Differential Splicing of the Lectin Domain of an O‐glycosyltransferase Influences Charged Peptide Preferences

Abstract

Mucin‐type O‐glycosylation is an essential post‐translational modification that is required for secretion, extracellular matrix composition and organ growth. O‐glycosylation is initiated by a large family of enzymes (GalNAcTs in mammals and PGANTs in Drosophila) that catalyze the addition of the sugar N‐acetylgalactosamine (GalNAc), onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes have distinct domains, including the catalytic domain and the C‐terminal ricin‐like lectin domain. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing the transfer of GalNAc, while the lectin domain is thought to recognize extant GalNAc on previously glycosylated substrates to position the catalytic domain for further glycosylation of unmodified serines and threonines. Previous work from our group has demonstrated that one member of this family in Drosophila undergoes differential splicing within the lectin domain, leading to variants that contain either a positively or negatively charged subdomain. Interestingly, while this splicing event affects only a subdomain of the lectin region, we find that it influences peptide specificity. We demonstrate that each charged enzyme variant has a preference for oppositely charged peptide substrates, and that this preference can be predictably altered by altering peptide charge. We further show that the expression of each splice variant is associated with the expression of appropriately charged mucins in secretory tissues of Drosophila, suggesting that differential splicing may be employed in vivo to ensure O‐glycosylation of highly charged substrates. Our results elucidate a mechanism for modulating substrate preferences of O‐glycosyltransferases based on the needs of the cell through alternative splicing within a specific subregion of the non‐catalytic domain.