Recombinant Mouse Serine palmitoyltransferase 3 (Sptlc3)

What is Serine Palmitoyltransferase 3 (Sptlc3) and what is its role in sphingolipid metabolism?

Sptlc3 is a subunit of the serine palmitoyltransferase (SPT) complex, which catalyzes the rate-limiting step in sphingolipid de novo synthesis. Sptlc3 shows approximately 68% identity to Sptlc2 and contains a pyridoxal phosphate consensus motif critical for its enzymatic activity . While traditionally SPT was described as a heterodimer composed of Sptlc1 and Sptlc2 subunits, the discovery of Sptlc3 has expanded our understanding of the complex's composition and functional diversity .

Sptlc3 significantly alters the substrate specificity of the SPT complex. The presence of Sptlc3 allows the enzyme to utilize a broader range of acyl-CoAs as substrates, particularly medium-chain acyl-CoAs like myristoyl-CoA (C14-CoA), resulting in the generation of unique sphingoid base species .

How does Sptlc3 differ from other SPT subunits in terms of substrate specificity?

The Sptlc1-Sptlc2-Sptssb complex shows a strong preference for C18-CoA substrate, whereas the Sptlc1-Sptlc3-Sptssb isozyme displays the ability to use a broader range of acyl-CoAs without apparent preference . Specifically:

• Sptlc3-expressing cells demonstrate approximately 5-fold higher activity with myristoyl-CoA compared to control cells .

• Sptlc3 enables the formation of C16-sphinganine and C16-sphingosine, which are not significantly produced by Sptlc2-containing complexes .

• Sptlc3 also facilitates the synthesis of C17 and C20 sphingoid bases, with particularly high efficiency for C20 sphingoid bases when compared to Sptlc2 .

This substrate flexibility of Sptlc3 results in a more diverse sphingolipid profile, potentially affecting membrane properties and cellular signaling events .

What are the known genetic variants and aliases for mouse Sptlc3?

Mouse Sptlc3 is known by several aliases in scientific literature and databases:

• Serine Palmitoyltransferase Long Chain Base Subunit 3

• C20orf38

• Sptlc2L

• Hlcb2b

• Lcb2b

• Long Chain Base Biosynthesis Protein 2b

• Long Chain Base Biosynthesis Protein 3

• Serine-Palmitoyl-CoA Transferase 3

• Serine Palmitoyltransferase 3

It's important to be aware of these alternative names when conducting literature searches or database queries to ensure comprehensive coverage of research related to this protein .

What are the recommended protocols for overexpressing recombinant mouse Sptlc3 in mammalian cell systems?

For successful overexpression of recombinant mouse Sptlc3 in mammalian cells, researchers typically use the following approach:

• Vector selection: The human or mouse Sptlc3 gene can be cloned into pcDNA3.1 expression vector, which has shown successful expression in HEK293 cells .

• Cell line selection: HEK293 cells are often used as they express low endogenous levels of Sptlc3 mRNA, providing a clean background for studying the effects of recombinant Sptlc3 expression .

• Transfection protocol: Standard transfection methods for HEK293 cells are appropriate, with expression typically confirmed by Western blot analysis .

• Expression verification: RT-qPCR can be performed using specific primers such as moSPTLC3fw: 5′-GGCTTGCAGGGAAATATG-3′ and moSPTLC3rv: 5′-GGATGACTGAAGTGTGGTTA-3′. Amplification conditions: 50 cycles of 10 s at 95°C, 10 s at 61°C, and 20 s at 72°C .

• Co-expression considerations: When studying the function of Sptlc3, researchers may want to co-express small subunits like ssSPTa or ssSPTb, as these can modify the activity and substrate preference of the SPT complex .

How can researchers measure Sptlc3 enzymatic activity in vitro?

Measuring SPT activity in Sptlc3-expressing cells requires specialized methods to detect the unique sphingoid bases produced:

• In vitro SPT activity assay:

  • Prepare microsomes from Sptlc3-expressing cells

  • Measure incorporation of radiolabeled L-serine into 3-ketodihydrosphingosine

  • For Sptlc3-specific activity, use myristoyl-CoA (C14-CoA) as the substrate

  • Optimal substrate concentration is typically in the range of 0.1-0.125 mM

• Kinetic measurements:

  • For palmitoyl-CoA, enzyme follows Michaelis-Menten kinetics up to approximately 0.1 mM

  • At concentrations above 0.15 mM, substrate inhibition is observed

  • For myristoyl-CoA, Sptlc3-expressing cells show about 5-fold higher activity compared to control cells

• Mass spectrometry analysis:

  • Liquid chromatography-mass spectrometry (LC-MS) is essential for identifying and quantifying the unique sphingoid bases produced by Sptlc3

  • Metabolic labeling with D3-15N-L-serine can be used to track newly synthesized sphingolipids

What methods can be used to detect and quantify the unique sphingoid bases produced by Sptlc3 activity?

Detection and quantification of Sptlc3-specific sphingoid bases require sophisticated analytical techniques:

• Sample preparation:

  • Extract total lipids from cells expressing recombinant Sptlc3

  • Perform base hydrolysis to release sphingoid bases from complex sphingolipids

  • Derivatize samples if necessary for improved detection sensitivity

• Analytical methods:

  • High-performance liquid chromatography (HPLC) coupled to mass spectrometry

  • Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)

  • Use of isotope-labeled standards (e.g., D3-15N-L-serine) for metabolic labeling

• Identification parameters:

  • C16-sphinganine and C16-sphingosine are signature metabolites of Sptlc3 activity

  • Additional Sptlc3-specific sphingoid bases include C17-sphingosine, methyl-branched C18-sphingosine (meC18SO), and C20-sphingosine

  • Retention time comparison with standards is crucial for identifying branched-chain sphingoid bases, which may be isobaric with straight-chain species

How does Sptlc3 contribute to the formation of branched-chain sphingolipids, and what are their functional implications?

Sptlc3 uniquely enables the formation of methyl-branched sphingoid bases, which has significant implications for membrane properties and cellular functions:

• Mechanism of formation:

  • Sptlc3 can utilize branched-chain fatty acids as substrates, particularly anteiso-methyl-palmitate (ante-mePA)

  • This leads to the formation of 16-(omega-3-) methyl-branched sphingosine (meC18SO)

  • The formation of meC18SO is primarily limited by substrate availability rather than enzymatic capacity

• Confirmation of identity:

  • The branched-chain nature of meC18SO has been confirmed through comparison with chemically synthesized standards

  • Despite being isobaric with C19-sphingosine, meC18SO has a distinct retention time (differing by 0.42 min)

• Metabolism to complex sphingolipids:

  • meC18SO is incorporated into complex sphingolipids including ceramides (meCer) and sphingomyelins (meSM)

  • There are significant differences in N-acylation patterns between C18SO-based and meC18SO-based complex sphingolipids

  • meC18SO-based ceramides and sphingomyelins preferentially contain longer N-acyl chains (C22:0, C24:0, and C24:1), while levels with conjugated C16:0 fatty acid are very low

• Functional implications:

  • Branched-chain sphingolipids may alter membrane fluidity and organization

  • This could affect membrane-dependent processes including signaling, trafficking, and protein function

  • The tissue-specific expression of Sptlc3, particularly in skin, suggests specialized roles for these unique sphingolipids

What is the physiological significance of Sptlc3-specific sphingolipids in plasma and different tissues?

The distribution and relative abundance of Sptlc3-specific sphingolipids in plasma and tissues provide insights into their physiological roles:

• Plasma distribution:

  • C18-sphingosine is the most abundant long-chain base in both human and mouse plasma

  • Among Sptlc3-specific long-chain bases, C16-sphingosine is most abundant in human plasma, while C17-sphingosine predominates in mouse plasma

  • Interestingly, methyl-branched C18-sphingosine (meC18SO) is detected only in human plasma and is absent in mouse plasma

• Lipoprotein association:

  • Sptlc3-specific sphingolipids are found in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL)

  • C16, C17, and meC18-sphingosine are relatively more abundant in LDL fractions compared to HDL

  • This distribution pattern may have implications for lipid transport and metabolism

• Tissue-specific expression and function:

  • Sptlc3 shows tissue-specific expression patterns, with particularly high abundance in skin

  • Changes in SPT activity involving Sptlc3 are associated with dermal pathologies

  • The unique sphingolipid profile generated by Sptlc3 may contribute to specialized membrane properties in different tissues

• Metabolic implications:

  • Genetic variants of Sptlc3 are associated with metabolic conditions such as dyslipidemia and atherosclerosis

  • The role of Sptlc3-specific sphingolipids in these conditions requires further investigation

How do the small subunits (ssSPTa and ssSPTb) modulate Sptlc3 activity and sphingolipid production?

The small subunits ssSPTa and ssSPTb interact with the SPT complex and modify its activity and substrate specificity in complex ways:

• Expression patterns:

  • Both ssSPTa and ssSPTb are expressed in mammalian cells, with ssSPTa typically being the predominant form

  • The expression of ssSPTa and ssSPTb is not significantly altered by the overexpression of Sptlc2 or Sptlc3

• Effects on standard C18-sphingolipid production:

  • Overexpression of ssSPTa in wild-type HEK293 cells has a minor stimulatory effect on C18-sphingosine synthesis

  • This effect is not observed in Sptlc2- or Sptlc3-overexpressing cells that already show increased C18-sphingosine formation

• Effects on Sptlc3-specific sphingolipids:

  • Overexpression of either ssSPTa or ssSPTb does not induce C16- or C17-sphingosine synthesis in Sptlc1-Sptlc2 expressing cells

  • There is a slight reduction trend in C16- and C17-sphingosine production in Sptlc3-expressing cells with ssSPTa or ssSPTb co-expression

  • Formation of methyl-branched C18-sphingosine in Sptlc3-expressing cells is only marginally decreased by ssSPTa or ssSPTb expression

• Stimulation of C20-sphingolipid production:

  • ssSPTb expression significantly stimulates the formation of C20-sphingosine

  • This effect is observed in wild-type cells but is stronger in cells co-expressing Sptlc2 or Sptlc3

  • Independent of ssSPTb expression, C20-sphingosine formation is generally higher in Sptlc3-expressing cells

What are the implications of Sptlc3 in metabolic disorders and how can researchers study this connection?

Sptlc3 has been linked to several metabolic conditions, providing important research directions:

• Associated metabolic conditions:

  • Genetic variants of Sptlc3 are associated with dyslipidemia

  • Sptlc3 polymorphisms have been linked to atherosclerosis risk

  • Changes in sphingolipid metabolism may contribute to metabolic syndrome pathogenesis

• Research approaches:

  • Genetic association studies examining Sptlc3 variants in patient populations

  • Lipidomic profiling of plasma from subjects with metabolic disorders

  • Analysis of Sptlc3-specific sphingolipids in different lipoprotein fractions

  • Animal models with Sptlc3 overexpression or knockout to study metabolic effects

• Methodological considerations:

  • Comprehensive sphingolipid profiling using mass spectrometry

  • Measurement of both free sphingoid bases and complex sphingolipids

  • Analysis of sphingolipid distribution in different lipoprotein fractions

  • Correlation of sphingolipid profiles with clinical parameters

How can researchers investigate the role of Sptlc3 in skin biology and dermatological conditions?

Given the high expression of Sptlc3 in skin and its association with dermal pathologies, several research approaches are relevant:

• Experimental models:

  • Primary keratinocyte cultures overexpressing or with reduced Sptlc3 expression

  • 3D skin equivalents to study the effects of altered Sptlc3 expression on skin barrier function

  • Mouse models with tissue-specific Sptlc3 modulation

• Analytical approaches:

  • Analysis of sphingolipid composition in skin samples

  • Evaluation of barrier function parameters

  • Assessment of keratinocyte differentiation markers

  • Measurement of transepidermal water loss and other skin physiology parameters

• Disease-relevant investigations:

  • Comparative sphingolipid profiling in healthy versus diseased skin

  • Analysis of Sptlc3 expression levels in different dermatological conditions

  • Evaluation of potential therapeutic approaches targeting Sptlc3 or its specific sphingolipid products

What experimental systems are most suitable for studying the interaction between Sptlc3 and other SPT subunits?

Understanding the complex interactions between Sptlc3 and other SPT subunits requires specialized experimental approaches:

• Cell-based co-expression systems:

  • Co-transfection of Sptlc1, Sptlc3, and small subunits in HEK293 or other suitable cell lines

  • Inducible expression systems to control the relative expression levels of different subunits

  • CRISPR-Cas9 genome editing to create knockout or knock-in cell lines

• Protein interaction studies:

  • Co-immunoprecipitation assays to detect physical interactions between subunits

  • Proximity ligation assays to visualize protein interactions in situ

  • Fluorescence resonance energy transfer (FRET) or bimolecular fluorescence complementation (BiFC) to study dynamic interactions

• Structural biology approaches:

  • Cryo-electron microscopy of reconstituted SPT complexes containing Sptlc3

  • X-ray crystallography of purified complexes or domains

  • Molecular dynamics simulations to predict interaction interfaces

• Functional assays:

  • In vitro reconstitution of SPT activity with purified components

  • Enzyme kinetics with various substrate combinations

  • Site-directed mutagenesis to identify critical residues for subunit interactions

Substrate Specificity and Sphingoid Base Production by Sptlc3

Data compiled from studies of recombinant SPTLC2 and SPTLC3 overexpression in HEK293 cells .

Comparative Analysis of Sphingoid Base Levels in Human and Mouse Plasma

Data derived from LC-MS analysis of plasma samples from humans and mice .

Effect of Small Subunits on Sphingoid Base Production in SPTLC3-expressing Cells

Data derived from co-expression studies of SPTLC3 with small subunits in HEK293 cells .