INVESTIGATING POST-TRANSLATIONAL MODIFICATIONS OF TETRASPANINS: PALMITOYLATION OF CD81 AND GLYCOSYLATION OF TSPAN-2

Members of the protein superfamily of tetraspanins are best defined by a simple structure comprising four transmembrane domains, two extracellular loops of unequal size, and short cytoplasmic regions.Despite their small size, tetraspanins are able to participate in multiple functions, as diverse as B cell activation, cancer metastasis, and viral infection.To compensate for a lack of intrinsic enzymatic activity, tetraspanins have gained the fascinating ability of associating with numerous different proteins.In addition, tetraspanins interact with each other forming a network on the plasma membrane: the tetraspanin web.In this way, functionally related proteins binding to different tetraspanins can be brought into close vicinity, thereby enhancing signaling pathways.The tetraspanin web is a dynamic environment and its regulation has grasped the attention of several research groups in the past few years.Particularly, several tetraspanins have been found to be palmitoylated, a post-translational modification attaching a palmitic acid to cysteine residues in a reversible manner.Palmitoylation is thought to be important for the integrity of the tetraspanin web.We examined the effect of disrupting putative palmitoylation sites on the tetraspanin CD81 by mutating its juxtamembrane cysteines.By flow cytometry, we observed a decrease in the detection of mutant CD81 at the cell surface.This was not due to defects in protein trafficking or antibody affinity, and might reflect an abnormal CD81 distribution in a membrane environment that prevents the exposure of the epitope recognized by the CD81 antibody.Immunoblotting analysis revealed a novel CD81 processing event that was impaired in the mutant CD81 proteins compared to wild-type.Finally, co-immunoprecipitation assays showed a reduction in binding of tetraspanin CD9 and Ig superfamily member EWI-2 to mutant CD81.Taken together, these results suggest the importance of juxtamembrane cysteines (via palmitoylation or protein conformational changes) in protein interactions of CD81 within the tetraspanin web.Although 33 tetraspanins are expressed in humans, less than half of them have been well studied.Among the "orphan" tetraspanins awaiting further examination is Tspan-2.Here, we provide the first elements for the characterization of mammalian Copyright