The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

中文翻译：

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鞘脂去饱和酶 DEGS1 变体的功能丧失和水平降低均与疾病机制相关

从头神经酰胺生物合成的最后一步包括在鞘脂 Δ4-去饱和酶 DEGS1 的催化下将二氢神经酰胺转化为神经酰胺。研究发现 DEGS1 变异导致了低髓鞘性脑白质营养不良家族的异质性临床表现。为了研究此类变异致病的机制，我们设计了一种使用 LC-MS/MS 体外有效检测去饱和酶活性的程序，并通过 CRISPR-Cas9 基因组编辑 (KO- DES-HEK）。用DEGS1变体转染KO-DES-HEK细胞，我们发现它们的转录本均与WT转录本一样过度表达，而同源蛋白的水平却低40%–80%。除了 L175Q 和 N255S 之外，许多变体都丧失了体外去饱和酶活性，它们的催化效率保持接近 WT 酶的 12%。用氘代棕榈酸酯对 KO-DES-HEK 进行代谢标记，然后对形成的鞘脂进行 LC-MS/MS 分析，结果显示神经酰胺/二氢神经酰胺和鞘磷脂/二氢鞘磷脂比率较低，并且可以通过 WT DEGS1 和 WT DEGS1 的过表达来恢复。 L175Q 和 N255S 变体，但不是所有其他变体的过度表达。对 N255S 变体杂合患者的成纤维细胞进行的类似分析显示，尽管在高底物和匀浆蛋白浓度下测量到了残余活性，但代谢标记后形成的相同不饱和和饱和鞘脂之间的变异 DEGS1 水平非常低，并且相同的不饱和和饱和鞘脂之间的比率较低。我们得出的结论是，功能丧失和蛋白质水平降低都与疾病发病机制相关。