Evolutionary analyses of the animal glycosyltransferase family 54 reveals two β1,4-N-acetylglucosaminyltransferase families

<i>N</i>-acetylglucosaminyltransferases involved in branched <i>N</i>-glycans synthesis, a major post-translational modification, are gathered in the CAZy glycosyltransferase family 54. To date, the origin and evolution of this biosynthetic pathway are unknown, and the functional organization of the Golgi enzymes remains elusive. Over 230 metazoan GT54-related genes were identified, and sequence-based analysis of vertebrate MGAT4 proteins shed light on evolutionary conserved peptide motifs and structural features like the lectin domain (CBM94). Molecular phylogeny analyses disentangled their evolutionary relationships, revealing the deep ancestry of two metazoan clusters, and unveiled the existence of seven vertebrate MGAT4 subfamilies. Comparative genomics and sequence-based analyses identified an evolutionarily conserved subgroup of GT54 gathering MGAT4A, MGAT4B, and MGAT4D, whereas the other subgroup comprised of MGAT4C, MGAT4E, MGAT4F, and MGAT4G evolved faster. Biochemical analyses conducted with representatives of each subgroup revealed the existence of two acceptor substrate specificities and suggested their intricate functional organization with the other Golgi branching enzymes.