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

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory 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 trafficking 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 trafficking 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 trafficking 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 finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.

Overproduction, Purification, and Stability of the Functionally Active Human Carnitine Acetyl Transferase.

Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 °C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 °C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications.

Low temperature bacterial expression of the neutral amino acid transporters SLC1A5 (ASCT2), and SLC6A19 (B0AT1).

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.