Cloning of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylserine Synthase (PSS) II, Overexpression of Which Suppresses the Phosphatidylserine Biosynthetic Defect of a PSS I-lacking Mutant of CHO-K1 Cells

Osamu Kuge,Kyoko Saito,Masahiro Nishijima

doi:10.1074/jbc.272.31.19133

Osamu Kuge, Kyoko Saito + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.31.19133

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Abstract

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the exchange of free L-serine for the polar head group (base) of preexisting phospholipid. We previously showed the presence of two different enzymes catalyzing the serine base exchange in Chinese hamster ovary (CHO) cells and isolated the cDNA of one of the enzymes, PtdSer synthase (PSS) I, which also catalyzes the exchange of the base moiety of phospholipid(s) for ethanolamine and choline. In this study, we cloned a CHO cDNA, designated as pssB, which encodes a protein exhibiting 32% amino acid sequence identity with CHO PSS I. Introduction of the pssB cDNA into CHO-K1 cells resulted in striking increases in both the serine and ethanolamine base exchange activities. In contrast to the PSS I cDNA, the pssB cDNA was incapable of increasing the choline base exchange activity. The expression of the pssB gene in Sf9 insect cells also results in striking increases in both serine and ethanolamine base exchange activities. The pssB cDNA was found to transform a PtdSer-auxotrophic PSS I-lacking mutant of CHO-K1 cells to PtdSer prototrophy. The PtdSer content of the resultant transformant grown without exogenous PtdSer for 2 days was 4-fold that of the mutant and similar to that of CHO-K1 cells, indicating that the pssB cDNA complemented the PtdSer biosynthetic defect of the PSS I-lacking mutant. These results suggested that the pssB cDNA encoded the second PtdSer synthase PSS II, which catalyzed the serine and ethanolamine base exchange, but not the choline base exchange.

Highlights

Phosphatidylserine (PtdSer)1 is one of the major membrane phospholipids in mammalian cells, comprising about 10% of the total phospholipids
These findings led us to conclude that PtdSer in Chinese hamster ovary (CHO) cells is synthesized through serine base exchange, which is catalyzed by two kinds of PtdSer synthase (PSS): one, PSS I, which is absent in mutant PSA-3, can use PtdCho as a substrate; and the other, PSS II, uses PtdEtn but not PtdCho as a substrate
A cDNA fragment of the CHO counterpart for human expressed sequence tag (EST) F11951 was generated from a CHO cDNA library, using a polymerase chain reaction and primers corresponding to parts of the EST sequence

Summary

Phosphatidylserine Synthase II of CHO Cells

The cDNA synthesis and construction of a cDNA library in a plasmid vector, pSPORT1, were performed with a SuperScript௢ plasmid system (Life Technologies, Inc.) according to the manufacturer’s instructions. The 0.3kilobase pair product of the second round of amplification was subcloned into a plasmid, pBluescript II SKϩ (Stratagene), sequenced, and used as a hybridization probe for screening of the CHO cDNA library, after enrichment of hybridizing clones with a GeneTrapper௢ cDNA-positive selection system (Life Technologies, Inc.). Fresh monolayers of Sf9 cells (75-cm flask) were infected with the transfection supernatant containing pssB recombinant virus or control virus and cultivated in TC-100 insect medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum. Protein was measured according to Lowry et al (11), using bovine serum albumin as a standard

RESULTS

Substrate Ethanolamine

Othera Total

DISCUSSION

PS PE