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J Biol Chem, Vol. 274, Issue 53, 38131-38139, December 31, 1999

Function of the Cloned Putative Neutral Sphingomyelinase as Lyso-platelet Activating Factor-Phospholipase C^*

Hirofumi Sawai^§, Naochika Domae^§, Narasimhan Nagan^¶, and Yusuf A. Hannun

From the  Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, the ^§ Department of Medicine, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573, Japan, and the ^¶ Department of Medical Laboratory Sciences, Medical University of South Carolina, Charleston, South Carolina 29425

Sphingolipids such as ceramide and sphingosine have been regarded as novel signal mediators in cells. However, the mechanisms of generation of these lipids upon various stimulation remain to be elucidated. Neutral sphingomyelinase (N-SMase) is one of the key enzymes in the generation of ceramide, and recently the cloning of a putative N-SMase was reported. Because the function of the protein was unclear in the previous report, we investigated the role it plays in cells. N-SMase activity in cells overexpressing the protein with hexa-histidine tag was immunoprecipitated with anti-hexa-histidine antibody. The metabolism of ceramide and SM was not apparently affected in overexpressing cells. Radiolabeling experiments using [³H]palmitic acid or [³H]hexadecanol demonstrated an accumulation of 1-O-alkyl-sn-glycerol and a corresponding decrease of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine in overexpressing cells. In vitro studies showed that both 1-acyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) and 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-platelet activating factor (lyso-PAF)) are good substrates of the protein. In further radiolabeling experiments, 1-acyl-lyso-PC was predominantly and equally metabolized into diacyl-PC in both vector and overexpressing cells. On the other hand, 1-O-alkyl-lyso-PC (lyso-PAF) was metabolized into both diradyl-PC and 1-O-alkyl-glycerol in overexpressing cells but only into diradyl-PC in vector cells. These results suggest that the protein acts as lyso-PAF-PLC rather than lyso-PC-PLC or N-SMase in cells.

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^* This work was supported in part by National Institutes of Health Grant GM43825 (to Y. A. H.) and CHP-Seed Money for Education and Research Reform Grant (to N. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-4921; Fax: 843-792-4322, E-mail: hannun@musc.edu.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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