A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

Suh, Jung Hee; Eltanawy, Abeer; Rangan, Apoorva; Saba, Julie D.

doi:10.1016/j.chemphyslip.2015.09.006

Cited by 6 publications

(7 citation statements)

References 43 publications

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“…SPL assays: SPL activity was quantified by measuring formation of (2E)-hexadecenal by quantification of its hydrazine derivative as described (57).…”

Section: Despite Lymphopenia In Splis Patients T Cell Function Persimentioning

confidence: 99%

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome

Zhao¹,

Tassew²,

Lee³

et al. 2021

JCI Insight

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Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in SGPL1, which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome (SRNS) and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first two years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1 KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated pro-inflammatory and pro-fibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared to untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.

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“…SPL assays: SPL activity was quantified by measuring formation of (2E)-hexadecenal by quantification of its hydrazine derivative as described (57).…”

Section: Despite Lymphopenia In Splis Patients T Cell Function Persimentioning

confidence: 99%

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome

Zhao¹,

Tassew²,

Lee³

et al. 2021

JCI Insight

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“…SGPL1 activity in whole cell extracts of patient fibroblasts and HEK293T cells expressing WT and mutant SGPL1 cDNA constructs was measured by quantifying the amount of hexadecenal formed over time as described (46). 3D protein structure.…”

Section: Methodsmentioning

confidence: 99%

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Lovric¹,

Gonçalves²,

Gee³

et al. 2017

Journal of Clinical Investigation

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Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of diseaseassociated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

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“…By using nicotinylhydrazide (Isoniazid) modification of the 2E-HEX, they obtained the first data on purified recombinant human S1PL ( K M = 5.2 ± 0.9 μM). Most recently, Suh et al (53) developed a stable isotope dilution method using an internal D-labeled standard to also determine the values for recombinant human S1PL in good agreement with the value determined by Novartis ( K M = 4.67 μM, V max = 2.6 pmol/min/μg).…”

Section: Discussionmentioning

confidence: 65%

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

McLean

Marles‐Wright

Custódio

et al. 2017

Journal of Lipid Research

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Sphingolipids (SLs) are ubiquitous elements in eukaryotic membranes and are also found in some bacterial and viral species. As well as playing an integral structural role, SLs also act as potent signaling molecules involved in numerous cellular pathways and have been linked to many human diseases. A central SL signaling molecule is sphingosine-1-phosphate (S1P), whose breakdown is catalyzed by S1P lyase (S1PL), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the cleavage of S1P to (2E)-hexadecenal (2E-HEX) and phosphoethanolamine. Here, we show that the pathogenic bacterium, Burkholderia pseudomallei K96243, encodes two homologous proteins (S1PL2021 and S1PL2025) that display moderate sequence identity to known eukaryotic and prokaryotic S1PLs. Using an established MS-based methodology, we show that recombinant S1PL2021 is catalytically active. We also used recombinant human fatty aldehyde dehydrogenase to develop a spectrophotometric enzyme-coupled assay to detect 2E-HEX formation and measure the kinetic constants of the two B. pseudomallei S1PL isoforms. Furthermore, we determined the X-ray crystal structure of the PLP-bound form of S1PL2021 at 2.1 Å resolution revealing that the enzyme displays a conserved structural fold and active site architecture comparable with known S1PLs. The combined data suggest that B. pseudomallei has the potential to degrade host SLs in a S1PL-dependent manner.

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A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

Cited by 6 publications

References 43 publications

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome

Efficacy of AAV9-mediated SGPL1 gene transfer in a mouse model of S1P lyase insufficiency syndrome

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Characterization of homologous sphingosine-1-phosphate lyase isoforms in the bacterial pathogen Burkholderia pseudomallei

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