Scramblases for protein glycosylation

蛋白质糖基化在所有真核生物中都是必不可少的，从引起疾病的原生生物如疟疾，到酵母和 哺乳动物分泌蛋白是N-糖基化、O-和C-甘露糖基化和/或糖基化磷脂酰肌醇 当它们进入内质网（ER）腔时，它们被（GPI）锚定。酵母不能合成N- 糖蛋白或GPI蛋白是不能存活的，具有相同缺陷的小鼠在胚胎时死亡。糖基化是 在登革热和SARS-CoV-2病毒感染中很重要，糖基化缺陷导致人类疾病。因此，在本发明中， 肌营养不良蛋白聚糖的O-甘露糖基化缺陷是肌营养不良和GPI缺陷的原因， 造血干细胞是溶血性疾病阵发性睡眠性血红蛋白尿症的基础。 先天性糖基化障碍（CDG）是一种严重的遗传性疾病，具有神经系统症状。 蛋白质糖基化反应需要糖脂甘露糖基-和葡糖基-磷酰基多萜醇（MPD， GPD）在ER的管腔中充当糖供体。由于这些脂质在细胞质侧合成， 它们必须翻转穿过内质网膜才能在管腔中发挥作用，这一过程需要特异性的 被称为加扰酶的转运蛋白，尚未被识别。微粒体中两种乱序酶的测定 和重组囊泡，使用天然脂质和短链类似物作为报告，揭示了运输是 双向、ATP非依赖性和高度结构特异性，可区分结构异构体。 我们将使用化学蛋白质组学和生物信息学方法鉴定MPD和GPD扰码酶。 部署由Häner小组（伯尔尼大学）合成的新型光点击探针，我们将确定 MPD和GPD相互作用基因组，我们假设它将包括扰码。我们的初步结果 验证了该方法：MPD探针在ER甘露糖基化中起作用，并且光识别特定的酵母 微粒体蛋白将通过定量蛋白质组学鉴定光加合蛋白质，并进行检测。 我们的重组检测中发现了扰乱酶的活性有希望的候选物将在体内进行验证， 评估酵母突变体的表型。对于GPD加扰酶，我们还将通过系统发育鉴定候选物。 Profiling是一种用于蛋白质功能分配的生物信息学方法。这种方法补充了照片- 该公司已经制定了一个GPD加扰酶候选名单，以供测试。 这是发现ER蛋白糖基化的关键参与者的相应建议。我们广泛 研究扰码的经验，使我们有能力达到这个目标。我们发现了 图1显示了A类GPCR的脂质扰乱酶活性，并且是第一个通过TMEM 16离子通道显示脂质扰乱的。 我们现在部署在硅片，生物化学和生物物理方法来阐明其机制。我们将使用这个 专业知识在未来的工作，以揭示结构特异性脂质扰乱介导的分子机制， 我们预测MPD和GPD扰乱酶与目前已知的磷脂扰乱酶不同， 加扰在生物学水平上，我们的发现将揭示与CDG相关的新遗传基因座。

Protein glycosylation is essential in all eukaryotes, from disease-causing protists such as malaria, to yeast and mammals. Secretory proteins are N-glycosylated, O- and C-mannosylated, and/or glycosylphosphatidylinositol (GPI)-anchored as they enter the lumen of the endoplasmic reticulum (ER). Yeast that cannot synthesize N- glycoproteins or GPI-proteins are inviable, and mice with the same defects die as embryos. Glycosylation is important in dengue and SARS-CoV-2 viral infections, and defects in glycosylation cause human disease. Thus, deficient O-mannosylation of dystroglycan is a cause of muscular dystrophy and GPI deficiency in hematopoietic human stem cells underlies the hemolytic disease paroxysmal nocturnal hemoglobinuria. Congenital Disorders of Glycosylation (CDGs) are severe inherited diseases with neurological symptoms. Protein glycosylation reactions require the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD, GPD) to act as sugar donors in the lumen of the ER. As these lipids are synthesized on the cytoplasmic side, they must be flipped across the ER membrane to function in the lumen, a process requiring specific transporters, termed scramblases, that have yet to be identified. Assays of the two scramblases in microsomes and reconstituted vesicles, using natural lipids and short-chain analogs as reporters, reveal that transport is bidirectional, ATP-independent, and highly structure specific, discriminating between structural isomers. We will identify the MPD and GPD scramblases using chemo-proteomic and bioinformatic approaches. Deploying novel photo-clickable probes synthesized by the Häner group (University of Bern) we will determine the MPD and GPD interactomes, that we hypothesize will include the scramblases. Our preliminary results validate this approach: the MPD probe functions in ER mannosylation and photo-identifies specific yeast microsomal proteins. Photo-adducted proteins will be identified by quantitative proteomics and tested for scramblase activity in our reconstitution-based assays. Promising candidates will be validated in vivo by evaluating phenotypes of yeast mutants. For GPD scramblase we will also identify candidates via phylogenetic profiling, a bioinformatics method for assignment of protein function. This approach complements the photo- identification strategy and has already yielded a list of GPD scramblase candidates for testing. This is a consequential proposal to discover critical players in ER protein glycosylation. Our extensive experience in studying scramblases puts us in a strong position to tackle this objective. We discovered the scramblase activity of Class A GPCRs and were the first to show lipid scrambling by a TMEM16 ion channel. We now deploy in silico, biochemical and biophysical methods to elucidate their mechanism. We will use this expertise in future work to reveal the molecular mechanism of structure-specific lipid scrambling mediated by the MPD and GPD scramblases that we predict to be distinct from that of the currently known phospholipid scramblases. At a biological level, our discoveries will reveal new genetic loci associated with CDGs.