METTL9-Mediated His107 N1 Methylation Of S100A9 Affected The Host’s Anti-Bacterial Response

Most research in the past decades has been focused on lysine and arginine methylation modification.However,protein histidine methylation is widespread and represents～13%of mammalian protein methylome events.Among them,METTL9 is the first Nl-methyltransferase discovered in mammals.Unlike the N3-methylation of SETD3 and METTL18,METTL9 processes with broad-specificity,specifically methylates a variety of substrates with a common x-His-x-His motif(xHxH)and catalyzes methyl transfer to the last histidine.METTL9,as a recently discovered protein methyltransferase responses to many substrates,no report upon the physiological function of its modification in vivo has been made.This new research direction is worthy of further in-depth discussion and expansion.In this study,we focused on S100A9 protein and its histidine methylated form S100A9me protein.We found that METTL9 could catalyzes His 107 N1-methylation of murine mS100A9 and His 105 N1-methylation of human hS100A9 in vitro by methylation and autoradiography experiments.Subsequently,we also demonstrated that METTL9 catalyzes the N1-methylation of S100A9 histidine in humans and mice in vivo by the preparation of polyclonal antibodies specific for human and mouse S100A9 histidine methylation sites.Meanwhile,we creatively purified mS100A9me and mS100A8/A9me recombinant proteins by co-transferring GSTmMETTL9 and pET28a-mS100A9 plasmids.Isothermal calorimetric titration(ITC)assay conducted over the mS100A9me recombinant protein showed that the His107 site of mS100A9 was successfully methylated,and the binding strength of mS100A9me protein to Zn(Ⅰ)site has decreased.The antibacterial activity of recombinant mS100A8/A9me protein was detected in vitro,and methylation at His107 of S100A9 reduced the antibacterial activity of mS100A8/A9.In vivo,wild-type(WT)C57BL/6J mice were subcutaneously injected with mS100A8/A9,mS100A8/A9me and mS100A8/A9 H107G recombinant proteins mixed with Staphylococcus aureus(S.aureus),respectively.The results showed that the recombinant mS100A8/A9 protein could resist the growth of Staphylococcus aureus in vivo.Consisting with the in vitro results,the antibacterial effect of mS100A8/A9 protein was reduced when the His 107 site of mS100A9 protein was methylated or mutated to Gly.Previous studies on Mettl9 in the laboratory revealed that WT and Mettl9 knockout(KO)mice had similar number and percentage of major cell populations,under steady state,and the deficient of Mettl9 embodied no obviously affect to the normal development of major cell populations.Mettl9 KO mice were healthy and had no obvious weight abnormality.After induced S.aureus skin infection model on WT and Mettl9 KO mice,we found that the complete loss of S100A9 His107me in Mettl9 deficient mice would lead to an increased resistance to Staphylococcus aureus infection.Taken together,our results elucidated that the METTL9-S100A9 His 107 methylation axis suppresses the zinc binding ability and zinc-dependent anti-S.aureus activity of S100A8/A9 in vitro and in vivo,and sheds light on the physiological significance of METTL9 and its mediated N1-methylation modification in the host’s antibacterial function.Our data will provide profound and inspiring perspectives for future research on the importance of histidine methylation of proteins in eukaryotes.