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 Tafesse, Joost C.M. Holthuis

Research output: Contribution to journalArticle

7 Citations (Scopus)

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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp 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)962-973
Number of pages12
JournalJournal of Lipid Research
Volume58
Issue number5
DOIs
StatePublished - May 1 2017

Fingerprint

Sphingolipids
Biosynthesis
Catalytic Domain
Enzymes
Cell membranes
Cells
Cell Membrane
Mutagenesis
Molecular Models
Phosphorylcholine
Molecular interactions
Mechanical stability
Ceramides
Site-Directed Mutagenesis
ceramide phosphoethanolamine
phosphatidylcholine-ceramide phosphocholine transferase
Phosphatidylcholines
Sorting
Histidine
Endoplasmic Reticulum

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

Cite this

Kol, M., Panatala, R., Nordmann, M., Swart, L., Van Suijlekom, L., Cabukusta, B., ... Holthuis, J. C. M. (2017). Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases. Journal of Lipid Research, 58(5), 962-973. https://doi.org/10.1194/jlr.M076133

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases. / Kol, Matthijs; Panatala, Radhakrishnan; Nordmann, Mirjana; Swart, Leoni; Van Suijlekom, Leonie; Cabukusta, Birol; Hilderink, Angelika; Grabietz, Tanja; Mina, John G.M.; Somerharju, Pentti; Korneev, Sergei; Tafesse, Fikadu; Holthuis, Joost C.M.

In: Journal of Lipid Research, Vol. 58, No. 5, 01.05.2017, p. 962-973.

Research output: Contribution to journalArticle

Kol, M, Panatala, R, Nordmann, M, Swart, L, Van Suijlekom, L, Cabukusta, B, Hilderink, A, Grabietz, T, Mina, JGM, Somerharju, P, Korneev, S, Tafesse, F & Holthuis, JCM 2017, 'Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases', Journal of Lipid Research, vol. 58, no. 5, pp. 962-973. https://doi.org/10.1194/jlr.M076133
Kol, Matthijs ; Panatala, Radhakrishnan ; Nordmann, Mirjana ; Swart, Leoni ; Van Suijlekom, Leonie ; Cabukusta, Birol ; Hilderink, Angelika ; Grabietz, Tanja ; Mina, John G.M. ; Somerharju, Pentti ; Korneev, Sergei ; Tafesse, Fikadu ; Holthuis, Joost C.M. / Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases. In: Journal of Lipid Research. 2017 ; Vol. 58, No. 5. pp. 962-973.
@article{8a22c7eab09d487c8d5e1d061e4af02e,
title = "Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases",
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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp 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.",
keywords = "Cell-free expression, Ceramide phosphoethanolamine, Click chemistry, Enzyme mechanisms, Golgi apparatus, Lipid biochemistry, Lipidomics, Model membranes, Protein engineering",
author = "Matthijs Kol and Radhakrishnan Panatala and Mirjana Nordmann and Leoni Swart and {Van Suijlekom}, Leonie and Birol Cabukusta and Angelika Hilderink and Tanja Grabietz and Mina, {John G.M.} and Pentti Somerharju and Sergei Korneev and Fikadu Tafesse and Holthuis, {Joost C.M.}",
year = "2017",
month = "5",
day = "1",
doi = "10.1194/jlr.M076133",
language = "English (US)",
volume = "58",
pages = "962--973",
journal = "Journal of Lipid Research",
issn = "0022-2275",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "5",

}

TY - JOUR

T1 - Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

AU - Kol, Matthijs

AU - Panatala, Radhakrishnan

AU - Nordmann, Mirjana

AU - Swart, Leoni

AU - Van Suijlekom, Leonie

AU - Cabukusta, Birol

AU - Hilderink, Angelika

AU - Grabietz, Tanja

AU - Mina, John G.M.

AU - Somerharju, Pentti

AU - Korneev, Sergei

AU - Tafesse, Fikadu

AU - Holthuis, Joost C.M.

PY - 2017/5/1

Y1 - 2017/5/1

N2 - 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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp 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.

AB - 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, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the 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 Glu permitting SMS-catalyzed CPE production and Asp 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.

KW - Cell-free expression

KW - Ceramide phosphoethanolamine

KW - Click chemistry

KW - Enzyme mechanisms

KW - Golgi apparatus

KW - Lipid biochemistry

KW - Lipidomics

KW - Model membranes

KW - Protein engineering

UR - http://www.scopus.com/inward/record.url?scp=85018900200&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85018900200&partnerID=8YFLogxK

U2 - 10.1194/jlr.M076133

DO - 10.1194/jlr.M076133

M3 - Article

C2 - 28336574

AN - SCOPUS:85018900200

VL - 58

SP - 962

EP - 973

JO - Journal of Lipid Research

JF - Journal of Lipid Research

SN - 0022-2275

IS - 5

ER -