Mapping of pikachurin heparan sulfate modification to a region dispensable for synaptic localization in photoreceptors.

Rod and cone photoreceptors (PRs) are specialized neurons that detect light, then transmit information to bipolar cells at synapses in the outer plexiform layer of the retina. Pikachurin (PIKA) is a multi-domain heparan sulfate (HS) proteoglycan expressed in PRs. It is secreted into the synaptic cleft, where it binds to presynaptic dystroglycan, postsynaptic orphan receptor GPR179, and postsynaptic adhesion molecule LRRTM4. Knockout of PIKA results in ultrastructural and functional defects at rod synapses. However, the role of HS in PIKA function at synapses is unknown. Using a panel of deletion and truncation mutants expressed in heterologous cells, we determined that a.a. 240-342, a region of unknown function between the last fibronectin III domain and the first EGF-like domain, is necessary and sufficient for PIKA HS modification. Site-directed mutagenesis of S244, identified as a likely candidate based on neighboring sequences, abolished HS modification, indicating that this residue is the sole HS attachment site. Deletion of the 240-342 region or mutation of S244 also abolished co-immunoprecipitation of LRRTM4, consistent with the previously reported HS-dependent binding. To assess requirements for synaptic localization, PIKA mutants were expressed in mouse rods. Deletion of the HS-containing region or the fibronectin III domains did not prevent localization at rod presynapses. In contrast, deletion of the first EGF-like and laminin G domains severely impaired localization, suggesting an important role for these domains in mediating trafficking or synaptic enrichment.