A single spliced catalytic domain residue regulates mucin-type O-glycosyltransferase activity in salivary gland function

Abstract Human GalNAc-T1 is a mucin-type O-glycosyltransferase that modulates immunity, extracellular matrix formation, and salivary gland development and function. However, its substrates and regulatory mechanisms remain unclear. Here, we investigate the Drosophila melanogaster GalNAc-T1 ortholog PGANT5 in salivary glands and provide details into its mechanism of O-glycosylation. Loss of pgant5 causes irregular secretory granule morphology, alters mucin packaging, and disrupts secretion. Alternative splicing of pgant5 yields pgant5A and pgant5B , with higher expression of pgant5A in salivary glands. Rescue with pgant5A , but not pgant5B , partially restores secretory granule morphology and secretion. A single residue difference within the substrate binding pockets of PGANT5A and PGANT5B increases the in vitro activity and specificity of PGANT5A towards salivary gland mucins relative to PGANT5B, corroborating the role of pgant5A in salivary glands. Our studies illustrate how alternative splicing of an O-glycosyltransferase resulting in modest amino acid changes can influence substrate specificity to regulate tissue-specific biological functions.