Phylogenetic Analysis Of Gene Structure And Regulatory Elements, Expression In Vitro And Function Identification Of Apolipoprotein D Gene From Amphioxus Branchiostoma Belcheri

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. Its genetic information on gene sequence and expression pattern has been widely used for interspecies comparative genome studies and developmental homology analysis.Apolipoprotein D (ApoD) is a member of the lipocalin family. ApoD can bind small hydrophobic molecules such as cholesterol and arachidonic acid, etc. It also plays a role in many physiological and pathological processes such as development and differertiation of embryo, apoptosis of cancer cells, immune defense, and so on. Study of ApoD has been largely focused on its expression and physiological function, and information regarding its genomic orgnization and regulatory elements is extremely limited.In this study, the genomic structure and regulatory elements of ApoD from 10 representive species including protozoan, invertebrate(protostomes and deuterostomes), protochordate and vertebrate were analyzed and compared. The genomic structure of ApoD is less conserved in organisms from protostome to deuterostome invertebrates, while it is highly conserved among chordates including amphioxus and verebrates. All four conserved cysteine residues are present in amino acid sequence of deuterostome ApoDs, while there are only two cysteine residues in amino acid sequence of protostomes ApoD. Structure divergence between protostome and deuterostome ApoD proteins suggests their function difference. The majority of regulatory elements are present in nearly all organism ApoD genes ranging from unicellular protozoan to mammals, suggesting that ApoD plays a very fundamental role, and possesses a conserved regulatory mechanism. However, there also exist some specific regulatory elements, which are present only in certain species and may perform some special roles.ApoD mRNA expression in murine NIH/3T3 fibroblasts exposed to various stresses such as as hydrogen peroxide and UV light shows dose-dependent increase. And a fly homolog of ApoD, Glial Lazarillo (GLaz), whose overexpression results in increased resistance to hyperoxia as well as a extension of lifespan under normoxia and resistance to starvation without altering lipid or protein content. These suggsest that ApoD is a nonspecific response to different oxidative stimuli and may be part of an antioxidant defense system.Prokaryotic expression plasmid pET32a-BbApoD has been constructed to determine whether BbApoD also has antioxidation, and recombinant BbApoD expressed in E. coli BL21 successfully. Antioxidation experiment in vitro shows that inhibition rate of hydroxyl radical by recombinant BbApoD manifests a concentration-dependent manner within dose range of 60μg/ml-420μg/ml. When concentration of recombinant BbApoD is between 60μg/ml and 180μg/ml, it will accelerate production of hydroxyl radical, but this acceleration shows decreasing trend. And when concentration of recombinant BbApoD is about above 200μg/ml, it begins to manifest inhibitory effect towards hydroxyl radical. Furthermore, the inhibition rate gradually increases associated with higher concentration and can reach 45.28%. This shows that scavenging effect of hydroxyl radical by higher concentration of recombinant BbApoD is significant. Metal-catalyzed oxidation (MCO) assay shows that the addition of recombinant BbApoD at a concentration between 100 and 400μg/ml to the MCO system, prevents nicking of the supercoiled DNA by the reactive oxygen species generated in the assay and about 50% of pUC19 keep supercoiled at the concentration of 200μg/ml. But lower concentration of recombinant BbApoD shows no protection of pUC19 DNA, and instead it will accelerate the degradation of DNA. This two antioxidant assay show that recombinant BbApoD inhibits production of free radicals of system at higher concentration and accelerates their production at lower concentration. At present, studies about function of ApoD expressed in vitro are still very little. A concentration-dependent manner of inhibition rate scavenging of hydroxyl radical and prevention nicking of the supercoiled DNA by recombinant BbApoD suggests that BbApoD probably takes part in antioxidation process in vivo widely. This is the first report about recombinant BbApoD of invertebrate has function of antioxidation. Protection effect of recombinant BbApoD towards lipid peroxidationl are still in study. And antioxidative mechanism of BbApoD in vivo need further research.