Mapping of residues in leishmanial glyceraldehyde-3-phosphate dehydrogenase crucial for binding with 3'-UTR of TNF-alpha mRNA

Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from Leishmania major (LmGAPDH) was present in extracellular vesicles and it inhibited host TNF- expression during infection via post-transcriptional repression. The LmGAPDH binding with the AU-rich elements in 3-untranslated region of TNF- mRNA (TNF- ARE) is sufficient for limiting this cytokine production, but the TNF- ARE binding residues in LmGAPDH are still unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurement revealed that the inhibition by TNF- ARE was competitive with respect to cofactor NAD+ in LmGAPDH. To identify the TNF- ARE binding residues of the LmGAPDH, we exploited a systematic mutational analysis of its NAD+ binding domain. Catalytic activity measurement indicates that both R13 and N336 amino acids in the NAD+ binding site are absolutely required for activity whereas other mutants including I14A, R16A, D39A and T112A showed higher Km (lower affinity) value for NAD+ binding and lower catalytic activity. REMSA studies revealed that the replacement of Arg-13 with Ala/Lys or Asn-336 with Ala resulted in complete loss of binding with the TNF- ARE. I14A, R16A, D39A and T112A residues at or near NAD+ binding site showed lower binding with the TNF- ARE compared to the wild-type protein. The protein induced fluorescence enhancement (PIFE) studies and in vitro protein translation assay further confirmed the REMSA results. Based on our findings, the NAD+ binding residues in LmGAPDH are important for TNF ARE binding.