An unusual dual sugar-binding lectin domain controls the substrate specificity of a mucin-type O-glycosyltransferase

N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex post-translational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a C-terminal lectin domain consisting of three homologous repeats (, , where and can potentially interact with O-GalNAc on substrates to enhance activity towards a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates diverse biological functions, including heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both and in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The repeat directs O-glycosylation to acceptor sites C-terminal to an existing GalNAc, while the repeat directs O-glycosylation to N-terminal sites. Additionally, GalNAc-T1 incorporates and binding into various substrate binding modes to cooperatively increase the specificity towards an intermediate acceptor site. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and inform on identifying the biological substrates affected by disruptions in GalNAc-T1 function.