The single EGF-like domain of mouse PAMR1 is modified by O-Glucose, O-Fucose and O-GlcNAc

• Published in: Glycobiology (Oxford) Vol. 31; no. 1; pp. 55 - 68
• Main Authors: Pennarubia, Florian, Germot, Agnès, Pinault, Emilie, Maftah, Abderrahman, Legardinier, Sébastien
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 09.01.2021
• Subjects: Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Animals; Databases, Protein; Epidermal Growth Factor - chemistry; Epidermal Growth Factor - isolation & purification; Epidermal Growth Factor - metabolism; Fucose - chemistry; Fucose - metabolism; Glucose - chemistry; Glucose - metabolism; Humans; Mice; N-Acetylglucosaminyltransferases - chemistry; N-Acetylglucosaminyltransferases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serine Proteases - chemistry; Serine Proteases - metabolism; POFUT1; PAMR1; POGLUT; EGF-LD; EOGT
• ISSN: 1460-2423; 1460-2423
• DOI: 10.1093/glycob/cwaa051

Abstract Epidermal growth factor-like domains (EGF-LDs) of membrane and secreted proteins can be modified by N-glycans and/or potentially elongated O-linked monosaccharides such as O-glucose (O-Glc) found at two positions (O-Glc 1 and O-Glc2), O-fucose (O-Fuc) and O-N-acetylglucosamine (O-GlcNAc). The presence of three O-linked sugars within the same EGF-LD, such as in EGF-LD 20 of NOTCH1, has rarely been evidenced. We searched in KEGG GENES database to list mouse and human proteins with an EGF-LD sequence including one, two, three or four potential O-glycosylation consensus sites. Among the 129 murine retrieved proteins, most had predicted O-fucosylation and/or O-GlcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and near 5% to three modifications. Among these latter, we focused on the peptidase domain-containing protein associated with muscle regeneration 1 (PAMR1), having only one EGF-LD. To test the ability of this domain to be glycosylated, a correctly folded EGF-LD was produced in Escherichia coli periplasm, purified and subjected to in vitro incubations with the recombinant O-glycosyltransferases POGLUT1, POFUT1 and EOGT, adding O-Glc1, O-Fuc and O-GlcNAc, respectively. Using click chemistry and mass spectrometry, isolated PAMR1 EGF-LD was demonstrated to be modified by the three O-linked sugars. Their presence was individually confirmed on EGF-LD of full-length mouse recombinant PAMR1, with at least some molecules modified by both O-Glc1 and O-Fuc. Overall, these results are consistent with the presence of a triple O-glycosylated EGF-LD in mouse PAMR1.