磷酸甘露聚糖变位酶2(PMM2)将甘露糖-6-磷酸转化为甘露糖-1-磷酸;GDP-甘露糖的底物,糖基化生物合成途径的组成部分。已显示PMM2基因中的致病变体与导致PMM2-先天性糖基化障碍(PMM2-CDG)的蛋白质低糖基化相关。虽然甘露糖补充在体外改善糖基化,但不是在体内,我们假设脂质体递送甘露糖-1-磷酸可以增加活化糖进入细胞靶向区室的稳定性和递送。因此,我们研究了脂质体包裹的甘露糖-1-P(GLM101)对来自PMM2-CDG个体的皮肤成纤维细胞的整体蛋白质糖基化和细胞蛋白质组的影响,以及在通路早期具有两个N-糖基化缺陷的个体中,即ALG2-CDG和ALG11-CDG。我们在来自不同个体的成纤维细胞中利用多重蛋白质组学和N-糖蛋白质组学,在PMM2,ALG2和ALG11基因中具有各种致病变体。蛋白质组学数据显示,在GLM101处理后,所有CDG成纤维细胞中一些蛋白质的丰度发生了中等但显着的变化。另一方面,N-糖蛋白质组学显示,GLM101治疗提高了PMM2和ALG2基因缺陷个体中多种细胞蛋白的几种高甘露糖和复合/杂合糖肽的表达水平。PMM2-CDG和ALG2-CDG都表现出携带Man6和更高级聚糖的糖肽增加了几倍,Man5和更小的聚糖部分减少。这表明GLM101有助于成熟糖型的形成。在所有个体中观察到蛋白质糖基化的这些变化,而与他们的遗传变异无关。ALG11-CDG成纤维细胞在治疗后也显示高甘露糖肽的增加;然而,改善不如其他两个CDG显着。总的来说,我们的研究结果表明,GLM101治疗克服了糖基化途径中的遗传阻滞,可作为在蛋白N-糖基化早期阶段存在酶缺陷的CDG的潜在治疗方法.
Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man6 and higher glycans and a decrease in Man5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation.