Characterization of SynCAM surface trafficking using a SynCAM derived ligand with high homophilic binding affinity

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Abstract

In order to better probe SynCAM function in neurons, we produced a fusion protein between the extracellular domain of SynCAM1 and the constant fragment of human IgG (SynCAM-Fc). Whether in soluble form or immobilized on latex microspheres, the chimera bound specifically to the surface of hippocampal neurons and recruited endogenous SynCAM molecules. SynCAM-Fc was also used in combination with Quantum Dots to follow the mobility of transfected SynCAM receptors at the neuronal surface. Both immobile and highly mobile SynCAM were found. Thus, SynCAM-Fc behaves as a high affinity ligand that can be used to study the function of SynCAM at the neuronal membrane.

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Materials and methods

Plasmid construction. The SynCAM ECD hFc construct was generated by inserting the extracellular domain of SynCAM in pREP10 vector containing the Fc domain of human IgG (a gift from RM Mege, INSERM, Paris). The extracellular domain of SynCAM (aa 1–377) was amplified from a pCMV-SynCAM1 (a gift from T. Biederer, UT Southwestern) with primers sense 5′-AGATCTGCAGGTGCCCGACATGGCGAGT-3′ and anti-sense 5′-AGATCTACTTACCTGCGTGGTCCACTGCCCCAAT-3′, each containining a BglII site. This 1160 pb PCR product was

Production and characterization of a SynCAM-Fc chimera

Because SynCAM extracellular domains seem to form quite robust homophilic interactions, both in affinity chromatography [4] and cell aggregation assays [3], we decided to label SynCAM receptors in neurons using SynCAM itself as a ligand. We first produced a plasmid containing the SynCAM extracellular sequence fused in C-terminal with the constant fragment of human immunoglobulin G (Fig. 1A). We expressed this construct in HEK cells, harvested the conditioned medium, and purified the secreted

Acknowledgments

We thank M. Lambert and R.M. Mège for the purification protocol, T. Biederer for the gift of the SynCAM plasmid, D. Bouchet and B. Tessier for help with cell culture, Grégory Giannone, M. Heine and C. Bats for advice on Quantum Dots coupling protocol, L. Cognet and M. Renner for the Mathlab program.

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