Recombinant bovine SPTSSA is a bioengineered protein derived from the gene encoding the small subunit A of serine palmitoyltransferase (SPT), an enzyme critical for de novo sphingolipid biosynthesis. SPT catalyzes the condensation of serine with fatty acyl-CoA to form 3-ketosphinganine, initiating the synthesis of sphingolipids—lipids essential for cellular membranes, signaling, and stress response . The recombinant form is typically expressed in E. coli or mammalian systems, often with a His-tag for purification .
SPTSSA is part of the heterotrimeric SPT complex, which includes:
Large subunits: SPTLC1 (essential for enzymatic activity) and SPTLC2/3 (regulatory subunits).
Small subunits: SPTSSA and SPTSSB (modulate substrate specificity and enzyme activity) .
Enzymatic Regulation: SPTSSA interacts with SPTLC1 and SPTLC2/3 to form the active SPT complex. Mutations in SPTSSA or its homologs (e.g., SPTSSB) alter substrate affinity, leading to aberrant LCB chain lengths (e.g., elevated C20 LCBs in Stellar mutant mice) .
Pathway Involvement:
Recombinant bovine SPTSSA is synthesized using heterologous expression systems:
Parameter | Details |
---|---|
Host System | E. coli, mammalian cells, or cell-free expression |
Tag | N-terminal His-tag for affinity purification |
Purity | ≥85% (SDS-PAGE validated) |
Sequence | Full-length (1–68 aa) or partial |
Activity: Requires co-expression with SPTLC1 and SPTLC2/3 for functional SPT activity .
Substrate Specificity: Preferentially binds palmitoyl-CoA (C16:0) but can utilize other fatty acyl-CoAs when SPTSSA is mutated .
Vascular Development: Endothelial SPTSSA deficiency in mice disrupts retinal vascularization and VEGF signaling, highlighting its role in angiogenesis .
Neurodegeneration: Mutations in SPTSSB (a homolog of SPTSSA) elevate C20 LCBs, causing axon degeneration and protein aggregation .
Cancer and Metabolic Disorders: Altered sphingolipid profiles linked to cancer progression and obesity. Recombinant SPTSSA aids in studying these pathways .
Lipid Raft Dynamics: SPTSSA-derived sphingolipids stabilize lipid rafts, critical for receptor signaling (e.g., VEGFR2) .
Species-Specific Functions: Comparative studies of bovine SPTSSA versus human/mouse homologs to identify conserved/regulatory roles.
Therapeutic Targets: Exploring SPTSSA inhibitors to modulate sphingolipid levels in diseases like cancer or neurodegeneration.
Interactome Mapping: Identifying binding partners (e.g., ORMDL proteins) that regulate SPTSSA activity .
Catalog Number | Host | Purity | Sequence | Source |
---|---|---|---|---|
RFL13073BF | E. coli | >90% | Full-length (1–68 aa) | Creative BioMart |
SPTSSA-BOV-01 | Cell-free | ≥85% | Partial | MyBioSource |
Subunit | Function | Key Interactions |
---|---|---|
SPTLC1 | Catalytic activity; essential for sphingolipid synthesis | SPTSSA, SPTLC2/3, ORMDL |
SPTSSA | Regulates substrate affinity; LCB chain length | SPTLC1, SPTLC2/3 |
SPTLC2/3 | Modulates enzyme activity; substrate specificity | SPTSSA, SPTLC1 |
SPTSSA serves as an activating subunit of serine palmitoyltransferase (SPT), the enzyme that catalyzes the rate-limiting reaction in sphingolipid synthesis. It plays a critical role in stabilizing the catalytic complex and enhancing enzymatic activity. Sphingolipids are essential components of cell membranes, particularly abundant in the nervous system and myelin membranes . As part of the SPT complex, SPTSSA influences the synthesis of sphingoid long-chain base backbones that form the foundation for all sphingolipids .
The functional importance of SPTSSA becomes evident through pathogenic variants that disrupt sphingolipid homeostasis, leading to neurological disorders such as hereditary spastic paraplegia. These variants impair the negative regulation of SPT by ORMDL proteins, resulting in excessive sphingolipid synthesis .
SPTSSA expression appears to be tightly regulated as part of the sphingolipid homeostatic mechanism. In normal conditions, SPTSSA works in concert with ORMDL proteins that mediate feedback inhibition of SPT enzymatic activity when sphingolipid levels become excessive . This careful balance is crucial since sphingolipids are both essential for cellular function and potentially cytotoxic at high concentrations.
In pathological conditions, particularly in glioblastoma, SPTSSA has been found to be significantly upregulated compared to normal tissues . Gene Set Enrichment Analysis (GSEA) has identified several biological processes associated with differential SPTSSA expression, including negative regulation of response to oxidative stress, negative regulation of mitotic cell cycle, and neuron death in response to oxidative stress .
For investigating SPTSSA interactions with other proteins, several methods have proven effective:
Co-immunoprecipitation: As demonstrated in research with SPTLC1-Flag, this approach can be used to isolate protein complexes. Cells expressing tagged proteins can be lysed, solubilized with appropriate detergents (such as 1% GDN), and immunoprecipitated using anti-tag antibodies. Interacting proteins can then be detected by immunoblotting .
Fluorescence microscopy with fusion proteins: Research has utilized SPT1-GFP fusion proteins to track localization and translocation upon specific stimuli. This approach revealed that exposure to 3-methylcholanthrene resulted in the translocation of SPT1 from cytoplasmic domains to focal adhesion complexes .
Indirect ELISA: This technique has been employed to analyze antibody titers in the development of monoclonal antibodies against SPTSSA, which are essential tools for studying protein expression and localization .
Pathogenic variants in SPTSSA have been shown to disrupt sphingolipid homeostasis with significant neurological consequences. Two specific variants have been well-characterized:
p.Thr51Ile (T51I) variant - This de novo monoallelic missense variant affects a highly conserved residue of SPTSSA. Functional studies revealed that this variant impairs the negative regulation of SPT by ORMDL proteins, leading to excessive sphingolipid synthesis .
p.Gln58AlafsTer10 (58fs) variant - This homozygous frameshift variant also results in dysregulated sphingolipid synthesis .
The neurological consequences of these variants include a complex form of hereditary spastic paraplegia (HSP) characterized by progressive motor impairment, spasticity, and variable language/cognitive dysfunction. In Drosophila models, excessive sphingolipid synthesis caused severe neurological defects and shortened lifespan .
The mechanistic basis appears to be the inability of ORMDL proteins to properly regulate SPT activity when these SPTSSA variants are present, leading to a toxic accumulation of sphingolipids that disrupts normal neuronal function and development.
SPTSSA expression has emerged as a significant prognostic marker in glioblastoma. Analysis using the GEPIA and CGGA databases has revealed that SPTSSA expression is significantly upregulated in diffuse glioma compared to normal tissues, and high expression is associated with poor survival outcomes .
Both univariate and multivariate analyses have confirmed that high SPTSSA expression is significantly associated with poor survival in glioma patients. In multivariate analysis, PRS-type, grade, IDH-mutation, 1p19q-codeletion, and SPTSSA expression were all independent prognostic factors .
The biological mechanisms connecting SPTSSA to cancer progression may involve:
Altered sphingolipid metabolism affecting cell membrane composition and signaling
Impact on cellular responses to oxidative stress
Influence on tumor-infiltrating immune cells
Gene Set Enrichment Analysis has identified several pathways differentially regulated based on SPTSSA expression levels, including negative regulation of mitotic cell cycle and cellular catabolic processes , suggesting multiple mechanisms through which SPTSSA may influence tumor biology.
SPTSSA appears to have significant connections to oxidative stress response pathways. Gene Set Enrichment Analysis comparing low and high SPTSSA expression datasets revealed enrichment of pathways related to:
Negative regulation of response to oxidative stress
Neuron death in response to oxidative stress
These pathways were enriched in the low SPTSSA expression phenotype, suggesting that higher SPTSSA expression may suppress normal oxidative stress responses. This could have important implications for understanding how altered sphingolipid metabolism influences cellular resilience to oxidative damage.
Additionally, research using recombinant cell lines with SPT1-GFP fusion proteins has shown that SPT1 may modulate the interaction between heat shock proteins (Hsp90) and the aryl hydrocarbon receptor (AhR), potentially affecting downstream events including oxidative stress response pathways .
Based on established methods for working with human SPTSSA, the following approach can be adapted for bovine SPTSSA:
Expression system selection:
Construct design:
Include affinity tags (HA-tag or Flag-tag) for easier purification and detection.
Consider co-expression with other SPT subunits (SPTLC1, SPTLC2) when studying the complete complex.
Purification protocol:
Lyse cells by sonication in appropriate buffer (e.g., 50 mM HEPES, pH 8.0, 150 mM NaCl with protease inhibitors).
Solubilize membrane proteins with suitable detergents (1% GDN has been effective).
Use affinity chromatography with anti-tag beads (e.g., anti-Flag beads).
Elute with specific peptides (e.g., 200 μg/ml Flag peptide).
Several cellular assays have proven valuable for investigating SPTSSA function:
Sphingolipid profile analysis:
Mass spectrometry-based methods to quantify sphingolipid species
Radiolabeled precursor incorporation assays to measure synthesis rates
Protein-protein interaction assays:
Functional assays:
In vivo models:
Proper validation of antibodies is crucial for reliable SPTSSA detection. The following approach has been documented:
Monoclonal antibody generation:
Antibody validation methods:
Western blot analysis using recombinant SPTSSA and tissue lysates
Immunohistochemistry (IHC) on tissue microarrays
Immunofluorescence (IF) to confirm subcellular localization
Testing in knockout/knockdown models to confirm specificity
Applications in tissue analysis:
SPTSSA has significant potential as a prognostic biomarker, particularly in glioma research:
Expression analysis approaches:
Survival analysis methods:
Correlation with immune infiltration:
Research has consistently shown that high SPTSSA expression is significantly associated with poor survival in glioma patients, making it a promising biomarker for risk stratification and potential therapeutic targeting .
To investigate how SPTSSA regulates sphingolipid synthesis, researchers should consider:
In vitro enzymatic assays:
Reconstitution of SPT complexes with different SPTSSA variants
Measurement of enzymatic activity using appropriate substrates
Analysis of how ORMDL protein interactions affect activity
Cellular sphingolipid profiling:
Lipidomic analysis using liquid chromatography-mass spectrometry (LC-MS)
Comparison of sphingolipid profiles in cells expressing wild-type versus variant SPTSSA
Investigation of how SPTSSA knockdown/overexpression affects sphingolipid homeostasis
Regulatory interaction studies:
Structure-function analysis to identify critical residues for ORMDL interaction
FRET or BRET assays to monitor protein-protein interactions in real-time
Systems biology approaches to model sphingolipid homeostasis regulation
The evidence from studies on pathogenic variants strongly suggests that SPTSSA plays a crucial role in mediating ORMDL-based negative regulation of SPT activity . Disruption of this regulatory mechanism leads to excessive sphingolipid synthesis with significant pathological consequences, highlighting the importance of understanding these interactions.