Cloning and Expression of Mouse Liver Phosphatidylserine Synthase-1 cDNA

Abstract

In eukaryotic cells, phosphatidylserine (PtdSer) is synthesized by two distinct synthases on the endoplasmic reticulum by a base-exchange reaction in which the polar head-group of an existing phospholipid is replaced with serine. We report the cloning and expression of a cDNA for mouse liver PtdSer synthase-1. The deduced protein sequence is >90% identical to that of PtdSer synthase-1 from Chinese hamster ovary cells and a sequence from a human myeloblast cell line. PtdSer synthase-1 cDNA was stably expressed in M.9.1.1 cells which are mutant Chinese hamster ovary cells defective in PtdSer synthase-1 activity, are ethanolamine auxotrophs, and have a reduced content of PtdSer and phosphatidylethanolamine (PtdEtn). The growth defect of M.9.1.1 cells was eliminated, and a normal phospholipid composition was restored in the absence of exogenous ethanolamine, implying that the cloned cDNA encoded PtdSer synthase. Mouse liver PtdSer synthase-1 was also expressed in McArdle 7777 rat hepatoma cells. In addition to a 3-fold higher in vitro serine-exchange activity, these cells also exhibited enhanced choline-and ethanolamine-exchange activities and incorporated more [ 3 H]serine into PtdSer than did control cells. However, the levels of PtdSer and PtdEtn in cells overexpressing PtdSer synthase-1 activity were not increased. Excess PtdSer produced by the transfected cells was rapidly decarboxylated to PtdEtn and the degradation of PtdSer, and/or PtdEtn derived from PtdSer, was increased. Moreover, the CDP-ethanolamine pathway for PtdEtn biosynthesis was inhibited. These data suggest that (i) cellular levels of PtdSer and PtdEtn are tightly controlled, and (ii) the metabolism of PtdSer and PtdEtn is coordinately regulated to maintain phospholipid homeostasis.

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

“…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 . In addition, in the majority of PSS1-deficient 21 or PSS2-deficient 22 mouse tissues (including the brain), the levels of phosphatidylserine and phosphatidylethanolamine are not reduced, despite the 65-98% reduction in total serine-exchange activity 21 .…”

Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2

“…85ATGs : ATGCGGAGGGCCGAGC-GCAGAGTC ; 1516TAGas : ATCATGAGGCGGCTGAGGC-CCCCT. cDNA from a λgt11 mouse liver expression library (Clontech, Palo Alto, CA, U.S.A.) was isolated [7] and used as a template for PCR. The 50 µl PCR reaction mixture contained 5 µl of 10-fold concentrated reaction buffer (250 mM Taps, pH 9.3, 500 mM KCl, 10 mM β-mercaptoethanol ; supplied by Panvera, Madison, WI, U.S.A.), 2.5 mM MgCl # , 0.25 mM of each nucleoside triphosphate, 200 ng of λ phage cDNA, 10 pmol each of 85ATGs and 1516TAGas gene-specific primers, and 2.5 units of TaKaRa Ex Taq DNA polymerase (Panvera).…”

“…The substrate specificities of PSS1 and PSS2 from CHO cells were confirmed when their cDNAs were cloned and expressed [5,6]. Expression of PSS1 cDNA from CHO cells in PSA3 cells [5], or expression of the cDNA encoding murine PSS1 in M.9.1.1 cells [7], increased the base-exchange activity and complemented the PtdSer\ethanolamine auxotrophy of these Abbreviations used : CHO, Chinese hamster ovary ; CMV, cytomegalovirus ; Mc/PSS2, McArdle 7777 cells expressing murine phosphatidylserine synthase-2 ; PSA, phosphatidylserine auxotroph ; PSS, phosphatidylserine synthase ; PtdCho, phosphatidylcholine ; PtdEtn, phosphatidylethanolamine ; PtdSer, phosphatidylserine. 1 To whom correspondence should be addressed (e-mail jean.vance!ualberta.ca).…”

“…Some evidence indicates that these two PtdEtn biosynthetic pathways are co-ordinately regulated so that PtdSer and PtdEtn homoeostasis is maintained. For example, when PtdEtn production from PtdSer decarboxylation was increased by overexpression of PSS1 in hepatoma cells, synthesis of PtdEtn via the CDP-ethanolamine pathway was inhibited [7]. Moreover, in M.9.1.1 cells in which PtdEtn production from PtdSer decarboxylation was reduced, the incorporation of [$H]ethanolamine into PtdEtn was approx.…”

“…Our studies provide evidence that murine PSS1 and PSS2 are differently regulated and that expression of murine PSS1 and PSS2 in these cells differentially modulates phospholipid metabolism. [7].…”