Recombinant Bovine Serine palmitoyltransferase small subunit A (SPTSSA)

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Description

Overview of Recombinant Bovine Serine Palmitoyltransferase Small Subunit A (SPTSSA)

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 .

Protein Structure

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) .

SubunitRoleKey Interaction
SPTLC1Catalytic core; rate-limiting step in sphingolipid synthesis SPTSSA/SPTSSB
SPTSSARegulates substrate affinity; specifies long-chain base (LCB) chain length SPTLC1, SPTLC2/3
SPTLC2/3Modulates enzyme activity and substrate preference SPTSSA, ORMDL proteins

Functional Mechanisms

  • 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:

    • Ceramide biosynthesis: SPTSSA contributes to the production of dihydrosphingosine (dhSph) and dihydroceramide (dhCer) .

    • Lipid raft formation: Sphingolipids derived from SPTSSA activity are enriched in lipid rafts, enabling VEGF signaling and endothelial cell function .

Recombinant Production

Recombinant bovine SPTSSA is synthesized using heterologous expression systems:

ParameterDetails
Host SystemE. coli, mammalian cells, or cell-free expression
TagN-terminal His-tag for affinity purification
Purity≥85% (SDS-PAGE validated)
SequenceFull-length (1–68 aa) or partial

Biochemical Characteristics

  • 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 .

Role in Sphingolipid Metabolism

  • 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 .

Disease Relevance

  • 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) .

Future Directions

  1. Species-Specific Functions: Comparative studies of bovine SPTSSA versus human/mouse homologs to identify conserved/regulatory roles.

  2. Therapeutic Targets: Exploring SPTSSA inhibitors to modulate sphingolipid levels in diseases like cancer or neurodegeneration.

  3. Interactome Mapping: Identifying binding partners (e.g., ORMDL proteins) that regulate SPTSSA activity .

Table 1: Recombinant Bovine SPTSSA Products

Catalog NumberHostPuritySequenceSource
RFL13073BFE. coli>90%Full-length (1–68 aa)Creative BioMart
SPTSSA-BOV-01Cell-free≥85%PartialMyBioSource

Table 2: SPT Complex Subunits and Functions

SubunitFunctionKey Interactions
SPTLC1Catalytic activity; essential for sphingolipid synthesisSPTSSA, SPTLC2/3, ORMDL
SPTSSARegulates substrate affinity; LCB chain lengthSPTLC1, SPTLC2/3
SPTLC2/3Modulates enzyme activity; substrate specificitySPTSSA, SPTLC1

Product Specs

Form
Lyophilized powder
Note: We prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order, and we will fulfill your request.
Lead Time
Delivery time may vary depending on the purchasing method and location. Please consult your local distributors for specific delivery details.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly before opening to concentrate the contents at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard final glycerol concentration is 50%. Customers can use this as a reference.
Shelf Life
The shelf life is influenced by various factors including storage conditions, buffer composition, storage temperature, and the inherent stability of the protein.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Storage Condition
Store at -20°C/-80°C upon receipt. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
The tag type will be determined during the production process. If you have a specific tag type requirement, please inform us, and we will prioritize its development.
Synonyms
SPTSSA; SSSPTA; Serine palmitoyltransferase small subunit A; Small subunit of serine palmitoyltransferase A; ssSPTa
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-68
Protein Length
full length protein
Species
Bos taurus (Bovine)
Target Names
SPTSSA
Target Protein Sequence
MALARAWKQMSWFYYQYLLVTALYMLEPWERTVFNSMLVSIVGMALYTGYVFMPQHIMAI LHYFEIVQ
Uniprot No.

Target Background

Function
Stimulates the activity of serine palmitoyltransferase (SPT). The composition of the serine palmitoyltransferase (SPT) complex determines the substrate preference. The SPTLC1-SPTLC2-SPTSSA complex exhibits a strong preference for C16-CoA substrate, while the SPTLC1-SPTLC3-SPTSSA isozyme utilizes both C14-CoA and C16-CoA as substrates, with a slight preference for C14-CoA. Plays a role in MBOAT7 localization to mitochondria-associated membranes (MAMs), and may be involved in fatty acid remodeling of phosphatidylinositol (PI).
Database Links
Protein Families
SPTSS family, SPTSSA subfamily
Subcellular Location
Endoplasmic reticulum membrane; Multi-pass membrane protein.

Q&A

What is the functional role of SPTSSA in sphingolipid biosynthesis?

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 .

How is SPTSSA expression regulated in normal and pathological conditions?

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 .

What experimental approaches are recommended for studying SPTSSA protein interactions?

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 .

How do SPTSSA variants affect sphingolipid homeostasis and what are the neurological consequences?

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.

What is the relationship between SPTSSA expression and cancer progression, particularly in glioblastoma?

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.

How does SPTSSA interact with stress response pathways, particularly oxidative stress?

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

  • Positive regulation of cellular catabolic processes

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 .

What are optimal protocols for expressing and purifying recombinant bovine SPTSSA?

Based on established methods for working with human SPTSSA, the following approach can be adapted for bovine SPTSSA:

  • Expression system selection:

    • Human embryonic kidney (HEK) cells have been successfully used for expressing SPTSSA and studying its interactions .

    • For higher protein yields, insect cell expression systems may be considered.

  • 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).

    • Verify purity by immunoblotting using the Odyssey system .

What cellular assays are most suitable for studying SPTSSA function?

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:

    • Co-immunoprecipitation to identify binding partners

    • Fluorescence microscopy with GFP-tagged proteins to visualize subcellular localization and translocation

  • Functional assays:

    • Cell proliferation assays to assess the impact of SPTSSA variants or expression levels

    • Cyp1A1 transactivation assays when studying interactions with Hsp90 and AhR pathways

  • In vivo models:

    • Drosophila models have been effective for studying neurological consequences of altered SPTSSA function

    • Knockdown or knockout approaches in appropriate cell lines

How can researchers effectively validate antibodies for SPTSSA detection?

Proper validation of antibodies is crucial for reliable SPTSSA detection. The following approach has been documented:

  • Monoclonal antibody generation:

    • Immunize SPF (specific pathogen-free) mice with SPTSSA-derived polypeptides.

    • Administer multiple subcutaneous injections (approximately 60 μg polypeptide at 3.0 mg/mL).

    • Analyze IgG antibody titers using indirect ELISA.

    • Perform cell fusion for hybridoma production .

  • 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:

    • Construction of tissue microarrays (TMAs) from paraffin-embedded, formalin-fixed slides

    • Selection of tissue areas containing >50% tumor for cancer studies

    • Transfer of representative tumor cores (approximately 1 mm) to recipient TMA blocks

How can SPTSSA be utilized as a prognostic biomarker in clinical research?

SPTSSA has significant potential as a prognostic biomarker, particularly in glioma research:

  • Expression analysis approaches:

    • Immunohistochemistry (IHC) on tissue microarrays has successfully assessed SPTSSA expression in glioma tissues .

    • Gene expression analysis through platforms like GEPIA and CGGA databases provides valuable prognostic information .

  • Survival analysis methods:

    • Kaplan-Meier survival analysis with log-rank tests to determine the relationship between SPTSSA expression and patient outcomes.

    • Both univariate and multivariate analyses to assess SPTSSA as an independent prognostic factor alongside established markers like IDH-mutation and 1p19q-codeletion .

  • Correlation with immune infiltration:

    • Tools like CIBERSORT and TIMER have been used to investigate correlations between SPTSSA expression and tumor-infiltrating immune cells .

    • These findings suggest potential connections between sphingolipid metabolism and tumor immunology.

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 .

What techniques are recommended for studying SPTSSA-mediated regulation of sphingolipid synthesis?

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.

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