Abstract
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 2.3.1.50) 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.
Highlights
Lysenin, a hemolytic protein derived from the earthworm Eisenia foetida, has a high affinity for sphingomyelin
Sphingolipid biosynthesis is initiated by condensation of L-serine with palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) to generate 3-ketodihydrosphingosine. 3-Ketodihydrosphingosine is converted to dihydrosphingosine, which is N-acylated and dehydrogenated to form ceramide at the endoplasmic reticulum
We have demonstrated that expression of the hamster LCB1 cDNA in SPB-1 strain, a temperature-sensitive Chinese hamster ovary (CHO) cell mutant defective in SPT, restored SPT activity to the mutant cells and that a hexahistidine (His6)-tagged hamster LCB1 protein but not an untagged protein produced in SPB-1 cells caused SPT activity to adsorb to a Ni2ϩ-immobilized resin, indicating that the hamster LCB1 protein is a component of the SPT enzyme [7]
Summary
Materials—Lysenin purified from the coelomic fluid of Eisenia foetida [8] and rabbit anti-lysenin antiserum were gifts from Drs Y. After blocking the blot membrane with 10% skim milk in PBS containing 0.1% Tween 20, the LCB1 and LCB2 proteins on the membrane were detected by using the anti-cLCB1 antibody and anti-cLCB2 antibody (0.5 g/ml), respectively, as the primary antibody and a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad; 1:2500 dilution) as the secondary antibody with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech), unless otherwise noted. The membranes (2 mg of protein) were incubated in 1 ml of a solubilization buffer consisting of 0.1 M sodium phosphate buffer (pH 8.0), 50 mM NaCl, 10 mM imidazole, 0.1% soybean phospholipids, and 1% sucrose monolaurate (Mitsubishi Kasei Shokuhin Inc., Tokyo, Japan) for 10 min. Membranes (0.8 mg of protein) prepared from CHO-K1 cells were incubated in 0.4 ml of 50 mM Hepes-Na (pH 7.5) containing 1% sucrose monolaurate for 10 min, and after centrifugation (105 ϫ g, 30 min) the supernatant fluid was recovered as the solubilized membrane fraction. Protein Determination—Protein concentrations were determined by the method of Lowry et al [19] using BSA as the standard
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