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 posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (alpha, beta, and gamma), where alpha and beta can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both alpha and beta in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The alpha repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the beta repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates alpha and beta into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.