Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism

Norma Marchesini,Chiara Luberto,Yusuf A Hannun

doi:10.1074/jbc.m212262200

Abstract

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.

Highlights

Sphingolipid metabolites are recognized as important components in signal transduction, in mammalian cells and in yeast where they are implicated in heat stress responses
Expression of nSMase2 in S. cerevisiae—In order to establish that nSMase2 is a structural gene for neutral sphingomyelinase (N-SMase), we expressed nSMase2 into the JK9-3d yeast (MATa/␣ trp1 leu2-3 his4 ura3 ade2rme1) strain deleted in ISC1 that encodes for an endogenous inositol phosphosphingolipid phospholipase C (Isc1p), as Isc1p shows robust SMase activity and its deletion removes SMase activity from yeast [37]
In this study we demonstrate that the cloned nSMase2 functions as a neutral Mg2ϩ-dependent sphingomyelinase in cells and that the protein exerts SMase activity both in vitro and in cells

Summary

Introduction

Sphingolipid metabolites are recognized as important components in signal transduction, in mammalian cells and in yeast where they are implicated in heat stress responses. A major sphingolipid metabolite, has been shown to play important roles in apoptosis, cell cycle arrest, and differentiation Sphingomyelinases (SMases) are enzymes that cleave the phosphodiester linkage of sphingomyelin into ceramide and phosphocholine, and they are implicated in several pathways of signal transduction and cell regulation. We demonstrated that, in cells, nSMase functions as a lysophospholipase C and not as a SMase [21], and this is supported by multiple studies showing that the. This paper is available on line at http://www.jbc.org nSMase and Sphingolipid Metabolism enzyme shows similar activity in vitro toward SM and lysophosphatidylcholine or lyso-platelet-activating factor (lysoPAF) [21, 23,24,25]. The only published study on nSMase demonstrates that it is regulated by PS, and it is abundant in brain [20], consistent with the basic properties of the partially purified N-SMase from rat [27] and bovine brain [28]

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