A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity

Kenneth Gable,Sita D Gupta,Gongshe Han,Somashekarappa Niranjanakumari,Jeffrey M Harmon,Teresa M Dunn

doi:10.1074/jbc.m110.122259

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.

Highlights

Serine palmitoyltransferase (SPT)3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis
Expression and Activities of Wild-type and Mutant SPT Heterodimers—It was previously shown that the yeast Lcb1pC180W mutant, which is analogous to the hLCB1C133W mutant, heterodimerizes with yeast Lcb2p, but that the mutant heterodimers are catalytically inactive [8]
To determine whether the hLCB1C133W mutation abolishes the activity of hLCB1/hLCB2 heterodimers, hLCB1 or hLCB1C133W was coexpressed with hLCB2a in CHO-LyB cells, and the activities of the human mutant and wild-type heterodimers were compared

Summary

Introduction

Serine palmitoyltransferase (SPT)3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis. Our recent discovery of the ssSPT subunits and our ability to express the human isozymes in yeast cells lacking endogenous SPT [2] has allowed us to directly test the possibility that the trimeric human SPT isozymes containing the most common HSAN1 mutant, hLCB1C133W, are enzymatically active and capable of utilizing alanine to synthesize the deoxy-longchain base, 1-deoxySa. Our results demonstrate that this is the case and provide additional evidence that these unusual products may be causally related to the loss of neurons in the dorsal root ganglia of patients with HSAN1.

Results

Conclusion