N-glycosylation is crucial for trafcking and stability of SLC3A2 (CD98)

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of infammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafcking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafcking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafcking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This fnding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafcking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.