Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein for serine palmitoyltransferase

J Biol Chem. 1998 Dec 11;273(50):33787-94. doi: 10.1074/jbc.273.50.33787.


Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin. Chinese hamster ovary (CHO) cells exhibited a high cytolytic sensitivity to lysenin, but treatment with sphingomyelinase rendered the cells resistant to lysenin. Temperature-sensitive CHO mutant cells defective in sphingolipid synthesis were resistant to lysenin, and this lysenin resistance was suppressed by metabolic complementation of sphingolipids. Selection of lysenin-resistant variants from mutagenized CHO cells yielded two types of sphingomyelin-deficient mutants, both of which showed less lysenin binding capability than wild-type cells. One mutant strain was severely defective in sphingomyelin synthesis but not glycosphingolipid synthesis, and another strain (designated LY-B) was incapable of de novo synthesis of any sphingolipid species and had no activity of serine palmitoyltransferase (SPT; EC catalyzing the first step of sphingolipid biosynthesis. LY-B cells lacked the LCB1 protein, a component of SPT, and transfection of LY-B cells with the hamster LCB1 cDNA restored both SPT activity and sphingolipid synthesis to the cells. Expression of an affinity peptide-tagged LCB1 protein in LY-B cells caused the endogenous LCB2 protein to adsorb to a tag affinity matrix. In addition, an anti-hamster LCB2 protein antibody co-immunoprecipitated both SPT activity and the wild-type LCB1 protein with the LCB2 protein. Thus, cell surface sphingomyelin is essential for lysenin-induced cytolysis, and lysenin is a useful tool for isolation of sphingomyelin-deficient mutants. Moreover, these results demonstrate that the SPT enzyme comprises both the LCB1 and LCB2 proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / biosynthesis
  • Acyltransferases / genetics
  • Acyltransferases / metabolism*
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cricetinae
  • Cytotoxins / metabolism*
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Proteins / pharmacology
  • Serine C-Palmitoyltransferase
  • Sphingomyelin Phosphodiesterase / metabolism
  • Sphingomyelins / metabolism*
  • Toxins, Biological


  • Cytotoxins
  • Proteins
  • Sphingomyelins
  • Toxins, Biological
  • lysenin
  • Acyltransferases
  • Serine C-Palmitoyltransferase
  • Sphingomyelin Phosphodiesterase