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

Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from Leishmania major (LmGAPDH) was present in extracellular vesicles and it inhibited host TNF-α expression during infection via post-transcriptional repression. The LmGAPDH binding with the AU-rich elements in 3’-untranslated region of TNF-α mRNA (TNF-α ARE) is sufficient for limiting this cytokine production, but the TNF-α ARE binding residues in LmGAPDH are still unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurement revealed that the inhibition by TNF-α ARE was competitive with respect to cofactor NAD ⁺ in LmGAPDH. To identify the TNF-α 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-α ARE. I14A, R16A, D39A and T112A residues at or near NAD ⁺ binding site showed lower binding with the TNF-α 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 TNFα ARE binding.