Mapping of Residues in Leishmanial Glyceraldehyde-3-phosphate Dehydrogenase Crucial for Binding to 3'-UTR of TNF-α mRNA.

Abstract

Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from Leishmania major (LmGAPDH) is present in extracellular vesicles and inhibits host TNF-α expression during infection via post-transcriptional repression. LmGAPDH binding to AU-rich elements in the 3'-untranslated region of TNF-α mRNA (TNF-α ARE) was sufficient for limiting cytokine production, but the TNF-α ARE binding residues in LmGAPDH remain unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurements revealed that the inhibition by the TNF-α ARE was competitive with respect to the cofactor NAD⁺ in LmGAPDH. To identify the TNF-α ARE binding residues of LmGAPDH, we performed a systematic mutational analysis of its NAD⁺ binding domain. Catalytic activity measurements indicated 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 K_m (lower affinity) values for NAD⁺ binding and lower catalytic activity. REMSA studies revealed that the replacement of Arg13 with Ala/Lys or Asn336 with Ala resulted in a complete loss of binding to the TNF-α ARE. I14A, R16A, D39A, and T112A residues at or near the NAD⁺ binding site showed lower binding to the TNF-α ARE compared to the wild-type protein. Protein-induced fluorescence enhancement (PIFE) studies and in vitro protein translation assays further confirmed the REMSA results. Based on our findings, the NAD⁺ binding residues in LmGAPDH are important for TNF-α ARE binding.