A disease-causing mutation in the active site of serine palmitoyltransferase causes catalytic promiscuity

Abstract

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has approximately 10-20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.

FIGURE 1.

Activity of SPT containing the hLCB1^C133W mutant subunit. hLCB1 or hLCB1^C133W were expressed with or without hLCB2a in CHO-LyB cells, and microsomes were prepared for immunoblotting to ascertain the expression of transfected genes (A) or used to assay SPT activity (B). Alternatively, a constant amount of hLCB1 (0.25 μg) was expressed with increasing amounts of hLCB1^C133W and microsomal SPT activity was determined (C).

FIGURE 2.

Coexpression of the ssSPT subunits with the hLCB1^C133W/hLCB2a heterodimer results in significant catalytic activity. A, yeast lcb1Δlcb2Δ mutants were transformed with plasmids expressing hLCB2a, wild-type, or mutant hLCB1, and empty vector (top row), HA-tagged ssSPTa (middle row), or HA-ssSPTb (bottom row). Transformants were tested for growth on YPD plates at 26° or 37 °C or with phytosphingosine. B, microsomal proteins obtained from the lcb1Δlcb2Δ yeast cells expressing hLCB2a with hLCB1 or hLCB1^C133W without (lanes 1 and 4) or with HA-ssSPTa (lanes 2 and 5) or HA-ssSPTb (lanes 3 and 6) were fractionated by SDS-PAGE, and subunit expression was visualized by immunoblotting using anti-hLCB1, anti-hLCB2a, and anti-HA antibodies. The lower of the two bands seen in immunoblots of yeast microsomal proteins using the anti-hLCB2a antibody is a nonspecific protein. C, microsomal SPT activities from yeast cells expressing various combinations of human subunits as in panel B were determined according to “Experimental Procedures.” D, microsomal protein obtained from CHO-LyB cells expressing hLCB2a, wild-type, or C133W mutant hLCB1, and HA-ssSPTa (lanes 1 and 3) or HA-ssSPTb (lanes 2 and 4) were fractionated by SDS-PAGE, and subunit expression was visualized by immunoblotting using anti-hLCB1, anti-hLCB2a, and anti-HA. E, microsomal SPT activities from CHO-LyB cells expressing various combinations of human subunits as in panel D were determined.

FIGURE 3.

Mutant human SPT heterotrimers synthesize 1-deoxySa (doxSA). Long-chain bases were analyzed from yeast lcb1Δlcb2Δ mutants expressing wild-type (broken line) or mutant (solid line) hLCB1/hLCB2a/ssSPTa (A) or hLCB1/hLCB2a/ssSPTb (B) heterotrimers or from CHO-LyB cells expressing wild-type (broken line) or mutant (solid line) hLCB1/hLCB2a/ssSPTa (C) or hLCB1/hLCB2a/ssSPTb (D) heterotrimers. The C18-1-deoxySa (C18 DoxSa) eluted at ∼36 min and the C20 DoxSa at ∼73 min.

FIGURE 4.

Properties of SPT enzymes containing wild-type and mutant LCB1 subunits. The serine K_m and the V_max of the wild-type (A) and HSAN1 C133W mutant (B) SPT enzymes were determined using [³H]serine as described under “Experimental Procedures.” The alanine K_m and V_max of the mutant enzyme was determined using [³H]alanine (C). The K_i values for alanine inhibition of serine utilization by the mutant and wild-type enzymes were determined by measuring the apparent K_m for serine in the presence of varying concentrations of unlabeled alanine (D).

FIGURE 5.

Synthesis of 1-deoxySa is modulated by the extracellular concentrations of the amino acid substrates. A, the indicated LCB standards (stds) were analyzed by high-performance liquid chromatography as described under “Experimental Procedures.” CHO-LyB cells expressing hLCB1^C133W/hLCB2a/ssSPTa were grown in Ham's F-12 with 5% Dulbecco's modified Eagle's medium (B), or in the same media supplemented with 1 mm alanine (C), 1 mm alanine and 1 mm serine (D), or 1 mm alanine and 10 mm serine (E), and long-chain bases were extracted and analyzed as described under “Experimental Procedures.”

FIGURE 6.

1-deoxySa induces ER stress. A, model of the hLCB2-ssSPT-LCB1 triple fusion SPT in which ssSPT (stippled) is inserted between hLCB2 and hLCB1. Topologies are based on hydropathy analyses as well as preliminary experimental data as described in the text. Cys-133, which when mutated to tryptophan causes HSAN, is indicated (C). The pyridoxal phosphate-binding lysine in hLCB2, which in the crystal structure of S. paucimobilis lies directly across the dimer interface from Cys-133 (9), is also indicated (K). B, CHO-LyB cells expressing mutant human LCB2a-ssSPTa-LCB1^C133W (bottom panel), but not wild-type LCB2-ssSPTa-LCB1 (top panel), fusion SPT accumulate 1-deoxySa (DoxSA). C, cells transfected with vector (lanes 1–3), or plasmids expressing hLCB2a-ssSPTa-LCB1 (WT; lanes 4–6), or hLCB2a-ssSPTa-LCB1^C133W (C133W; lanes 7–9) were grown in Ham's F-12 with or without 10 mm serine or 10 mm alanine for 48 h. As controls, untransfected cells were treated with vehicle (lane 10) or 20 μg/ml tunicamycin for 5 h (lane 11). LCB2a-ssSPTa-LCB1, GADD153, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were visualized by immunoblotting following fractionation of total cellular proteins by SDS-PAGE. D, untransfected CHO-LyB cells were treated with vehicle (0.4% bovine serum albumin/phosphate-buffered saline/ethanol) (lane 1), 1 μm 1-deoxySa (lane 2), 1 μm dihydrosphinganine (lane 3), 1 μm sphingosine (lane 4), 0.25% DMSO (lane 5), or 20 μg/ml tunicamycin in DMSO (lane 6). GADD153 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were visualized by immunoblotting as above. Results are representative of data obtained in three independent experiments.