The impact of N-glycosylation on the functions of polysialyltransferases

Poly-alpha -2,8-sialic acid (polysialic acid) is a post-translational modif ication of the neural cell adhesion molecule (NCAM) and an important regula tor of neuronal cell-cell interactions. The synthesis of polysialic acid de pends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understandin g the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions medi ated by polysialic acid. We recently demonstrated that polysialyltransferas es are bifunctional enzymes exhibiting auto- and NCAM polysialylation activ ity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylat ion variants lacking sialic acid and galactose were found to be inactive fo r both auto- and NCAM polysialylation. In the present study, we have analyz ed the number and functional importance of N-linked oligosaccharides presen t on polysialyltransferases. We demonstrate that autopolysialylation depend s on specific N-glycans attached to Asn(74) in ST8SiaIV and Asn(89) and Asn (219) in ST8SiaII. Deletion of polysialic acid acceptor sites by site-direc ted mutagenesis rendered the polysialyltransferases inactive in vitro and i n vivo. The inactivity of autopolysialylation-negative polysialyltransferas es in vivo was not caused by the absence or default targeting of the enzyme s. The data presented in this study clearly show that active polysialyltran sferases are competent to perform autopolysialylation and provide strong ev idence for a tight functional link between the two catalytic functions.