Polysialic acid is an anti-adhesive carbohydrate polymer that is expressed in a developmentally- regulated manner. In mammals, the main carrier of polysialic acid is the neural cell adhesion molecule, NCAM. Polysialylated NCAM has important functions in nervous system development, synaptic plasticity, and neuronal generation. Moreover, deregulated polysialic acid expression is associated with cancer metastasis and with the development of neuropsychiatric disorders.;Polysialylation is catalyzed by the polysialyltransferase enzymes, and is a protein-specific glycosylation event. The FN1 domain of NCAM is critical for the polysialylation of N-glycans located on the adjacent Ig5 domain. Our laboratory proposed that polysialyltransferases bind to the NCAM FN1 domain and in this way are positioned to polysialylate Ig5 N-glycans. Furthermore, the structure of the FN1 domain revealed the presence of a novel surface acidic patch and alpha-helix with roles in NCAM polysialylation.;In this study, the protein-specific polysialylation of NCAM was further investigated. We solved the crystal structure of the NCAM Ig5-FN1 tandem. The sites of polysialylation on Ig5 were found to be on the opposite face as the FN1 acidic patch and alpha-helix. However, the polysialylation of engineered N-glycans on the Ig5 domain revealed some flexibility in the polysialylation process. Furthermore, this analysis revealed that the polysialyltransferases must engage the Ig5 domain to prevent a redirection of polysialylation to O-glycans.;An alignment of the FN1 sequences of NCAM and the unpolysialylated protein, olfactory cell adhesion molecule (OCAM), revealed two unconserved sequences within NCAM FN1, Pro510-Tyr511-Ser512 (PYS), and Gln516-Val517-Gln 518 (QVQ). We found that the acidic patch and PYS are required for O-glycan polysialylation, while QVQ has a positioning role similar to the alpha-helix. Analysis of chimeric proteins revealed that OCAM FN1 allows partial polysialylation of NCAM Ig5 N-glycans, while the Ig5 domain of OCAM does not permit polysialylation.;Finally, a conserved polybasic region within the polysialyltransferases was identified. Specific residues within this sequence are required for NCAM polysialylation, but not enzyme activity, suggesting they may be required for the initial interaction with NCAM. We propose that altering this interaction would be an attractive method of regulating polysialic acid expression in disease states.