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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sergey Vakhrushev  

LAB HEAD: Sergey Vakhrushev

PROVIDER: PXD029885 | Pride | 2022-05-20

REPOSITORIES: Pride