Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates.

Polysialic acid (polySia) is a large glycan polymer that is added to some glycoproteins by two polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. As polySia modulates cell adhesion and signaling, immune cell function, and tumor metastasis, it is of interest to determine how the polySTs recognize their select substrates. We have recently identified residues within the ST8Sia-IV polybasic region (PBR) that are required for neural cell adhesion molecule (NCAM) recognition and subsequent polysialylation. Here, we compared the PBR sequence requirements for NCAM, neuropilin-2 (NRP-2), and synaptic cell adhesion molecule 1 (SynCAM 1) for polysialylation by their respective polySTs. We found that the polySTs use unique but overlapping sets of PBR residues for substrate recognition, that the NCAM-recognizing PBR sites in ST8Sia-II and ST8Sia-IV include homologous residues, but that the ST8Sia-II site is larger, and that fewer PBR residues are involved in NRP-2 and SynCAM 1 recognition than in NCAM recognition. Noting that the two sites for ST8Sia-IV autopolysialylation flank the PBR, we evaluated the role of PBR residues in autopolysialylation and found that the requirements for polyST autopolysialylation and substrate polysialylation overlap. These data together with the evaluation of the polyST autopolysialylation mechanism enabled us to further identify PBR residues potentially playing dual roles in substrate recognition and in polySia chain polymerization. Finally, we found that ST8Sia-IV autopolysialylation is required for NRP-2 polysialylation and that ST8Sia-II autopolysialylation promotes the polymerization of longer polySia chains on SynCAM 1, suggesting a critical role for polyST autopolysialylation in substrate selection and polySia chain elongation.