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

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-Glc1 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 observed. We searched in KEGG GENES database to identify 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-GIcNAcylation sites. Around 68% of EGF-LDs were subjected to only one O-linked sugar modification and nearly 5% to three modifications. Among these latter proteins, 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 the 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.

Automated summary

EGF-LDs can be modified by various O-linked sugars, with most mouse proteins experiencing O-fucosylation and/or O-GIcNAcylation. The majority of EGF-LDs undergo only one O-linked sugar modification, while a small percentage undergoes three modifications. The study demonstrated triple O-glycosylation in mouse PAMR1 EGF-LD, indicating complex glycosylation patterns in this protein domain.