TY - JOUR

T1 - Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells

AU - Konstantinidi, Andriana

AU - Nason, Rebecca

AU - Čaval, Tomislav

AU - Sun, Lingbo

AU - Sørensen, Daniel M

AU - Furukawa, Sanae

AU - Ye, Zilu

AU - Vincentelli, Renaud

AU - Narimatsu, Yoshiki

AU - Vakhrushev, Sergey Y

AU - Clausen, Henrik

N1 - Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Animals

KW - Glycosylation

KW - HEK293 Cells

KW - Humans

KW - Mucins/metabolism

KW - Polysaccharides/genetics

KW - Protein Domains

KW - Recombinant Proteins/genetics

KW - Sheep

U2 - 10.1016/j.jbc.2022.101784

DO - 10.1016/j.jbc.2022.101784

M3 - Journal article

C2 - 35247390

VL - 298

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 4

M1 - 101784

ER -