3C-O2-1620（3P-45） PDF
A proteomic approach to elucidate the role of metalloproteases during neuronal cells differentiation
Many membrane proteins are known to be subject to limited proteolysis at their juxtamembrane regions, which is called as ectodomain shedding. Ectodomain shedding membrane bounded-ligands results in activation of downstream signalings, while that of cell adhesion molecules causes loss of cell-cell contacts.
In order to know what kind of proteins are secreted during differentiation of neuronal cells in metalloprotease-dependent manners, secreted proteome (secretome) analyses of culture medium in which neuroblastoma N1E-115 were differentiated to neurons with or without GM6001, a broad-spectrum metalloprotease inhibitor, under non-serum condition were conducted. Because most of membrane-bounded proteins are known to have N-glycosylation, we included N-glycosylated peptides enrichment1,2) into our secretomics workflow.
Most of peptides declined in the presence of GM6001 were derived from cell adhesion molecules including Nectin-2, MUC18, and L1CAM. Surprisingly, GM6001-sensitive peptides also included those derived from protein tyrosine kinase receptors and multiple transmembrane proteins. These results indicate that differentiation of N1E-115 neuronal cells accompanies cell-autonomously activated release of more various kinds of proteins than it has been noted so far, in metalloprotease-dependent manners. We further showed that ADAM19 enhances cleavage of L1CAM in HEK293T, suggesting that secretome analyses shown here is a valuable method to screen a set of membrane-bounded proteins shed in some contexts.