Biophysical and Biochemical Determinants of Protein-Specific Polysialylation

Protein-specific polysialylation is a unique post-translational modification found only on a small set of cell-surface glycoproteins. This glycan, synthesized by the two Golgi-localized polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV, is crucial for proper development of the nervous system and also plays a role in maintenance of synaptic plasticity, tissue regeneration, and cancer metastasis. Unlike most other glycosyltransferases, polySTs are selective toward carrier proteins they modify. In order to understand the molecular mechanism of protein-specific polysialylation, I have investigated protein-protein interactions between polySTs and various substrates. In this work, I have demonstrated a direct interaction between a specific acidic patch in the first fibronectin type III (FN1) repeat of the Neural Cell Adhesion Molecule (NCAM) and the ST8Sia-IV polybasic region (PBR), which likely serves as an initial step for protein-specific polysialylation. Biophysical characterization of this interaction has led to more refined understanding of the relationship between the NCAM FN1 domain and the adjacent fifth immunoglobulin like (Ig5) domain that carries polysialylated N-glycans. I have also defined sequence requirements for Neuropilin-2 (NRP-2) polysialylation and have shown that it follows the ‘two-domain paradigm’, wherein initial interaction of ST8Sia-IV with the NRP-2 meprin-A5 antigen-mu tyrosine phosphatase (MAM) domain leads to polysialylation of the O-glycans in the adjacent linker region. I have further shown that NRP-1 can be a novel polyST substrate because of its recycling to the polyST-expressing compartments, despite containing a suboptimum recognition domain. I have extended my study to include ST8Sia-II, to understand the biochemical basis governing its polysialylation of NCAM and Synaptic Cell Adhesion Molecule (SynCAM 1). Finally, my work sheds light on the role and mechanism of autopolysialylation in substrate polysialylation. Taken together, these results have contributed to precise understanding of the molecular mechanism of protein-specific polysialylation in order to devise strategies to enhance or block polysialylation to obtain therapeutic benefits.