title: Inhibition of mucin-type O-glycosylation through metabolic processing and incorporation of N-thioglycolyl-D-galactosamine peracetate (Ac5GalNTGc).
abstract: Mucin-type O-glycans form one of the most abundant and complex post-translational modifications (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. Among 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 an 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 the 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.
authors.affiliation: Laboratory of Chemical Glycobiology (CGB), National Institute of Immunology (NII) , Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Kavita
authors.initials: K
authors.lastname: Agarwal
authors.forename: Rachna
authors.initials: R
authors.lastname: Kaul
authors.forename: Monika
authors.initials: M
authors.lastname: Garg
authors.forename: Asif
authors.initials: A
authors.lastname: Shajahan
authors.forename: Saroj Kumar
authors.initials: SK
authors.lastname: Jha
authors.forename: Srinivasa-Gopalan
authors.initials: SG
authors.lastname: Sampathkumar
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D005688:Galactosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D019169:Jurkat Cells; D051917:Leukosialin / Q000378:metabolism; D009077:Mucins / Q000378:metabolism*
chemical_list: D051917:Leukosialin; D009077:Mucins; C000588872:N-thioglycolyl-D-galactosamine peracetate; D005688:Galactosamine; D000116:Acetylgalactosamine