Biological Chemistry Section and Developmental Glycobiology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
Developmental Glycobiology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
J Biol Chem. 2024 Sep;300(9):107628. doi: 10.1016/j.jbc.2024.107628. Epub 2024 Aug 2.
The UDP-N-acetylgalactosamine polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes initiates O-linked glycosylation by catalyzing the addition of the first GalNAc sugar to serine or threonine on proteins destined to be membrane-bound or secreted. Defects in individual isoforms of the GalNAc-T family can lead to certain congenital disorders of glycosylation (CDG). The polypeptide N-acetylgalactosaminyltransferase 3 (GALNT)3-CDG, is caused by mutations in GALNT3, resulting in hyperphosphatemic familial tumoral calcinosis due to impaired glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) within osteocytes of the bone. Patients with hyperphosphatemia present altered bone density, abnormal tooth structure, and calcified masses throughout the body. It is therefore important to identify all potential substrates of GalNAc-T3 throughout the body to understand the complex disease phenotypes. Here, we compared the Galnt3 mouse model, which partially phenocopies GALNT3-CDG, with WT mice and used a multicomponent approach using chemoenzymatic conditions, a product-dependent method constructed using EThcD triggered scans in a mass spectrometry workflow, quantitative O-glycoproteomics, and global proteomics to identify 663 Galnt3-specific O-glycosites from 269 glycoproteins across multiple tissues. Consistent with the mouse and human phenotypes, functional networks of glycoproteins that contain GalNAc-T3-specific O-glycosites involved in skeletal morphology, mineral level maintenance, and hemostasis were identified. This library of in vivo GalNAc-T3-specific substrate proteins and O-glycosites will serve as a valuable resource to understand the functional implications of O-glycosylation and to unravel the underlying causes of complex human GALNT3-CDG phenotypes.
UDP-N-乙酰氨基半乳糖胺：N-乙酰氨基半乳糖转移酶（GalNAc-T）家族的酶通过催化将第一个 GalNAc 糖添加到注定要成为膜结合或分泌的蛋白质上的丝氨酸或苏氨酸，启动 O-连接糖基化。GalNAc-T 家族中个别同工酶的缺陷可导致某些先天性糖基化紊乱（CDG）。多肽 N-乙酰氨基半乳糖基转移酶 3（GALNT）3-CDG 是由于 GALNT3 突变引起的，导致成骨细胞中磷酸盐调节激素成纤维细胞生长因子 23（FGF23）的糖基化受损，从而导致高磷血症家族性肿瘤性钙化症。高磷血症患者的骨密度改变、牙齿结构异常和全身钙化肿块。因此，重要的是要确定体内所有潜在的 GalNAc-T3 底物，以了解复杂的疾病表型。在这里，我们比较了部分模拟 GALNT3-CDG 的 Galnt3 小鼠模型与 WT 小鼠，并使用化学酶法条件、使用 EThcD 触发扫描构建的依赖产物的方法、定量 O-糖蛋白组学和全局蛋白质组学，从 269 种糖蛋白中鉴定出 663 种 Galnt3 特异性 O-糖基化位点，分布在多个组织中。与小鼠和人类表型一致，包含 GalNAc-T3 特异性 O-糖基化位点的糖蛋白功能网络涉及骨骼形态、矿物质水平维持和止血。这个体内 GalNAc-T3 特异性底物蛋白和 O-糖基化位点的文库将作为一个有价值的资源，以了解 O-糖基化的功能意义，并揭示复杂的人类 GALNT3-CDG 表型的潜在原因。
