Study On The Metabolism And Function Of Tryptophan Mediated By IL4I1

In recent years,reasonable diet has gradually become an economic,safe and efficient anti-cancer and anti-allergy strategy.As a component of protein,tryptophan is widely present in various foods.Moreover,the metabolites of tryptophan are involved in diverse life activities such as growth,circadian rhythm,metabolism,and immune response.Therefore,tryptophan metabolism attracted extensive attention.IL4I1 encodes an enzyme capable of catalyzing L-amino acids and has high catalytic activity for aromatic amino acids,namely phenylalanine,tryptophan and tyrosine.In humans,IL4I1 is mainly expressed in immune cells and has an important immunomodulatory effect.However,the catalytic effect of IL4I1 on tryptophan has not been reported.In order to explore the products and function of IL4I1 catalyzed tryptophan and understand the relationship between food components and immune regulation at the molecular level,we detected the products of IL4I1 catalyzed tryptophan by LC-MS/MS and researched the function of this metabolic pathway in IL4I1-knockout mice.The main results are as follows:1.Identify the enzyme activity of IL4I1Human and mouse IL4I1 genes were cloned into Plenti vector,and then transfected into 293T cells with lentiviral vector.The 293T cells that stably expressing IL4I1 were selected to establish IL4I1-293T cell line.Biochemical experiments show that the overexpressed ILAI1 protein can catalyze tryptophan,implying that IL4I1 expressed by 293T cells is a complete fuctional protein.The mouse Il4i1 protein was purified from the medium of 293F cells,and the enzymatic reaction conditions were further optimized by using the purified protein,that IL4I1 has the highest catalytic activity under neutral pH conditions.2.IL4I1 catalyzes tryptophan into indole-3-acetic acid(IAA)and indole-3-carboxaldehyde(IAld),and the endogenous indole pyruvate pathway was discovered for the first time.We detected the products of purified mI14i1 catalyzing tryptophan by LC-MS/MS and compared with the standard,and then we determined that IL4I1 catalyzes tryptophan to produce indole-3-acetic acid and indole-3-carboxaldehyde.Based on the properties that IL4I1 catalyzes amino acids to produce pyruvate,the process of IL4I1 catalyzing tryptophan was speculated as:tryptophan?indole-3-pyruvate?indole-3-acetaldehyde?indole-3-acetic acid?indole-3-carboxaldehyde.This process is consistent with the indole pyruvate pathway mediated by intestinal flora.Therefore,we considered that the indole pyruvate pathway also exists in the host cell and regulated by IL4I1.3.The expression and function of IL4I1 in vivoIt was confirmed that Il4il is highly expressed in migratory dendritic cells by single-cell sequencing and RT-PCR.And the products of IL4I1 metabolizing tryptophan indole-3-pyruvate(IPyA),indole-3-acetic acid,and indole-3-carboxaldehyde can activate the AHR signaling pathway.We constructed Il4i1-knockout mice.Compared with the wild type,the growth of melanoma in Il4i1-knockout mice is significantly slower.The above results indicate that the tryptophan metabolism mediated by IL4I1 plays an important role in tumor immunity,which may be achieved by the activation of the AHR signaling pathway stimulated by tryptophan metabolites to regulate the migration and function of dendritic cells.