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

Deirdre A Foley,Kristin G Swartzentruber,Matthew G Thompson,Shalu Shiv Mendiratta,Karen J Colley

doi:10.1074/jbc.m110.170209

Deirdre A Foley, Kristin G Swartzentruber + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.170209

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Abstract

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro(510)-Tyr(511)-Ser(512) (PYS) and Gln(516)-Val(517)-Gln(518) (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro(510)-Tyr(511) eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.

Highlights

Further dissection of the contribution of inserted neural cell adhesion molecule (NCAM) FN1 residues demonstrated that the FN1 ␣-helix and QVQ sequences are critical for polyST positioning and Ig5 N-glycan polysialylation, whereas the acidic patch and PYS sequences play a primary role in FN1 O-glycan polysialylation
In this study we have used the FN1 domain from the unpolysialylated olfactory cell adhesion molecule, OCAM, for both sequence comparisons and gain-of-polysialylation experiments
Modeling of the OCAM FN1 domain based on the structure of NCAM FN1 predicts that OCAM FN1 lacks the acidic patch, and ␣-helix that we have shown play roles in the polysialylation of NCAM Ig5 N-glycans

Summary

Introduction

Inserting NCAM FN1 sequences (acidic patch, ␣-helix, PYS, and QVQ) does not substantially enhance the polysialylation of the chimeras, but instead it shifts the addition of polysialic acid to O-glycans. Further dissection of the contribution of inserted NCAM FN1 residues demonstrated that the FN1 ␣-helix and QVQ sequences are critical for polyST positioning and Ig5 N-glycan polysialylation, whereas the acidic patch and PYS sequences play a primary role in FN1 O-glycan polysialylation.

Results

Conclusion