Investigating post-translational modifications of Tetraspanins: palmitoylation of CD81 and glycosylation of Tspan-2
Members of the protein super family 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
Tspan-2 by investigating expression patterns, N-glycosylation status, and association
with other tetraspanins.
School:Kansas State University
School Location:USA - Kansas
Source Type:Master's Thesis
Keywords:tetraspanin tspan 2 palmitoylation glycosylation cd81 biology molecular 0307
Date of Publication:01/01/2008