Functional Study Of Amine Oxidase Gene From Scylla Paramamosain

Amine oxidase is mainly involved in the metabolism of amines in organisms,and it plays an important role in regulation the nerve conduction,metabolism and cells growth and differentiation of organisms.Monoamine oxidase is a flavoproteinase,and widely present in various tissues of living organisms,and is mainly distributed in the mitochondrial outer membrane and presynaptic membrane of the central nervous system and peripheral nervous tissues.Based on the differences in selectivity and sensitivity to substrates and inhibitors,MAOs are divided into two subtypes,MAO-A and MAO-B,and are encoded by different genes located on the X chromosome.MAO-A has high affinity for serotonin,norepinephrine and dopamine,and can be specifically inhibited by chloriline.MAO-B has a strong affinity for the substrates phenylethylamine,benzylamine and tryptamine,and it can be suppressed by selegilan.In addition,a large number of studies have shown that MAO is closely related to mammals’aggressive behavior and human anti-social behavior.As a recently discovered polyamine oxidase,spermine oxidase can oxidize and decomposesperminedirectlytoproducespermidine,phenylpropionaldehyde and reactive oxygen species H₂O₂ without acetylamine.Polyamines regulate the proliferation,differentiation,and apoptosis of cells by binding to nucleic acid substances,receptor proteins,and recognition molecules on the cell membrane.The recent SMO research focuses on the relationship between its mediated inflammatory response and tumors.Scylla paramamosain is widely distributed in tropical or subtropical offshore estuarine areas.It is an important marine aquaculture crab in China.The study was aimed to study the green crabs as a research object,and successfully constructed a cDNA library of A.acutus,and then cloned the full-length cDNA sequence of the target gene using RACE technology for bioinformatics analysis.The experimental crabs were treated with quercetin,a typical inhibitor of monoamine oxidase,and the expression pattern of the amine oxidase gene was detected by realtime quantitative PCR at different concentrations of quercetin.The pCold-I-SpAMO recombination plasmid was constructed to express it in Escherichia coli.The function of the recombinant protein was verified.The main finding of this article are summarized as follows:1.The full-length cDNA sequence of the amine oxidase gene was cloned successfully by RACE method and named as SpAMO after comprehensive analysis.The NCBI online comparison showed that it has high similarity with SMO genes of other species.The full-length cDNA of SpAMO is 2599 bp,it contains a 1521 bp open reading frame（ORF）,a 26bp 5’-untranslated region,and a 1052 bp 3’-untranslated region,encoding a total of 506 amino acids.The first 26 residues of the amino acid sequence are the signal peptide region,and a polyadenylation signal（AATAAA）was found 11 bp upstream of poly-A,which is the termination signal.A number of ATTTA（G）motifs related to transcriptional stability were found in the3’-UTR region of the SpAMO gene.The predicted protein has a molecular weight of 56.96 kDa and a theoretical isoelectric point of 5.04.SpAMO contains a conserved FAD binding domain,and the other three conserved regions are PLN0568,YobN,and proto_IX_ox.Phylogenetic analysis revealed that the crabs of A.mimicus SpAMO were clustered with SMO and formed a pair of insect invertebrates.The tertiary structure prediction results showed that the SpAMO was based on the tertiary structure of N（1）-acetyl-spermine/spermidine in the rat’s rat model,with a similarity of28.25%.And both of the FAD binding domain tertiary structure is highly similar,both containing anα-helix and two extended backbones.2.The detection of qRT-PCR found that in vitro injection of quercetin inhibited the transcripton level of SpAMO.When the injection dose was 5mg/kg/d,the transcriptional level of SpAMO showed a fluctuating tendency.In the first 24 hours,the expression levels in both tissues decreased,and the subsequent 2 days showed a significant increase in expression levels.On the 2nd day,the expression level decreased again,and the inhibitory effect was stronger than that in the first 24 h.The inhibition of SpAMO in brain tissue by quercetin was stronger than that in hemolymph.Quercetin is prone to autooxidation,the reversibility of inhibition and weak inhibition of SpAMO may be the cause of fluctuating changes.When the injection dose was 50 mg/kg/d,the inhibitory effect was more significant,the expression level of SpAMO gene in hemolymph and brain tissue decreased to about 10%of the original level,and the expression level of SpAMO stabilized at a low level,and the decrease in mRNA levels in the hemolymph is even more pronounced.3.By constructing pCold-I-SpAMO recombination expression vector and transforming the expression strain,the obtained recombinant protein SpAMO was identified,purified,renatured and functionally verified.It can be seen that both the SpAMO and the four substrates can generate reactive oxygen species（H₂O₂）by oxidative deamination.The average fluorescence values of the four substrates are 18.4,12.8,1.3 and 1.4,respectively.It was found that the oxidative deamination of 5-HT and dopamine by SpAMO was significantly stronger than that of spermine and phenethylamine.Therefore,the activity of SpAMO was stronger in 5-HT and dopamine than in spermine and phenethylamine.Therefore,it can be inferred that the function of SpAMO mainly catalyzes 5-HT and dopamine in S.paramamosain.In conclusion,a bioinformatics analysis of SpAMO showed that it has molecular characteristics of spermine oxidase（SMO）,but an inhibition test and enzyme activity study indicated that it also functions like monoamine oxidase（MAO）.It is speculated that SpAMO might be a novel amine oxidase in S.paramamosain that has the functions of both SMO and MAO.