Localization And Enzyme Kinetics Of Aminopepetidase N3 From Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasitic protozoa that belongs to the top complex,which can cause toxoplasmosis in humans and animals.This parasite can infect almost all nucleated cells.According to serological statistics,about one-third of the world's population is infected with Toxoplasma gondii.For immunocompromised patients and pregnant women,Toxoplasma infection can cause serious complications and can be transmitted vertically to the fetus.Currently,there are no effective drugs to treat toxoplasmosis.Therefore,it is urgent to find effective drug targets and develop new drugs to treat toxoplasmosis.As a protease,aminopeptidase plays a key role in protein maturation,peptide degradation and protein stabilization.Many aminopeptidases have been widely studied as drug targets for the prevention and treatment of human and animal diseases.In particular,the prevention and treatment of malaria parasites have combined these aminopeptidases as effective drug targets.The study of Toxoplasma gondii aminopeptidase is currently rare.Aminopeptidase N is an important protease derived from the M1 family of metal peptidases.It is a broad-spectrum aminopeptidase capable of hydrolyzing neutral amino acid residues at the N-terminus of the peptide chain to form free amino acids.The aminopeptidase of the M1 family is more conservative and is present in many organisms.The enzyme is present in the worms such as coccidia,malaria parasite,and Cryptosporidium.Aminopeptidase N is a potential drug target and its properties have been extensively studied.In this study,Toxoplasma gondii was used as a research object to systematically analyze the subcellular localization and biochemical characteristics of Toxoplasma gondii aminopeptidase N3(TgAPN3).The homology alignment showed that the amino acid sequence of TgAPN3 was highly identical(97%)to H.serrata,and the homology to human aminopeptidase N was low(24%).Cell localization analysis revealed that TgAPN3 localizes in the cytoplasm of the worm and colocalizes with the marker proteins of dense granules.To analyze the enzymatic activity of TgAPN3,the recombinant Toxoplasma gondii aminoglyase N3(rTgAPN3)was expressed in vitro using E.coli,and the purified protein showed significant enzymatic activity on the H-Ala-MCA fluorescent substrate.The change in pH had a significant effect on the enzymatic activity of TgAPN3,which showed good activity in the acidic range and showed the maximum enzyme activity at pH 7.0.Different metal ions also affect the enzyme activity of TgAPN3.Compared with other metal ions,Co2+ions have the most obvious enhancement effect on enzyme activity.The identification of the substrate preference indicated that the enzyme preferentially selected a substrate containing an N-erminal Ala residue,followed by Tyr and Cys.The identification of the enzyme inhibitor showed that bestatin and phebestatin significantly inhibited the enzymatic activity of rTgAPN3.The localization analysis of TgAPN3 laid the foundation for its functional research,and the identification of TgAPN3 in vitro biochemical characteristics provided an important theoretical basis for the study of this enzyme as a drug target for Toxoplasma gondii.