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 their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the 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 the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom–up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.
Highlights
Present address for Zilu Ye: Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, Copenhagen, Denmark
The classification of mucins may in this respect not be consistent, for example, the P-selectin-glycoprotein ligand 1 (PSGL-1) contains an O-glycodomain with characteristic tandem repeat (TR) [28], whereas the cell membrane glycoprotein classified as MUC16 (CA125) has a very large N-terminal O-glycodomain without apparent TRs [29–31]
Our studies of recombinantly expressed reporters for mucin TRs and the isolated ovine submaxillary mucin (OSM) mucin suggest that essentially all Ser/Thr residues available in mucin TRs are O-glycosylated and with near complete occupancy, with the notable exception of lower occupancy in a few transmembrane mucin TRs with distinct TR sequences
Summary
Synthase itself, respectively, resulting in mucin TR reporters with only the simplest O-glycan structure, GalNAcα1-O-Ser/ Thr, designated Tn. We previously reported intact MS analysis of representative TR domains derived from several secreted (MUC2, MUC5AC, and MUC7) and transmembrane (MUC13 and MUC22) human mucins by the use of mucin TR reporters recombinantly expressed in glycoengineered HEK293 cells [34]. We used intact MS to analyze O-glycans occupancy of mucin TR reporters expressed in HEK293KO C1GALT1 cells with additional double KO of GALNT7 and T10.
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