Research Articles
Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases

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SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.

This article has been withdrawn by the authors. See the withdrawal notice here: April 1 erratum jlr. M068692ERR.

cell-free expression
ceramide phosphoethanolamine
click chemistry
enzyme mechanisms
Golgi apparatus
lipid biochemistry
lipidomics
model membranes
protein engineering

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This work was supported by a Marie Curie Intra-European Fellowship (J.G.M.M.), the European Union Seventh Framework Programme Marie-Curie ITN ‘Sphingonet’ Grant (289278), the Deutsche Forschungsgemeinschaft Sonderforschungsbereich/SFB944-P14 (J.C.M.H.), and an Incentive Award of the Faculty of Biology/Chemistry from the University of Osnabrück (M.K.). The authors declare that they have no conflicts of interest with the contents of this article.

    Abbreviations:

    Asp

    aspartic acid

    CPE

    ceramide phosphoethanolamine

    CPI

    ceramide phosphoinositol

    ER

    endoplasmatic reticulum

    Glu

    glutamic acid

    HA

    hemagglutinin

    His

    histidine

    IMDM

    Iscove modified Dulbecco medium

    NBD

    nitrobenzooxadiazole

    NBD-Cer

    NBD-C6-ceramide

    NBD-CPE

    NBD-C6-CPE

    ORF

    open reading frame

    PC

    phosphatidylcholine, PE, phosphatidylethanolamine

    PI

    phosphatidylinositiol

    SMS

    sphingomyelin synthase

    SMSr

    sphingomyelin synthase-related protein

1

M. Kol and R. Panatala contributed equally to this article.