Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

Harrison, Peter; Gable, Kenneth; Niranjanakumari, Somashekarappa; Kelly, Van; Clarke, David J.; Naismith, J.H.; Dunn, Teresa M.; Campopiano, Dominic J.

doi:10.1194/jlr.m089367

Cited by 8 publications

(9 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Formation of the KDS product was confirmed by MALDI-TOF-MS analysis. This allowed a comparison with another well-characterized bacterial SpSPT from S. paucimobilis (Harrison et al 2019). The PgSPT bound both substrates with a similar affinity to SpSPT, but in contrast to this isoform, PgSPT displayed much slower kinetics.…”

Section: Discussionmentioning

confidence: 99%

Porphyromonas gingivalisSphingolipid Synthesis Limits the Host Inflammatory Response

et al. 2020

View full text Add to dashboard Cite

Porphyromonas gingivalis, like other bacteria belonging to the phylum Bacteroidetes, synthesizes sphingolipids (SLs). However, their exact roles in microbial physiology and their potential role in mediating interactions with their eukaryotic host are unclear. Our working hypothesis for this study was that synthesis of SLs (host-like lipids) affords a mechanism that allows P. gingivalis to persist in homeostasis with its host. In a previous study, we deleted a gene (PG1780 in strain W83) predicted to encode a serine palmitoyl transferase (SPT)—the enzyme that catalyzes the first conserved step in the synthesis of SLs—and we determined that the mutant was unable to synthesize SLs. Here, we characterized the SPT enzyme encoded by PG1780, analyzed the impact of SPT deletion on P. gingivalis gene expression (RNA-Seq analysis), and began to define the impact of SL synthesis on its interactions with host cells. Enzymatic analysis verified that the protein encoded by PG1780 is indeed an SPT. RNA-Seq analysis determined that a lack of SL synthesis results in differential expression of extracytoplasmic function sigma factors, components of the type IX secretion system (T9SS), and CRISPR and cas genes. Our data demonstrate that when human THP1 macrophage-like cells were challenged with the wild type (W83) and the SL-null mutant (W83 ΔSPT), the SL-null strain elicited a robust inflammatory response (elevated IL-1β, IL-6, IL-10, IL-8, RANTES, and TNFα) while the response to the parent strain W83 was negligible. Interestingly, we also discovered that SLs produced by P. gingivalis can be delivered to host cells independent of cell-to-cell contact. Overall, our results support our working hypothesis that synthesis of SLs by P. gingivalis is central to its ability to manipulate the host inflammatory response, and they demonstrate the integral importance of SLs in the physiology of P. gingivalis.

show abstract

Section: Discussionmentioning

confidence: 99%

Porphyromonas gingivalisSphingolipid Synthesis Limits the Host Inflammatory Response

et al. 2020

View full text Add to dashboard Cite

show abstract

“…More recently, Harrison et al used differentially labeled L -serine isotopologs and either palmitoyl-CoA or pentadecanoyl-CoA as SPT substrates to study the impact of stable-isotope label number and position in L -serine on the kinetics of recombinant human and bacterial SPT. For this, they applied HPLC equipped with a fluorescence detector to quantify C17 LCBs, and direct infusion high-resolution MS for detection of 3KS products (Harrison et al, 2019). Both studies used state-of-the-art techniques but limited their scopes to the first two steps of sphingolipid de novo synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…From an analytical point of view, the labeling of both the fatty acid and amino acid precursor molecules with stable-isotopes is advantageous. Most published MS-based SPT assays used labeled serine only (Ren et al, 2018; Harrison et al, 2019). Since serine is a small molecule (chemical formula: C 3 H 7 NO 3 ), the number of introducible labels is limited.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Harrison and co-workers found that the presence of a serine α-deuterium significantly decreased the reaction rate of recombinant bacterial SPT, a point also applicable to L -serine (2,3,3-d 3 ) used in the present study. However, recombinant human SPT was unaffected by the deuterium label at Cα position (Harrison et al, 2019). We used rat liver-derived SPT, which has a greater homology to human SPT than that of bacterial SPT.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry

Wigger

Gulbins

Kleuser

et al. 2019

Front. Cell Dev. Biol.

View full text Add to dashboard Cite

Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C16 fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d3 and L-serine-d3. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B1). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates – naturally occurring or chemically tailored – as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.

show abstract

“…Serine palmitoyltransferase (SPT) is a heterodimeric membrane protein, that could catalyse the formation of 3-ketodihydrosphingosine (3-KDS) from serine and palmitoyl CoA through condensation reaction 17 . SPT is the initial and rate-limiting enzyme in the de novo biosynthesis of sphingolipids, directly affecting the synthesis of two main groups of sphingolipids in fungal cells, such as inositol phosphoryl ceramide (IPC) and glucosylceramide (GlcCer) 11,[18][19][20] . Some studies have shown that inhibiting the enzymes involved in the biosynthetic pathways of sphingolipids could decrease the virulence of fungal pathogens 21 .…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel SPT inhibitor WXP-003 by docking-based virtual screening and investigation of its anti-fungi effect

Wang

Xin

Sun

et al. 2021

Journal of Enzyme Inhibition and Medicinal Chemistry

View full text Add to dashboard Cite

Serine palmitoyltransferase (SPT) plays the key role on catalysing the formation of 3-ketodihydrosphingosine, which is the first step of the de novo biosynthesis of sphingolipids. SPT is linked to many diseases including fungal infection, making it a potential therapeutic target. Thus, a logical docking-based virtual screening method was used to screen selective SPT inhibitor against fungi, not human. We used myriocinsimilarity database to identify compounds with good binding with fungal SPT and poor binding with homology human SPT model. Preliminary bio-assay led to the discovery of a promising inhibitor WXP-003, which displayed good inhibitory activity against diversity fungi strains with MIC ranging from 0.78 to 12.5 lg/mL. WXP-003 could significantly reduce sphingolipids content in fungi and no effect on mouse fibroblast cell line L929. Molecular dynamics simulation depicted that SPT/WXP-003 complex formed the favoured interactions. Taken together, discovery of WXP-003 provided valuable guide for the development of novel anti-fungal agents.

show abstract

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

Cited by 8 publications

References 52 publications

Porphyromonas gingivalisSphingolipid Synthesis Limits the Host Inflammatory Response

Porphyromonas gingivalisSphingolipid Synthesis Limits the Host Inflammatory Response

Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry

Identification of a novel SPT inhibitor WXP-003 by docking-based virtual screening and investigation of its anti-fungi effect

Contact Info

Product

Resources

About