Hereditary sensory neuropathy type 1 mutations confer dominant negative effects on serine palmitoyltransferase, critical for sphingolipid synthesis

Abstract

Hereditary sensory neuropathy type 1 (HSN1) is a dominantly inherited degenerative disorder of the peripheral nerves. HSN1 is clinically and genetically heterogeneous. One form arises from mutations in the gene SPTLC1 encoding long-chain base 1 (LCB1), one of two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step of sphingolipid synthesis. We have examined the effects of the mutations C133Y and C133W, which we have identified in two HSN1 families, on the function of SPT. Although in HSN1 lymphoblasts, the C133Y and C133W mutations do not alter the steady-state levels of LCB1 and LCB2 subunits, they result in reduced SPT activity and sphingolipid synthesis. Moreover, in a mutant Chinese hamster ovary (CHO) cell strain with defective SPT activity due to a lack of the LCB1 subunit, these mutations impair the ability of the LCB1 subunit to complement the SPT deficiency. Furthermore, the overproduction of either the LCB1C133Y or LCB1C133W subunit inhibits SPT activity in CHO cells despite the presence of wild-type LCB1. In addition, we demonstrate that in CHO cells the mutant LCB1 proteins, similar to the normal LCB1, can interact with the wild-type LCB2 subunit. These results indicate that the HSN1-associated mutations in LCB1 confer dominant negative effects on the SPT enzyme.

Figure 1

Comparison of SPT-specific activity between HSN1 patients and healthy controls. SPT activity was determined in microsomes isolated from lymphoblastoid cell lines of HSN1 patients (HSN1) and age- and gender-matched healthy controls (Cont). Each assay was performed in triplicate, and data are presented as means with SD. Cell line ID numbers correspond to those listed in Table 1.

Figure 2

Western immunoblot analysis of LCB1 and LCB2 proteins present in lymphoblastoid cells from HSN1 patients and healthy controls. Each lane contains an equal amount (5 μg) of microsomal proteins isolated from lymphoblastoid cells of healthy controls (Cont) and from HSN1 patients (HSN1). LCB1 and LCB2 proteins were detected by Western immunoblot analysis as described in Methods. The ID numbers of cell lines used are in parentheses.

Figure 3

Metabolic labeling of lipids with radioactive serine in lymphoblastoid cells from HSN1 and healthy controls. Cells were incubated with L-[³H(G)]serine for 2.5 hours. Lipids were isolated by high-performance TLC as described in Methods. Lipid synthesis was expressed as counts per minute per milligram of protein. The mean values with SD from triplicate experiments are shown. *P < 0.01 vs. the healthy control levels. TSLs, total sphingolipids.

Figure 4

Expression of wild-type hamster LCB1 and its C133Y or C133W mutant in LY-B cells. LY-B cells were transfected with various plasmids and analyzed. As controls, untransfected LY-B and wild-type CHO-K1 cells were also analyzed. Lanes 1 and 2 represent untransfected CHO-K1 and LY-B cells, respectively. Lanes 3, 4, 5, and 6 represent LY-B cells transfected with pSV-OK (empty vector), pSV-cLCB1, pSV-cLCB1C133Y, and pSV-cLCB1C133W, respectively. (a) Western blotting of the LCB1 subunit. Each lane was loaded with 20 μg protein of cell lysate. (b) Metabolic labeling of lipids. After incubation of cells with L-[¹⁴C(U)]serine for 2 hours, lipids were extracted from the cells and separated by TLC. The radioactive image of lipids on the TLC plate is shown. PE, phosphatidylethanolamine. (c) SPT activity in cell lysate. Data shown are from triplicate experiments.

Figure 5

Stable overexpression of wild-type hamster LCB1 and its C133Y or C133W mutant in CHO-K1 cells. (a) Western blotting for the LCB1 subunit. Each lane was loaded with 20 μg protein of cell lysate. (b) SPT activity in cell lysate. Data shown are from triplicate experiments. (c) Metabolic labeling of lipids in intact cells with L-[¹⁴C(U)]serine. Radioactivity of each lipid type was determined and normalized for protein. Data are shown as the percentage of the mean values obtained in CHO-K1 cells from four experiments. *P < 0.01 vs. the CHO-K1 levels.

Figure 6

Coimmunoprecipitation of the LCB2 subunit with the LCB1 subunit and its HSN1-like mutants. After transfection of CHO-K1 cells with expression plasmids encoding wild-type and mutated cLCB1 with or without the amino-terminal FLAG-tag, cell membranes were prepared. The membranes were solubilized with 1% (wt/vol) sucrose monolaurate, and the solubilized fraction was subjected to an anti-FLAG Ab–coupled matrix. After washing of the matrix, proteins bound to the matrix were eluted by incubation in the SDS-sample buffer at 70°C for 5 minutes, and analyzed by Western blotting with anti-cLCB2 Ab as described in Methods.