A Novel α-Helix in the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Is Critical for N-Glycan Polysialylation

Abstract

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...

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

“…We solved the crystal structure of NCAM FN1 (40). The structure confirmed the presence of the acidic patch (Asp 506 , Asp 520 , Glu 521 , and Glu 523 ) and also revealed a unique ␣-helix linking strands 4 and 5 of the FN1 ␤-sandwich (40). Replacing the ␣-helix with two threonine residues, as found in the Robo2 FN1 structure, or two alanine residues did not prevent polysialylation.…”

“…These results suggested that some or all of the acidic patch residues may be part of a larger polyST recognition region. We solved the crystal structure of NCAM FN1 (40). The structure confirmed the presence of the acidic patch (Asp 506 , Asp 520 , Glu 521 , and Glu 523 ) and also revealed a unique ␣-helix linking strands 4 and 5 of the FN1 ␤-sandwich (40).…”

“…Replacing the ␣-helix with two threonine residues, as found in the Robo2 FN1 structure, or two alanine residues did not prevent polysialylation. However, polysialic acid was found on FN1 O-glycans rather than on Ig5 N-glycans (40). These data suggested that the ␣-helix has a role in positioning the polySTs and/or mediating an Ig5-FN1 interaction that allows the polySTs to access Ig5 N-glycans.…”

“…1), is polysialylated exclusively on O-glycans (38). We also showed that replacing the acidic patch with either alanine or arginine residues substantially decreases or eliminates NCAM7 polysialylation, although replacing the ␣-helix has no effect (40). We evaluated whether replacing PYS and QVQ in the simpler NCAM7 protein might reveal roles for these sequences not observed in full-length NCAM.…”

“…Molecular modeling and structural analysis of the Ig5-FN1 unit has given us insights into the FN1 sequences that may play a role in NCAM recognition (39,40). Molecular modeling led to the identification of a unique acidic patch on the surface of the FN1 domain (39).…”