Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line

Mucin-type-O-glycosylation on proteins is integrally involved in human health and disease and is coordinated by an enzyme family of 20 N-acetylgalactosaminyltransferases (GalNAc-Ts). Detailed knowledge on the biological effects of site-specific O-glycosylation is limited due to lack of information on specific glycosylation enzyme activities and O-glycosylation site-occupancies. Here we present a systematic analysis of the isoform-specific targets of all GalNAc-Ts expressed within a tissue-forming human skin cell line, and demonstrate biologically significant effects of O-glycan initiation on epithelial formation. We find over 300 unique glycosylation sites across a diverse set of proteins specifically regulated by one of the GalNAc-T isoforms, consistent with their impact on the tissue phenotypes. Notably, we discover a high variability in the O-glycosylation site-occupancy of 70 glycosylated regions of secreted proteins. These findings revisit the relevance of individual O-glycosylation sites in the proteome, and provide an approach to establish which sites drive biological functions.

蛋白质的粘蛋白型O-糖基化（mucin-type-O-glycosylation）与人类健康和疾病密切相关，该过程由包含20种N-乙酰半乳糖胺转移酶（N-acetylgalactosaminyltransferases，GalNAc-Ts）的酶家族调控。由于缺乏特定糖基化酶活性及O-糖基化位点占据率的相关信息，学界对位点特异性O-糖基化的生物学效应的认知仍较为有限。本研究对表达于可构建组织的人类皮肤细胞系中的所有GalNAc-T同工型的特异性靶标开展了系统性分析，并证实了O-糖链起始过程对上皮组织形成具有显著生物学效应。我们在多种蛋白质上发现了超过300个独特的糖基化位点，这些位点均受某一种GalNAc-T同工型特异性调控，这与该酶对组织表型的影响相一致。值得注意的是，我们在分泌蛋白的70个糖基化区域中观察到O-糖基化位点占据率存在高度异质性。本研究结果重新审视了蛋白质组中单个O-糖基化位点的生物学相关性，并为确定哪些位点可驱动生物学功能提供了可行的研究路径。