Characterization of Membrane glycoprotein M6a endocyticrecycling pathway upon treatment with the neutralizing monoclonal antibody

MICAELA D GARCIA; KARINA FORMOSO; FRANCISCO J BARRANTES; ALBERTO CC FRASCH; CAMILA SCORTICATI

Mar del Plata

Congreso; XXX SAN; 2015

SAN

Neuronal glycoprotein M6a belongs to the proteolipid protein family PLP. M6a iscomposed of 278 amino acid residues that form two external loops, a minor EC1 and amajor EC2, and four transmembrane domains; the N- and C-terminal regions lye in thecytoplasm. M6a is involved in neurite and filopodia outgrowth and synaptogenesis throughunknown mechanisms. To date, no natural ligands or binding partners of M6a have beenfound. However, a monoclonal antibody against EC2 (mAb) has been used to block M6afunction through clustering and possibly by M6a internalization. The membrane proteincomposition depends on endocytic recycling mechanisms. Interestingly, the PLP endocyticsorting in the oligondendrocyte surface precedes cell differentiation and it wascharacterized with mAb-internalization assays. Here we used HEK-293 cells and neurons toinvestigate the subcelular localization of M6a under mAb treatment. We found that M6acolocalizes partly with clathrin in the cell surface. Deconvolution (3D) of confocal z-stacksof each condition showed that after 30 minutes of treatment most of the M6a-mAbcomplex was present in endosomes and a few of them were positive for Rab5 (earlyendosome marker). No positive codistribution was observed between M6a and LAMP1(lysosome marker) after 30 min or 1h. Therefore, we concluded that the reorganization ofM6a in the cell surface after mAb treatment leads to its endocytosis. This endocytosismight depend on clathrin and Rab5 GTPase coordination.