Co-stain with Sppl2a mRNA probes (ACD Bio, Cat No. 516171) to confirm protein-RNA colocalization.
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Preabsorption test: Incubate antibody with SPPL2B/SPPL2C peptides; signal should persist if specific .
Data example: In , only antibodies recognizing the N-terminal luminal domain (absent in SPPL2B/C) reliably detected SPPL2A in DC lysates.
Why do some SPPL2A antibodies fail in flow cytometry despite working in Western blot?
This stems from conformational epitopes masked in native membrane proteins. Solutions:
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Fixation/permeabilization: Use BD Cytofix/Cytoperm™ with 0.1% saponin to expose intracellular epitopes.
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Live cell staining: Engineer cells expressing SPPL2A extracellular tags (e.g., HA at N-terminus ).
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Competitive FACS: Mix KO and WT cells equally; specific antibodies yield two distinct populations .
Critical controls: Always include Sppl2a<sup>−/−</sup> cells and isotype-matched antibodies.
How to quantify SPPL2A activity in primary human B cells?
A modified reporter assay is required:
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Lentiviral transduction: Deliver TNFα-NTF-VP16/Gal4 reporter into CD19<sup>+</sup> B cells.
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SPPL2A-dependent readout: Luciferase activity correlates with proteolytic function (R² = 0.91 vs. CD74/p8 levels ).
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Pharmacological inhibition: Dose-response to SPL-707 (IC<sub>50</sub> = 5 nM ) confirms assay validity.
Troubleshooting: Use IL-4 + CD40L to maintain B cell viability during 48-hr assays .
How to reconcile increased MHC-II with reduced antigen presentation in SPPL2A-inhibited B cells?
Paradoxical data arise because SPPL2A:
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Regulates CLIP-HLA-DR complexes: KO cells have 3.2× more surface CLIP (Fig. 3B ), blocking antigenic peptide loading.
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Alters endosomal pH: Accumulated p8 increases vesicular pH from 4.5 → 5.2, impairing cathepsin S-mediated CLIP removal .
Experimental approach:
What metrics confirm successful SPPL2A targeting in autoimmune disease models?
Beyond B cell counts (e.g., 70% reduction in mature B cells ), assess:
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Serum autoantibodies: Anti-dsDNA IgG decreases 4.1× in lupus-prone MRL/lpr mice treated with SPL-707 .
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Germinal center B cells: SPPL2A inhibition reduces GL7<sup>+</sup> Fas<sup>+</sup> cells by 82% .
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T<sub>FH</sub> cells: Correlate with autoantibody reduction (r = −0.79 ).
Advanced model: Inducible KO in NZB/W F1 mice allows stage-specific SPPL2A targeting.
