The Impact of N-Glycosylation on the Functions of Polysialyltransferases

Abstract

Poly-␣-2,8-sialic acid (polysialic acid) is a posttranslational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto-and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both autoand NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends 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-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylationnegative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.

Engineering of complex protein sialylation in plants

“…The impact of polySia on polyST activities is not entirely clear. One study claims that autopolysialylation is not essential for the enzymes' activity (23); however, other experiments point to its importance for efficient polysialylation of NCAM (24,40). Our results indicate that (autopoly) sialylation positively affects the stability of the two polySTs in planta.…”

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

“…In agreement with in cellulo as well as in vitro experiments concerning the polysialylated glycosylation sites of ST8SiaII, we detected polySia chains on N-glycans of glycosylation site 5 in vivo (52). In addition to this glycosylation site, N-glycans of the third glycosylation site were discussed to be a target for autopolysialylation.…”

Polysialylation of the Synaptic Cell Adhesion Molecule 1 (SynCAM 1) Depends Exclusively on the Polysialyltransferase ST8SiaII in Vivo

“…Antibodies, Enzymes, and Reagents-The anti-polySia monoclonal antibody (mAb) 735 (IgG 2a ) (31) and endosialidase (endoN) were purified as described previously (32,33). The rabbit polyclonal antibody (pAb) directed against the C-terminal domain of SynCAM 1 was purchased from Sigma-Aldrich.…”