TY - ABST

T1 - Isoform-specific mucin type O-glycosylation maintain epithelial homeostasis

AU - I., Bagdonaite

AU - E.M.H., Pallesen

AU - K., Lavrsen

AU - S.Y., Vakhrushev

AU - L., Hansen

AU - H.J., Joshi

AU - E.P., Bennett

AU - S., Dabelsteen

AU - H.H., Wandall

N1 - M1 - (Bagdonaite I., ieva@sund.ku.dk; Pallesen E.M.H.; Lavrsen K.; Vakhrushev S.Y.; Hansen L.; Joshi H.J.; Wandall H.H.) University of Copenhagen, Department of Cellular and Molecular Medicine, Copenhagen N, Denmark M1 - (Bennett E.P.; Dabelsteen S.) University of Copenhagen, Department of Odontology, Copenhagen N, Denmark

PY - 2017

Y1 - 2017

N2 - Mucin type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Deficiencies of individual GalNAc-Ts cause subtle distinct phenotypes in insect and mu-rine models, and altered expression patterns of GalNAc-Ts have been identified as prognostic cancer markers. Yet, we have little understanding of the cell and tissue specific functions of the individual isoforms. We have recently used precise genetic engineering to target the human C1GalT1 chaperone COSMC to generate stable cells and tissue models with homogenous truncated GalNAc O-glycans, demonstrating a malignant phenotype. We now extend these studies to characterize the impact of site-specific glycosylation on epithe-lial differentiation and malignant transformation. Using an organotypic model system equipped with cells with and without GalNAc-T1,-T2, or-T3, we identify distinct pheno-types, and selective effects on specific cellular pathways identified via differential transcriptomic, phosphoproteomic, and proteomic analyses. In addition, we find non-redundant O-glycosylation performed by single isoforms using quantitative differential O-glycoproteomics and identify isolated sites on proteins involved in cell adhesion, differentiation, and stress response. Importantly, knock-out of GalNAc-T1,-T2, or-T3 does not lead to malignant transformation, in contrast to homogeneous truncation of O-glycans. While GalNAc-T1-T3 are abundantly expressed in most healthy tissues, we found that GalNAc-T6 was highly upregulated in colon adeno-carcinomas but absent in normal-appearing adjacent tissue samples, suggesting that it plays a role in colon carcinogenesis. GalNAc-T6 expression was associated with a cancer-like growth pattern, whereas GalNAc-T6 knockout cells showed more normal differentiation patterns, normalized cell-cell adhesion, and formed crypts in tissue cultures. In addition, several GalNAc-T6 specific targets were identified by O-glycoproteomic analysis. Taken together, these data strongly suggest that individual GalNAc-T isoforms glycosylate a subset of specific targets that play important roles in tissue differ-entiation, homeostasis and oncogenesis.

AB - Mucin type O-glycosylation is initiated by a large family of UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferases (GalNAc-Ts) that target different proteins and are differentially expressed in cells and organs. Deficiencies of individual GalNAc-Ts cause subtle distinct phenotypes in insect and mu-rine models, and altered expression patterns of GalNAc-Ts have been identified as prognostic cancer markers. Yet, we have little understanding of the cell and tissue specific functions of the individual isoforms. We have recently used precise genetic engineering to target the human C1GalT1 chaperone COSMC to generate stable cells and tissue models with homogenous truncated GalNAc O-glycans, demonstrating a malignant phenotype. We now extend these studies to characterize the impact of site-specific glycosylation on epithe-lial differentiation and malignant transformation. Using an organotypic model system equipped with cells with and without GalNAc-T1,-T2, or-T3, we identify distinct pheno-types, and selective effects on specific cellular pathways identified via differential transcriptomic, phosphoproteomic, and proteomic analyses. In addition, we find non-redundant O-glycosylation performed by single isoforms using quantitative differential O-glycoproteomics and identify isolated sites on proteins involved in cell adhesion, differentiation, and stress response. Importantly, knock-out of GalNAc-T1,-T2, or-T3 does not lead to malignant transformation, in contrast to homogeneous truncation of O-glycans. While GalNAc-T1-T3 are abundantly expressed in most healthy tissues, we found that GalNAc-T6 was highly upregulated in colon adeno-carcinomas but absent in normal-appearing adjacent tissue samples, suggesting that it plays a role in colon carcinogenesis. GalNAc-T6 expression was associated with a cancer-like growth pattern, whereas GalNAc-T6 knockout cells showed more normal differentiation patterns, normalized cell-cell adhesion, and formed crypts in tissue cultures. In addition, several GalNAc-T6 specific targets were identified by O-glycoproteomic analysis. Taken together, these data strongly suggest that individual GalNAc-T isoforms glycosylate a subset of specific targets that play important roles in tissue differ-entiation, homeostasis and oncogenesis.

KW - chaperone

KW - endogenous compound

KW - mucin

KW - n acetylgalactosaminyltransferase

KW - unclassified drug

KW - uridine diphosphate n acetylgalactosamine

KW - adenocarcinoma

KW - cancer model

KW - case report

KW - cell adhesion

KW - colon carcinogenesis

KW - differentiation

KW - ex vivo study

KW - genetic engineering

KW - glycosylation

KW - homeostasis

KW - human

KW - human cell

KW - human tissue

KW - intestine lymphatic tissue

KW - knockout gene

KW - malignant transformation

KW - phenotype

KW - phosphoproteomics

KW - stress

KW - tissue culture

U2 - 10.1007/s10719-017-9784-5

DO - 10.1007/s10719-017-9784-5

M3 - Conference abstract in journal

VL - 34

SP - S42

JO - Glycoconjugate Journal

JF - Glycoconjugate Journal

SN - 0282-0080

IS - Suppl. 1

M1 - Abstract 78

ER -