Cloning,Expression And Enzyme Characterization Of Lactate Dehydrogenase A And B From Echinococcus

ObjectiveBased on bioinformatics analysis,this study cloned the target gene of lactate dehydrogenase?LDH?and expressed its target protein,and fluorescence localized lactate dehydrogenase in different tissues of Echinococcus.Optimal reaction conditions for lactate dehydrogenase enzymatic reactions,determination of relevant enzyme kinetic parameters,and inhibitor testing.Because lactate dehydrogenase may be an important target molecule reflecting the infection of parasites and anti-infective treatment,the study of the properties and functions of lactate dehydrogenase of Echinococcus helps to further understand its growth and development characteristics,invasion and parasitic mechanisms.And it will lay the foundation for the development of new diagnostic and therapeutic tools.MethodsBioinformatics methods were used to analyze the LDH-A and LDH-B amino acid sequences of E.granulosus and E.multlocularis.The p ET28a-LDH recombinant plasmid was constructed and expressed in E.coli BL21/DE3.The four recombinant LDH were purified by Ni²⁺NTA column and were used to prepare antibodies.The distribution of four enzymes in the cyst of E.granulosus was analyzed by fluorescence immunolocalization technique.The in vitro catalytic kinetics of the four enzymes were analyzed using the cofactors?i.e.nicotinamide adenine dinucleotide?NAD⁺?,nicotinamide adenine dinucleotide coenzyme?NADH?and acetylpyridine-adenine dinucleotide?APAD⁺??,as well as the substrates?i.e.lactate,pyruvate,malic acid,2-ketobutyrate and 3-phenylpyruvate?.In addition,the inhibition of nicotinic acid,thionicotinamide,3-pyridinealdehyde and gossypol on the four enzymes were studied,respectively.ResultsSequencing and BLAST analysis showed that Eg LDH-A,Eg LDH-B,Em LDH-A and Em LDH-B all contained 331 amino acids with a conserved LDH domain.Eg LDH-A shares 94%homology with Em LDH-A,and Eg LDH-B shares 59%homology with Em LDH-B.The active sites of the four enzymes were all located at 189-195 amino acid,but compare with LDH-A,there was a mutation of Valine?Val?to Isoleucine?Ile?at 189aa in LDH-B.All the four enzymes have seven substrate binding sites;LDH-B have 12 NAD⁺binding sites,it has one more binding site than LDH-A?ie,Gly28-Val30?.In addition,the Asn53,Lys56,Ala94 binding site in LDH-B were switched to Asp53,Arg56,Thr94 in LDH-A.Four soluble recombinant proteins were obtained with an electrophoretic purity of more than 90%.Immunolocalization analysis showed that Eg LDH-A and Em LDH-A were mainly localized in the cytoplasmic part of protoscoleces,while Eg LDH-B and Em LDH-B were mainly located in the germinal layer of protoscolex.In vitro enzyme kinetics showed that in the reduction reaction,the optimum p H of the four enzymes was 7.5 and the optimal reaction temperature was 37°C.In the oxidation reaction,the optimum p H of the two LDH-A is 9.5,and the optimum temperature is 60°C;while the same value of the two LDH-Bs are 11.0 and 37°C,respectively.The Km values of r Eg LDH-A for NADH,NAD⁺,pyruvate,and lactic acid were 0.847 m M,0.207 m M,3.512 m M,and 119.3 m M,respectively;r Eg LDH-B was 0.286m M,1.32 m M,1.099 m M,and 41.7 m M,respectively;r Em LDH-A was 0.294 m M,0.299 m M,1.555 m M,and 182.6 m M,respectively;and r Em LDH-B was 0.345 m M,1.134 m M,0.958m M,and 40.213 m M,respectively.Following analysis of the Kcat/Km values for the four enzymes,it was found that pyruvate is the most suitable substrate in the reduction reaction while lactic acid is the most suitable substrate in oxidation reaction and APAD is the most suitable cofactor in oxidation reaction.The catalytic efficiency of LDH-B is higher than LDH-A.In addition,inhibitory experiments found that nicotinic acid,3-pyridinealdehyde,and gossypol have stronger effect on LDH-B than LDH-A.Conversely,thionicotinamide has a stronger inhibitory effect on LDH-A than LDH-B.ConclusionTwo novel LDH-B genes were identified from E.granulosus and E.multilocularis in this study.The sequences and the activities of four enzymes including Eg LDH-A,Eg LDH-B,Em LDH-A and Em LDH-B were identified and compared.The differences between two isozymes,LDH-A and LDH-B,were found in localization,enzyme kinetic in Echinococcus.Further more,this study revealed that LDH-B has higher catalytic efficiency than LDH-A.This study has provided the basis and insights for subsequent research on the biological function of Echinococcus LDH and related research on vaccines and drug targets against Echinococcus infection.