Differential glycosylation of Tractin and LeechCAM, two novel Ig-superfamily members, regulates neurite extension and fascicle formation
Specific carbohydrate structures on neural cell adhesion molecules are promising candidates for assisting in patterning neural connections during development. An excellent system to study the potential function of specific oligosaccharide modifications of neural proteins regulating pathway formation is in the projections of sensillar neurons in leech. At least five different monoclonal antibodies (Lan3-2, Lan2-3, Lan4-2, Laz2-369, Laz7-79) which recognize different glycoepitopes specific to the entire population as well as distinct subsets of these sensillar neurons have been identified. Their expression patterns and antibody perturbation studies are consistent with the hypothesis that a hierarchy of distinct carbohydrate structures expressed by specific neurons are involved in regulating neuronal pathway formation in this system. The further analysis of these aspects has been impeded by a lack of knowledge of the molecules carrying the glycosylated epitopes;I have used immunoaffinity purification techniques with the Lan3-2 antibody to identify the molecules carrying the Lan3-2 and Lan4-2 glycoepitopes. Both epitopes are expressed on two novel Ig-superfamily members, Tractin and LeechCAM, respectively. Tractin has a highly unusual structure: it contains six Ig-like domains, four FNIII-like domains, an acidic-domain, twelve repeats of a novel proline-and glycine-rich motif, a transmembrane domain, and an intracellular domain with an ankyrin binding motif. Tractin possesses a potential cleavage site and evidence suggests that the N-terminal half of the molecule is secreted. LeechCAM has five Ig-like domains, two FNIII-like domains, and a transmembrane domain and may be a leech homolog of NCAM, FasII, and ApCAM. Surprisingly, Tractin and LeechCAM are expressed by both peripheral and central neurons but are differentially glycosylated with the Lan3-2 and Lan4-2 epitopes only in the peripheral sensory neurons and their axonal projections. In vivo functional studies injecting purified Lan3-2 antibody into the germinal plate show that normal neurite extension and fascicle formation of the sensillar sensory neurons are impaired by the antibody while the development and projections of central neurons expressing Tractin and LeechCAM but not carrying the Lan3-2 glycoepitope are indistinguishable from controls. These findings suggest that differential glycosylation of widely expressed neural CAMs can functionally regulate neuronal outgrowth and fascicle formation of distinct neuronal subpopulations.