Enzymatic measurement of phosphatidylserine in cultured cells

Morita, Shin; Shirakawa, Sachimi; Kobayashi, Yukiko; Nakamura, Keiko; Teraoka, Reiko; Kitagawa, Shuji; Takato, Tsuyoshi

doi:10.1194/jlr.d021808

Cited by 32 publications

(27 citation statements)

References 40 publications

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“…Other methods. Immunoblotting was performed using polyclonal antibody to PTDSS1 (Y-19, sc-51410, Santa Cruz Biotechnology) 27 and standard methods. Cellular protein content was determined by the BCA method (Pierce) with BSA as the standard.…”

Section: Staining Of Neutral Lipids and Quantification Of Triacylglycmentioning

confidence: 99%

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Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome

Jenkins²,

et al. 2013

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Lenz-Majewski syndrome (LMS) is a syndrome of intellectual disability and multiple congenital anomalies that features generalized craniotubular hyperostosis. By using whole-exome sequencing and selecting variants consistent with the predicted dominant de novo etiology of LMS, we identified causative heterozygous missense mutations in PTDSS1, which encodes phosphatidylserine synthase (PSS ). PSS is one of two enzymes involved in the production of phosphatidylserine. Phosphatidylserine synthesis was increased in intact fibroblasts from affected individuals, and end-product inhibition of PSS by phosphatidylserine was markedly reduced. Therefore, these mutations cause a gain-of-function effect associated with regulatory dysfunction of PSS . We have identified LMS as the first human disease, to our knowledge, caused by disrupted phosphatidylserine metabolism. Our results point to an unexplored link between phosphatidylserine synthesis and bone metabolism.

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Section: Staining Of Neutral Lipids and Quantification Of Triacylglycmentioning

confidence: 99%

“…For example, PSS1 overexpression in HEK293 cells only slightly increases cellular phosphatidylserine levels 27 . Furthermore, overexpression of PSS1 or PSS2 in McArdle hepatoma cells does not increase cellular phosphatidylserine or phosphatidylethanolamine content 19,28 .…”

mentioning

confidence: 99%

Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome

Jenkins²,

et al. 2013

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“…reported the phosphatidylserine determination method using PLD from Strreptomyces chromofuscus (41). Moreover, it has been reported that plasmalogen level is correlated with dementia and cancer, so that plasmalogen can be a biomarker for them (42).…”

Section: Expression Purification and Characterization Of Pldmentioning

confidence: 99%

Substrate recognition mechanism of Streptomyces phospholipase D and enzymatic measurement of plasmalogen

Matsumoto

Sugimori

2015

Journal of Bioscience and Bioengineering

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The substrate recognition mechanism of phospholipase D and enzymatic measurement of choline plasmalogen were investigated. A phospholipase D (PLD684) from Streptomyces sp. strain NA684 was purified 184-fold from the culture supernatant with 23.7% recovery. Maximum activity for l-α-lysophosphatidylcholine (LPC) hydrolysis was found at pH 5.0 and 80°C. The hydrolytic activity was remarkably affected by the concentration of Triton X-100 in the reaction mixture. In the presence of 0.05-0.5% and 0.1-0.2% (wt/vol) Triton X-100, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and choline plasmalogen were efficiently hydrolyzed by PLD684, respectively. Hydrolysis of LPC and choline lysoplasmalogen did not require Triton X-100; rather, the hydrolytic activity was inhibited by more than 0.05% (wt/vol) Triton X-100. The enzyme preferred mixed micelle substrates to liposomal substrates and hydrolyzed 98.4% of mixed micelle POPC in 1 h. Kinetic analysis showed that the rate-limiting steps of hydrolysis of mixed micelle POPC and emulsified LPC by PLD684 were the bulk step and the surface step, respectively. These results suggest that PLD684 has at least two substrate recognition mechanisms to recognize various phospholipids that have considerably different physical properties derived from their head and tail groups. Understanding of how PLD684 recognizes substrate forms will be useful for elucidating roles of lipolytic proteins in nature. Moreover, we report an enzymatic measurement of choline plasmalogen using PLD684 and phospholipase B. This is the first enzymatic method for measuring choline plasmalogen.

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“…Measurement of PS content of the plasma membrane was performed using a three-reagent system as recently reported (45). Reagent S1 contained 600 U/ml PLD, 20 U/ml LAAO, 50 mM NaCl, and 50 mM Tris-HCl (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

Adu-Gyamfi

Johnson

Fraser

et al. 2015

J Virol

199

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Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. IMPORTANCEThe lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Lipid-enveloped viruses harbor a lipid membrane bilayer derived from their host cell during the budding process. This envelope provides the virus stability, protection of its genetic contents, and a reservoir for its transmembrane glycoprotein, which mediates entry into cells (1, 2). The viral lipid envelope may be a viable target for drug development, as particular alterations in the lipid coat or receptor-lipid interaction can inhibit viral entry (3-6). The lipid-dependent budding and egress of some lipid-enveloped viruses have been investigated. For example, it is well established that HIV-1 binds and utilizes 1,2-dioleoyl-sn-glycero-3-phospho-(1=-myo-inositol-4=,5=-bisphosphate) [PI(4,5)P 2 ] enriched in the plasma membrane (PM) inner leaflet for assembly and egress from the cell (7,8). Enteroviruses and flaviviruses use a phosphatidylinositol-4-phosphate [PI(4)P]-enriched organelle to replicate (9), and enteroviruses are packaged into phosphatidylserine (PS)-enriched vesicles, thereby enhancing the efficiency of viral transmission (10). The budding and egres...

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Enzymatic measurement of phosphatidylserine in cultured cells

Cited by 32 publications

References 40 publications

Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome

Gain-of-function mutations in the phosphatidylserine synthase 1 (PTDSS1) gene cause Lenz-Majewski syndrome

Substrate recognition mechanism of Streptomyces phospholipase D and enzymatic measurement of plasmalogen

Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

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