PA14 domain of glycosyltransferase B4GALNT3 is a lectin that binds to sulfated glycan ligands

Mammalian proteins are decorated with a variety of glycans, providing proteins with enormous functional diversity. GalNAcβ1-4GlcNAc (LacdiNAc or LDN), a unique sub-terminal glycan structure regulating the half-life of circulating glycoproteins, is biosynthesized by the dedicated glycosyltransferases, B4GALNT3 and B4GALNT4. We recently reported that B4GALNT3 contains a unique non-catalytic PA14 domain that is necessary for the enzyme activity, while the precise function of PA14 is unclear. Here we show that PA14 in B4GALNT3 is a lectin domain required for the activity of B4GALNT3 toward glycoprotein substrates. Glycan microarray experiments, together with surface plasmon resonance and molecular dynamics simulations, demonstrated the specific binding between the PA14 domain of B4GALNT3 and sulfated glycan ligands, such as Gal6Sβ1-4GlcNAc6S. Both addition of the sulfated disaccharide ligands and point-mutation at the putative sugar binding site in the PA14 domain inhibited the in vitro activity of B4GALNT3 particularly toward glycoprotein substrates. These data suggest that sulfated glycans negatively regulate the PA14-dependent catalytic activity of B4GALNT3 toward glycoproteins. Suppression of cellular glycan sulfation by knocking out the Golgi transporters for sulfation donor PAPS, SLC35B2, and SLC35B3, likely resulted in the enhanced biosynthesis of LDN by B4GALNT3 in cells. Moreover, overexpression of CHST8, which catalyzes sulfation of LDN, seemed to reduce B4GALNT3 activity in cells, suggesting negative feedback regulation of B4GALNT3 by the sulfated product. These findings indicate that recognition of sulfated glycan ligands by its PA14 domain negatively regulates B4GALNT3 activity, highlighting a novel regulation mechanism for LDN synthesis mediated by a lectin domain.