Identification of a buried β-strand as a novel disease-related motif in the human polysialyltransferases

The polysialyltransferases ST8SIA2 and ST8SIA4 and their product, polysialic acid (polySia), are known to be related to cancers and mental disorders. ST8SIA2 and ST8SIA4 have conserved amino acid (AA) sequence motifs essential for the synthesis of the polySia structures on the neural cell adhesion molecule. To search for a new motif in the polysialyltransferases, we adopted the in silico Individual Meta Random Forest program that can predict disease-related AA substitutions. The Individual Meta Random Forest program predicted a new eight-amino-acids sequence motif consisting of highly pathogenic AA residues, thus designated as the pathogenic (P) motif. A series of alanine point mutation experiments in the pathogenic motif (P motif) showed that most P motif mutants lost the polysialylation activity without changing the proper enzyme expression levels or localization in the Golgi. In addition, we evaluated the enzyme stability of the P motif mutants using newly established calculations of mutation energy, demonstrating that the subtle change of the conformational energy regulates the activity. In the AlphaFold2 model, we found that the P motif was a buried beta-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried beta-strand that regulates the full activity of polysialyltransferases from the inside of the molecule.