Polysialic acid is a linear homopolymer of N-acetyl or Nglycolyl neuraminic acid (Neu5Ac or Neu5Gc) occurring in ␣2, 5-, 8-, or 9-ketosidic linkages (reviewed in Ref. 1). Polysialic acid has been found widely distributed throughout nature, from the capsular polysaccharide of neuroinvasive bacteria, such as Escherichia coli K1 (2), to unfertilized rainbow trout eggs and the jelly coat of sea urchin eggs (3, 4), to human tumors and tissues (1, 5). While there has been a great deal of research on bacterial polysialylation, eukaryotic, especially mammalian, protein polysialylation has been the focus of intense investigation in recent years.Although polysialic acid has been found throughout the taxonomic spectrum, only the oligosaccharides of a few mammalian proteins bear this modification. While polysialic acid has been found on the ␣-subunit of the rat brain voltage-sensitive sodium channel (6) and unidentified proteins in breast cancer and basophilic leukemia cell lines (7), the most abundant carrier of polysialic acid is neural cell adhesion molecule (NCAM) 1 (8). Polysialylated NCAM has been observed in developing brain (9), kidney (10), heart, and muscle (11). It is widely postulated that the polysialylation of NCAM oligosaccharides during the development of the nervous system and other organ systems in the embryo and neonate leads to a general decrease in cell adhesion (12-17). It is believed that the presence of cell surface polysialic acid disrupts the homophilic binding properties of NCAM and facilitates cellular migration, neurite outgrowth, and synaptic plasticity (12-17). Interestingly, polysialylated NCAM is also reexpressed on some metastatic cancers such as neuroblastoma (18), small cell lung carcinoma (19), and the highly metastatic kidney tumor, Wilms tumor (5). As in development, cell surface-expressed polysialylated NCAM is thought to increase the migration of cancer cells, thereby enhancing their metastatic potential (18, 20 -23).Two recently cloned polysialyltransferases have been shown to be responsible for the polysialylation of NCAM. The first, STX (ST8Sia II), is a type II membrane protein with a predicted molecular mass of 42.5 kDa and six potential N-linked glycosylation sites. STX has been cloned from rat (24), mouse (25), and human (26) sources and was shown to have polysialyltransferase activity toward N-linked oligosaccharide structures (25-27). The second polysialyltransferase, PST (ST8Sia IV), is also a type II membrane protein with a predicted molecular mass of 41.2 kDa. PST has five potential N-linked glycosylation sites, with four reportedly being used (28). PST was cloned from human (29), hamster (30), mouse (31), rat (32), and chicken 2 sources and was also shown to have polysialyltransferase activity toward N-linked oligosaccharides (30,31,34). Interestingly, recent in vitro studies on PST revealed that the PST enzyme is itself modified by polysialic acid and that this modification may be important for activity (28).In light of the observation that PST is polysialylated in vitr...

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel ␣-helix that links strands 4 and 5 of its ␤-sandwich structure. Replacement of the ␣-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N-and O-glycans in the same way, while the ␣-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 ␣-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.Polysialic acid is a developmentally regulated, anti-adhesive glycan that is found predominantly on the neural cell adhesion molecule, NCAM.2 Long, negatively charged polysialic acid chains decrease NCAM-dependent and -independent adhesion processes (1), thereby facilitating axon guidance and pathfinding, neurite outgrowth, synaptic plasticity, and general cell migration in the central nervous system (2-5). Polysialic acid levels are high in the embryo and neonate and decrease in the adult, except in areas of the brain such as the hippocampus and olfactory bulb that require on-going cell migration and functional plasticity (2, 3). In addition, highly polysialylated NCAM is re-expressed on the surface of some cancer cells where it is believed to promote cancer cell growth and invasion (6 -11).The polysialyltransferases ST8Sia II (STX) and ST8Sia IV (PST) are responsible for NCAM polysialylation, and their expression levels mirror the abundance of polysialic acid during different developmental stages (12, 13). Recent studies using polysialyltransferase and NCAM knock-out animals highlight the importance of polysialic acid during brain development (14 -16). While deletion of NCAM results in relatively mild morphological and behavioral effects in mice (17), the simultaneous deletion of the two polysialyltransferases results in severe alterations in brain development and premature death (14, 15). Strikingly, animals lacking NCAM and the polysialyltransferases appear normal, suggesting that the presence of polysialic acid is critical for down-regulating NCAM-dependent adhesion at specific times during brain development (15).Polysialic acid is unique among glycan modifications in that it is added to a small subset of proteins including the ␣-subunit of t...