Ernst Daniela, Murphy Sinéad M, Sathiyanadan Karthik, Wei Yu, Othman Alaa, Laurá Matilde, Liu Yo-Tsen, Penno Anke, Blake Julian, Donaghy Michael, Houlden Henry, Reilly Mary M, Hornemann Thorsten
Institute for Clinical Chemistry, University Hospital Zurich, CH-8091, Zurich, Switzerland.
Neuromolecular Med. 2015 Mar;17(1):47-57. doi: 10.1007/s12017-014-8339-1. Epub 2015 Jan 8.
1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position.
1-脱氧鞘脂（1-deoxySL）是一类非典型鞘脂，由丝氨酸棕榈酰转移酶（SPT）通过对L-丙氨酸而非其典型底物L-丝氨酸的随意使用而形成。SPT中的几种突变与遗传性感觉和自主神经病变I型（HSAN1）相关。目前的假说认为，这些突变会导致亲和力从L-丝氨酸向L-丙氨酸发生永久性转变，从而导致HSAN1患者体内1-deoxySL的病理性生成增加。此外，野生型SPT在某些条件下也会形成1-deoxySL，并且在患有代谢综合征和糖尿病的个体中发现其水平升高。然而，控制野生型酶底物转变的分子机制尚不清楚。在此，我们报道了两个无关的HSAN1家族中存在一种新型的SPTLC2-S384F变体。受影响的患者血浆1-deoxySL水平升高，并且HEK293细胞中S384F突变体的表达增加了1-deoxySL的形成。此前，S384已被报道为SPTLC2中两个（S384和Y387）假定的磷酸化位点之一。通过等电聚焦证实了野生型SPTLC2的磷酸化。通过创建模拟组成型磷酸化（S384D、S384E）或非磷酸化（S384A、Y387F、Y387F+S384A）蛋白的突变体，测试了S384磷酸化对SPT活性的影响。S384D变体而非S384E变体与1-deoxySL形成增加相关。其他突变对活性和底物亲和力没有影响。总之，我们的数据表明S384F是HSAN1中的一种新型突变，并且野生型SPT的底物特异性可能受该位置磷酸化的动态调节。
