Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family

In the central nervous system, many cell adhesion molecules are known to pa rticipate in the establishment and remodeling of the neural circuit. Some o f the cell adhesion molecules are known to be anchored to the membrane by t he glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble memb rane fraction of low density or so-called "raft," In this study, we surveye d the GPI-anchored proteins in the Triton-insoluble low density fraction fr om 2-week-old rat brain by solubilization with phosphatidylinositol-specifi c phospholipase C, By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F 3/contactin, and T-cadherin was shown. In addition, one of the major protei ns, having an apparent molecular mass of 36 kDa after the peptide N-glycosi dase F digestion, was found to be a novel protein, The result of cDNA cloni ng showed that the protein is an immunoglobulin superfamily member with thr ee C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCA M, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a k indred of IgLON). Kilon-specific monoclonal antibodies were produced, and W estern blotting analysis showed that expression of Kilon is restricted to b rain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylam ide gel electrophoresis in its expressed form, In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases d uring development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on den drites and soma of pyramidal neurons.