Reelin is a secreted glycoprotein that plays essential roles in the brain development and functions. Reelin is specifically cleaved at two sites, called N-t and C-t. N-t cleavage can occur both in the extracellular space and in the endosomes. However, the physiological significance of N-t cleavage has not been investigated.
In this study, we determined the exact N-t site catalyzed by a protease secreted by cerebral cortical neurons. N-t cleavage occurred between Pro1,244 and Ala1,245 within Reelin repeat 3. Reelin mutant in which Pro1,244 was replaced with aspartate (Reelin-PD) was resistant to a protease secreted by cerebral cortical neurons and its biological activity stayed longer than that of wild type Reelin. Interestingly, Reelin-PD remained in the intracellular region longer than wild type Reelin and persistently activated downstream signaling. Therefore, N-t cleavage of Reelin is required for halting the signaling machinery in the extracellular space as well as within endosomes of target neurons. Then, we established a monoclonal antibody that recognizes only uncleaved Reelin and found that it is localized in the vicinity of Reelin-producing cells while the N-terminal fragment diffuses, or is transported, to distant regions. These data demonstrate that N-t cleavage of Reelin plays critical roles in regulating the duration and range of Reelin functions.
We also identified the protease in charge of N-t cleavage by purifying it from culture supernatant of primary cortical neurons by a series of column chromatographies. Recombinant protein of this protease cleaved Reelin at N-t site and the cleavage of Reelin is markedly decreased in the embryonic cerebral cortex of its knockout mouse. Importantly, the amount of Dab1 protein is decreased in the cerebral cortex of the knockout mouse, indicating that Reelin signal is hyperactivated. Therefore, it is proven in vivo that N-t cleavage by a specific protease is the major inactivation pathway of Reelin in the developing cerebral cortex. Further elucidation of Reelin proteolysis and trafficking will lead to the understanding of its regulatory mechanism.