Exploring glycosylation of mucins with O-glycodomain reporters expressed in glycoengineered HEK293 cells

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of resistance to proteolytic digestion, and knowledge of positions of O-glycans is particularly limited for these regions. Mucin O-glycodomains are believed to contain important binding cues for endogenous lectin receptors and the microbiota, and it is important to develop strategies to characterize and produce these abundant molecules. Here, we took advantage of our recently developed glycoengineered cell-based platform for display and production of mucin TR reporters with custom designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in MUC2, MUC20, MUC21, PSGL-1, and Syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in HEK293 SimpleCells (Tn-glycoform). Interestingly, while all the sites in TRs derived from secreted mucins were almost fully occupied, positions in TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of the StcE and BT4244 glycoproteases, revealing a more restricted substrate specificities than reported. Finally, we used these for bottom-up analysis of isolated ovine submaxillary mucin (OSM) and identified the gene. The study provides insight into O-glycosylation of mucins and mucin-like domains and the strategies developed open up for wider analysis of native mucins.

黏蛋白及带有黏蛋白样区域的糖蛋白，其序列中常包含密集O-糖基化结构域，这类结构域多存在于串联重复（tandem repeat, TR）序列内。传统上，这类O-糖基化结构域（O-glycodomains）因耐受蛋白水解酶解而难以进行表征，且针对这些区域的O-聚糖（O-glycans）位点信息尤为匮乏。人们认为黏蛋白O-糖基化结构域包含内源性凝集素受体与微生物群识别的关键结合基序，因此开发表征及制备这类丰度较高分子的策略具有重要意义。本研究借助我们近期开发的糖工程化细胞展示与生产平台，该平台可用于制备带有定制化O-糖基化修饰的黏蛋白TR报告分子，以此对源自黏蛋白及黏蛋白样糖蛋白的O-糖基化结构域进行表征。我们结合完整质谱质量分析与自下而上位点特异性分析（bottom-up site-specific analysis）技术，对MUC2、MUC20、MUC21、PSGL-1以及Syndecan-3中的O-糖基化位点（O-glycosites）进行定位。我们发现，当这些O-糖基化结构域在HEK293 SimpleCells（Tn糖型（Tn-glycoform））中表达时，其所有潜在的丝氨酸/苏氨酸位点均发生了O-糖基化修饰。值得注意的是，尽管源自分泌型黏蛋白的TR序列中所有位点几乎均被完全修饰，但部分跨膜黏蛋白的TR序列位点的糖基化修饰效率相对较低。我们进一步利用黏蛋白TR报告分子对StcE与BT4244糖蛋白酶（glycoproteases）的切割位点进行表征，结果显示其底物特异性较此前报道更为受限。最后，我们将该体系用于分离得到的羊颌下腺黏蛋白（ovine submaxillary mucin, OSM）的自下而上蛋白质组分析，并成功鉴定出其编码基因。本研究为黏蛋白及黏蛋白样结构域的O-糖基化修饰研究提供了新见解，所开发的策略也为天然黏蛋白（native mucins）的更广泛分析提供了可行路径。