In the present study we have clarified some of the key molecular aspects of mammalian SPT activity and have defined how the HSN1-associated LCB1 subunit mutations lead to diminished enzyme activity. We demonstrated that the C133Y and C133W mutations in the SPTLC1
gene result in decreased SPT activity in lymphoblasts from HSN1 patients. In addition, these mutations not only impaired the ability of the LCB1 subunit to support SPT activity in a CHO cell line that lacks the endogenous LCB1 subunit, but also reduced SPT activity when expressed in the wild-type CHO strain. Based on these results, we conclude that both HSN1-LCB1 mutations alter SPT activity through a dominant negative effect, explaining why this form of HSN1 is dominantly inherited. Moreover, we demonstrated in normal CHO cells that both mutant LCB1 proteins interact with the wild-type LCB2 subunit. Accordingly, we suggest that the competition between the mutated and wild-type LCB1 for interaction with LCB2 and formation of the SPT complex may account for the domi-nant negative effects of the mutations. The present results are consistent with the earlier observations showing that in yeast, HSN1-like mutations reduce SPT activity through a dominant negative effect (20
). However, our results differ from a previous study in which, in HSN1 lymphoblastoid cells, the SPTLC1
mutations resulted in increased levels of GlcCer (14
There might be a possibility that reduction of SPT activity in HSN1 lymphoblasts is due simply to a gene-dosage effect of wild-type SPTLC1
rather than to the dominant negative effects of the mutated alleles, although overproduction of mutated LCB1
products clearly gave negative effects on SPT activity in CHO cells. When one copy of yeast LCB1
having HSN1-like mutations is introduced to haploidal LCB1+
yeast cells, however, SPT activity is reduced by approximately 50%, whereas introduction of one copy of the wild-type LCB1
gene does not affect the activity (20
). These results can be produced by dominant negative effects of the HSN1 mutations, but not by a gene-dosage effect of wild-type LCB1
. Moreover, three SPTLC1
mutations have been detected in a total of 13 HSN1 families (13
), all of which are missense mutations. No deletions or nonsense mutations have been detected. Thus, HSN1-mutated SPTLC1
alleles probably provide dominant negative effects on SPT activity in patient-derived cells, although a reduced dosage of wild-type SPTLC1
might also contribute to the reduction of SPT activity. Further evaluation of the dominant negative versus haploinsufficiency mechanisms in dominantly inherited HSN1 should await analysis with the as yet unavailable LCB1 heterozygous knockout mouse model.
SPT belongs to a group of PLP-dependent α-oxoamine synthases (POASs) that catalyze the condensations of amino acids with carboxylic acid-CoA thioesters to generate α-oxoamines. In addition to the similarities of the chemical reactions catalyzed, members of the POAS enzyme family have strong amino acid sequence similarities. The three-dimensional structure of 8-amino-7-oxononanoate synthase (AONS) (33
), a member of the POAS family, has yielded important insights into other POAS members. For example, the PLP cofactor binds to the enzyme in a cleft between the two AONS subunits, and residues from both subunits participate in the cofactor binding (33
). Although the crystal structure of SPT is not yet available, the AONS structure would suggest that the active site of SPT is composed of adjacent residues from both LCB1 and LCB2 subunits. By analogy, in addition to the amino acid residues in the LCB2 subunit that contribute to the structure of the SPT catalytic site, including the PLP-binding lysine residue, several specific residues of the LCB1 subunit probably contribute, as well. In this scenario, the mutant LCB1C133Y and LCB1C133W subunits are perhaps unable either to contribute to the formation of the active catalytic site or to alter the active site structure to interfere with enzyme activity. Consistent with this hypothesis, the sequence motif around Cys133 in LCB1 is highly conserved from yeast to human, suggesting that this region plays a crucial role in SPT structure and/or catalytic activity. In addition, when a tertiary structure of the yeast LCB1/LCB2 heterodimer complex is modeled according to the AONS structure, Cys180 of yeast Lcb1p (which corresponds to Cys133 in mammalian LCB1 protein) resides at the interface between the two subunits in close proximity to the PLP-binding lysine residue of the Lcb2p (20
Based upon the critical role of sphingolipids in the skin (35
), it is interesting to note that skin wounds accompany the approximate onset of sensory neuropathy. Whether damage to sensory neurons represents an initiating or contributing factor in HSN1 disease progression is unknown. However, preliminary results from a single patient did not reveal significant changes in epidermal Cer content or composition, suggesting that sufficient sphingolipid production occurs in this disease despite the HSN1 LCB1 mutation(s). Cutaneous lipid production, barrier function, and disease progression are being studied.
It remains unclear why mutations in a protein widely expressed in all tissues trigger pathology that is highly restricted to specific subsets of cells within a tissue. One explanation for this incongruity could be an as yet undefined alternative splice form of LCB1 and/or LCB2 in the dorsal root ganglia (DRG) on which the HSN1 mutations exert specific action. Alternatively, degeneration of DRG in HSN1 may be associated with one or more unique features of sensory neurons that reveal a critical sensitivity to the status of sphingolipids. For example, a slight reduction in sphingolipid production/content over an extended duration, due to a partial deficiency in SPT activity, could result in a selective damage of a subset of sensory neurons, thereby inducing the slowly progressive and late-onset sensory neuropathy.
CHO mutant cells completely defective in SPT activity cannot grow in a sphingolipid-deficient medium (25
). This loss of viability is reconstituted upon addition of exogenous sphingolipids (25
). In addition, a complete loss of SPT activity also results in embryonic lethality in the fruit fly (38
). Interestingly, when SPT deficiency is partial, mutant flies grow into adults with abnormalities in various external organs, but such abnormalities are rescued upon feeding with sphingosine (38
). Thus, our finding that the HSN1 mutations negatively affect the activity of mammalian SPT might indicate the possibility that external supply of sphingolipids would prevent or delay the appearance of clinical symptoms in patients affected by the HSN1 mutations.
Neurodegeneration in autosomal dominant diseases has been often associated with the accumulation of insoluble protein aggregates. For example, amyloid-β and α-synuclein have been shown to aggregate in Alzheimer disease (39
) and Parkinson disease (41
), respectively. By analogy, it is conceivable that in addition to its dominant negative effect on SPT activity, mutant LCB1 might accumulate, introducing a new toxic function into neurons. Clearly, neuronal cell models and animal models of HSN1 will help answer these and other questions related to the pathogenic mechanism(s) of HSN1 and the development of possible prophylactic methods.