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

Matthijs Kol, Radhakrishnan Panatala, Mirjana Nordmann, Leoni Swart, Leonie Van Suijlekom, Birol Cabukusta, Angelika Hilderink, Tanja Grabietz, John G.M. Mina, Pentti Somerharju, Sergei Korneev, Fikadu G. Tafesse, Joost C.M. Holthuis

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1273-1285
Number of pages13
JournalJournal of lipid research
Volume57
Issue number7
DOIs
StatePublished - Jul 2016

Keywords

  • Cell-free expression
  • Ceramide phosphoethanolamine
  • Click chemistry
  • Enzyme mechanisms
  • Golgi apparatus
  • Lipid biochemistry
  • Lipidomics
  • Model membranes
  • Protein engineering

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Cell Biology

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  • Cite this

    Kol, M., Panatala, R., Nordmann, M., Swart, L., Van Suijlekom, L., Cabukusta, B., Hilderink, A., Grabietz, T., Mina, J. G. M., Somerharju, P., Korneev, S., Tafesse, F. G., & Holthuis, J. C. M. (2016). Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site engineering of sphingomyelin synthases. Journal of lipid research, 57(7), 1273-1285. https://doi.org/10.1194/jlr.M068692