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

Shalu Shiv Mendiratta,Nikolina Sekulic,Francisco G Hernandez-Guzman,Brett E Close,Arnon Lavie,Karen J Colley

doi:10.1074/jbc.m608073200

Shalu Shiv Mendiratta, Nikolina Sekulic + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m608073200

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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 alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-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 alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

Highlights

Migration in the central nervous system [2,3,4,5]
Polysialic acid is unique among glycan modifications in that it is added to a small subset of proteins including the ␣-subunit of the voltage-dependent sodium channel [18], the polysialyltransferases themselves, which are capable of autopolysialylation [19,20,21], a form of the CD36 scavenger receptor found in milk [22], and NCAM, the most abundant polysialylated protein
Overlay of the two fibronectin type III (Fn III) domains indicated that these two residues would suffice as a NCAM FN1 Possesses a Unique ␣-Helix That Links Strands 4 linker between strands 4 and 5 without perturbing the FN1 and 5 of Its ␤-Sandwich Structure—Fn III repeats contain ϳ100 ␤-sandwich fold (Fig. 2d)

Summary

Introduction

Migration in the central nervous system [2,3,4,5]. 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]. We found that replacing all four acidic patch residues with alanine did not further decrease the recognition and polysialylation of FL-NCAM (data not shown).

Results

Conclusion