Posttranslational processing and functional analysis of Tractin, an L1 family member in leech

摘要

During neurogenesis, glycosylated cell adhesion molecules (CAMs) mediate the growth cone navigation and synapse formation. The structure of neuronal CAMs of Ig superfamily is characterized by the variability and complexity of their extracellular region which contain multiple tandemly arranged domains. The diversity in the structure of the neuronal CAMs is generated by alternative splicing and posttranslational modification, such as differential glycosylation and proteolytic processing.;Tractin is a member of the L1-family of neuronal CAMs in leech. It contains six Ig domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif (PG/YG) with sequence similar to type IV collagen, a transmembrane domain and an intracellular tail with ankyrin and PDZ binding motifs. Tractin is constitutivly cleaved in vivo at a proteolytic site in the third FNIII-like domain. The COOH-terminal fragment is further cleaved at a site proximal to the transmembrane domain.;The sequence RKRRSR of the cleavage site I in the third FNIII-like domain of Tractin conforms to the consensus sequences for cleavage by members of the furin family of convertases. This proteolytic site is shared by most of the L1 family members. The results of furin specific inhibitor experiments, site-specific mutagenesis of Tractin constructs expressed in S2 cells, as well as by Tractin expression in furin deficient LoVo cells reveal that a furin convertase is the likely protease mediating this processing. Cross-immunoprecipitations with Tractin domain specific antibodies suggest that the resulting NH 2- and COOH-terminal cleavage fragments interact with each other. This interaction provides a means for the NH2-terminal fragment to be tether to the membrane. Furthermore, the S2 cell aggregation assays show that the NH2-terminal fragment is necessary for homophilic adhesion. The cells expressing only the transmembrane COOH-terminal fragment can functionally restore the adhesive properties of Tractin with the application of the NH 2-fragment. This novel proteinprotein interaction mechanism may be of general relevance of the processing of L1 family CAMs processed at this proteolytic site and its biological function.