A single catalytic domain residue regulates the substrate specificity of a mucin-type O-glycosyltransferase in salivary gland function

Human GalNAc-T1 is a ubiquitously expressed mucin-type O-glycosyltransferase that modulates various biological pathways, including immunity, extracellular matrix formation, and salivary gland development and function. However, substrates of GalNAc-T1 are mostly unknown and the mechanistic details of regulation are not well characterized. Here, we investigate the role of the <i>Drosophila melanogaster</i> GalNAc-T1 ortholog PGANT5 in salivary glands and provide structural and biochemical details into its mechanism of O-glycosylation. We find that loss of <i>pgant5</i> causes irregular secretory granule morphology, altered mucin packaging, and disruption of secretion. Alternative splicing of <i>pgant5</i> yields two isoforms, <i>pgant5A</i> and <i>pgant5B</i>, with higher expression of <i>pgant5A</i> in salivary glands. Rescue with <i>pgant5A</i>, but not <i>pgant5B,</i> partially restores secretory granule morphology and salivary gland function. A single residue difference within the substrate binding pockets of PGANT5A and PGANT5B increases the <i>in vitro</i> activity and specificity of PGANT5A towards salivary gland mucins relative to PGANT5B, corroborating the <i>in vivo</i> expression and role of <i>pgant5A</i> in salivary glands. Our data additionally provide new insights into a catalytic domain motif that regulates GalNAc-T activity. Overall, our studies illustrate how alternative splicing of an O-glycosyltransferase that results in modest amino acid changes can influence substrate specificity to regulate tissue-specific biological functions.