Inhibition of mucin-type O-glycosylation through metabolic processing and incorporation of N-thioglycolyl-D-galactosamine peracetate (Ac5GalNTGc).
Mucin-type O-glycans form one of the most abundant and complex post-translational modification (PTM) on cell surface proteins that govern adhesion, migration, and trafficking of hematopoietic cells. Development of targeted approaches to probe functions of O-glycans is at an early stage. Amongst several approaches, small molecules with unique chemical functional groups that could modulate glycan biosynthesis form a critical tool. Herein, we show that metabolism of peracetyl N-acyl-D-galactosamine derivatives carrying N-thioglycolyl- (Ac5GalNTGc, 1) moiety – but not N-glycolyl- (Ac5GalNGc, 2) and N-acetyl- (Ac4GalNAc, 3) – through the N-acetyl-D-galactosamine (GalNAc) salvage pathway induced abrogation of MAL-II and PNA epitopes in Jurkat cells. Mass spectrometry of permethylated O-glycans from Jurkat cells confirmed presence of significant amounts of elaborated O-glycans (sialyl-T and disialyl-T) which were inhibited upon treatment with 1.O-glycosylation of CD43, a cell surface antigen rich in O-glycans, was drastically reduced by 1 in a thiol-dependent manner. By contrast, only mild effects were observed for CD45 glycoforms. Direct metabolic incorporation of 1 was confirmed by thiol-selective Michael addition reaction of immunoprecipitated CD43-myc/FLAG. Mechanistically, CD43 glycoforms were unperturbed by peracetylated N-(3-acetylthiopropanoyl)- (4), N-(4-acetylthiobutanoyl)- (5), and N-methylthioacetyl- (6) galactosamine derivatives, N-thioglycolyl-D-glucosamine (7, C-4 epimer of 1), and α-O-benzyl 2-acetamido-2-deoxy-D-galactopyranoside (8), confirming the critical requirement of both free sulfhydryl and galactosamine moieties for inhibition of mucin-type O-glycans. Similar, yet differential, effects of 1 were observed for CD43 glycoforms in multiple hematopoietic cells. Development of small molecules that could alter glycan patterns in an antigen-selective and cell-type selective manner might provide avenues for understanding biological functions of glycans.
Mucin-type O-glycans form one of the most abundant and complex post-translational modification (PTM) on cell surface proteins that govern adhesion, migration, and trafficking of hematopoietic cells. Development of targeted approaches to probe functions of O-glycans is at an early stage. Amongst several approaches, small molecules with unique chemical functional groups that could modulate glycan biosynthesis form a critical tool. Herein, we show that metabolism of peracetyl N-acyl-D-galactosamine derivatives carrying N-thioglycolyl- (Ac5GalNTGc, 1) moiety – but not N-glycolyl- (Ac5GalNGc, 2) and N-acetyl- (Ac4GalNAc, 3) – through the N-acetyl-D-galactosamine (GalNAc) salvage pathway induced abrogation of MAL-II and PNA epitopes in Jurkat cells. Mass spectrometry of permethylated O-glycans from Jurkat cells confirmed presence of significant amounts of elaborated O-glycans (sialyl-T and disialyl-T) which were inhibited upon treatment with 1.O-glycosylation of CD43, a cell surface antigen rich in O-glycans, was drastically reduced by 1 in a thiol-dependent manner. By contrast, only mild effects were observed for CD45 glycoforms. Direct metabolic incorporation of 1 was confirmed by thiol-selective Michael addition reaction of immunoprecipitated CD43-myc/FLAG. Mechanistically, CD43 glycoforms were unperturbed by peracetylated N-(3-acetylthiopropanoyl)- (4), N-(4-acetylthiobutanoyl)- (5), and N-methylthioacetyl- (6) galactosamine derivatives, N-thioglycolyl-D-glucosamine (7, C-4 epimer of 1), and α-O-benzyl 2-acetamido-2-deoxy-D-galactopyranoside (8), confirming the critical requirement of both free sulfhydryl and galactosamine moieties for inhibition of mucin-type O-glycans. Similar, yet differential, effects of 1 were observed for CD43 glycoforms in multiple hematopoietic cells. Development of small molecules that could alter glycan patterns in an antigen-selective and cell-type selective manner might provide avenues for understanding biological functions of glycans.
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