Study On Molecular Evolution Of Primate Group ⅡA Phospholipase A₂

Objective:To analyse the information about the gene of the group IIA phospholipase A2 (PLA2) on several primates which have close genetic relationship but dividing into distinc species, to get the message on characteristics of molecular evolution about group IIA phospholipase A2.Methods:All known mRNA and genes data of group IIA PLA2 on primates, which including of Homo sapiens, Pan troglodytes and Macaca mulatta, were collected and compared with the data of group I B PLA2. To analyze base substitutions of transition and transversion, ratio of transition and transversion; and the change of base substitutions took place in coding region and non-coding region, position of the site of the triplet codon, etc.Results:When base replacement occurred in the gene coding region of Primate Phospholipase A2, the total number of base replacement of type IIA and type I B PLA2 was the minimum between Homo sapiens and Pan troglodytes. However, the base replacement increased significantly between Homo sapiens and Macaca mulatta, as well as between Macaca mulatta and Pan troglodytes, and it was about five times and three times to the former. The base replacement observed in site I and site III of type I B PLA2 codon between Homo sapiens and Pan troglodytes was at the most, each with fourty percentage, and it was twenty percentage at least on site II on codon; For Homo sapiens and Macaca mulatta, Macaca mulatta and Pan troglodytes, according to the bases replacing frequency from high to low, it wasⅢ>Ⅱ>Ⅰsite of codon in turn, the percentage of total base substitution occurred in siteⅠandⅡwas fourty-eight percent to fifty percent, lower than that occurred in the siteⅢfrom fifty percent to fifty-two percent. And the ratio of the base transition and transversion between Homo sapiens and Macaca mulatta, Macaca mulatta and Pan troglodytes, was more than one, but lower than one between Homo sapiens and Pan troglodytes. Compared with typeⅠB PLA2, the total number of base replacement of type IIA PLA2 among the three species of Primate biology was more (62 vs 54), and the proportion of bases replacement of codons is also higher; the base replacement was observed at most on siteⅢof codon for type IIA PLA2 among Homo sapiens, Macaca mulatta and Pan troglodytes, accounting from 40.7 percent to 50 percent, it was occurred in the siteⅠand siteⅡof codon from 25.9 percent to 33.3 percent, but the rate of total base replacement was from 55.5 percent to 59.2 percent for the two sites ofⅠandⅡof codon, it was higher significantly than the siteⅢof codon. For the characteristic of mutations, the bases transition was more than bases transversion between Homo sapiens and Pan troglodytes, as well as between Macaca mulattas and Pan troglodytes. In contrast, bases transversion was more than transition slightly between Homo sapiens and Macaca mulatta.Conclusions:The rate of evolution of group IIA PLA2 was moderate, and acclerated evolution occurs in the group IIA PLA2 gene on primates. It may be associated with concerted evolution. OBJECTIVE:To investigate the properties of cDNA and amino acid sequence of human group IIA secreted phospholipase A2 (phospholipase A2, PLA2) on different individuals of Chinese, and compared to those of the known foreign species, understanding the features on molecular evolution of human group IIA secreted PLA2.METHODS:12 patients due to acute appendicitis or chronic appendicitis with acute exacerbation of the purposes of appendectomy, whichever was the surgical removal of appendicitis inflammation 50-100mg, the total RNA extracted by Trizol reagent, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to amplify the cDNA of group IIA secreted PLA2, PCR product bands confirmed by agarose gel electrophoresis, and then sent the PCR product with clear electrophoretic bands to a professional sequencing company for forward and reverse DNA sequencing, analyzed DNA sequence obtained and its deduced amino acid sequence, and compared them with those of the known alien species, observed the base and amino acid replacement, and combined with DNA sequencing profiles, analyzed the characteristics of their molecular evolution.RESULTS:The cDNA of group II A secreted PLA2 amplified successfully by appendicitis symptoms organizations from 12 different individuals of Chinese, and deduced the corresponding amino acid sequence, when they were compared with the known alien species, It was found that:The amplified cDNA sequence of groupⅡA secreted PLA2 were basically the same as the known alien species with the exception of single nucleotide polymorphisms (Single Nucleotide polymorphism, SNPs). Analysis of the sequence Map, samples in 12 cases,2 cases of single mutant alleles of SNPs visible sites are located in codon 32 and codon 44, and mutations occured in hot spot locus of CpG dinucleotide with heterozygous (CG & CT) and homozygous phenomenon. The amino acid sequence deduced from group IIA secreted PLA2 cDNA has exactly the same structure as those of the foreign ethnic groups.CONCLUSIONS:The cDNA sequence of human group II A secreted phospholipase A2 in different individuals of Chinese was highly conserved, as well as those of other ethnic groups, polymorphism was observed in codon 32 and codon 44, and the mutations occured in hot spot locus of CpG dinucleotide. The Primary structure of human group IIA secreted phospholipase A2 molecule on Chinese ethnic has exactly the same structure as other ethnic groups, indicating that the molecular which plays an important role in the the host innate immune system was highly conserved among different ethnic. OBJECTIVE:To analyse the similarity between Homo sapiens phosphoilipase A2 cDNA and Leishmania infantum DNA and to understand the cause.METHODS:Searched from Gene Banks and obtained the cDNA data of human secretoryⅡA phospholipase A2 in the first step, then, by the tools of BLAST, aligned the gene fragments with every 20 bases together a group (for instance, from 1 to 20; 10 to 30;and 20 to 40···, by analogy with the others until the end of the cDNA sequence) from Homo sapiens phosphoilipase A2 cDNA to nucleotide database step by step, collecting and sorting all of the consensus sequences provide to analyze.RESULTS:There were 4 only consensus sequences between Homo sapiens phosphoilipase A2 cDNA and Leishmania infantum DNA when the length of the cDNA fragment was more or equal 17 bases, and it was differed from these that there were 9 consensus sequences between the two species when the number of the bases fragment≥16, and the sequences distributed mainly in the chromosome 29 and chromosome 30.CONCLUSIONS:There are some indenty DNA fragments between Homo sapiens phosphoilipase A2 and Leishmania infantum DNA, it may due to horizontal transfer from Leishmania to Homo sapiens. OBJECTIVE:To understand the relationship between molecular evolution and characteristics of amino acid replacement for bactericidal phospholipase A2 on primate mammals.METHODS:Amino acid sequences from Homo sapiens, Pan troglodytes and Macaca mulatta were administrated with paired comparison, and compared with group I B phospholipase A2, analyzed their location, frequency of occurrence and characteristics in their replacement of amino acid(including of their polarity, nonpolarity;acidity, neutrality, basic), respectively.Then, utilizing soft of Cn3d4.1 to build three dimensional crystal structure for their protein molecular, so as to understood their location and region of amino acid substitution on primate, made a conclusion about their change regularity, and correlated with the antibacterial activityRESULTS:There were 4,12 and 12 amino acid residues replacing among Homo sapiens, Pan troglodyte and Macaca mulatta;and the replacements of amino acid mainly ocuring in charged amino acides, especially in the positive charged ones (basic amino acid);on the other hand, amino acid replacement focused on four limited domains which play an importmant role in the enzyme function.CONCLUSIONS:Molecular evolution of group IIA phospholipase A2 is fast on primate mammals, consequently, there are more basic amino acids in IIA phospholipase A2, and it may be contributed to their powerful bactericidal activity. OBJECTIVE:To observe the bactericidal effect of the polypeptide which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2 (Group IIA PLA2) in vitro, and discuss its mechanism of the bactericidal effect.METHODS:According to C-terminal 26 amino acid residues sequence of Human Group IIA PLA2, and synthesized it as a piece of polypeptide P26. Incubated 6 bacterial strains (including of Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax, Escherichia coli, Bacillus proteus and Bacillus pyocyaneus) solution with different concentration of polypeptide at 37℃for 2 hours in a water bath respectively. Then diluted the reaction solution and poured agar plates. After incubated for 18-24 hours in the thermostated container at 37℃, Counted the colony formed unit and calculated the bactericidal rates.RESULTS:The polypeptide P26 possessed potent bactericidal activity to the gram-positive (G+) bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax; weak bactericidal activity to the gram-negative (G-) bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus.CONCLUSIONS:The polypeptide which derived from C-terminal 26 amino acid residues sequence of Human Group IIA PLA2possesses bactericidal activity. It is speculated that the polypeptide might have similar bactericidal mechanism as Human Group IIA PLA2 and the other antibacterial peptides. OBJECTIVE:To study the bactericidal activity of the polypeptide P26 and its variant due to modified and reconstructed on it, which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2 (Group IIA PLA2) in vitro, and analysis the cause leading to the difference of the bactericidal activity among them.METHODS:Synthesized linear polypeptide P26 corresponding to the C-terminal 26 amino acid residues sequence of Human Group IIA phospholipase A2, and then, modified it to be a cyclic polypeptide by cyclization on it, reconstructed it to be another new polypeptide by changing four basic amino acid residues of the linear polypeptide P26 into nonpolarity hydrophobic amino acid and polarity neutral amino acid.Incubated 6 bacterial species solution(including of gram-positive bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax;gram-negative bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus) with different concentration of polypeptide at 37℃for 2 hours in a water bath respectively. Then diluted the reaction solution and poured agar plates. After incubated for 18-24 hours in the thermostated container at 37℃, Counted the colony formed unit and calculated the bactericidal rates. RESULTS:The linear and cyclic polypeptide P26 possessed potent bactericidal activity on the gram-positive (G+) bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax; but weak bactericidal activity on the gram-negative (G-) bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus. And the cyclic polypeptide P26 possess potent action on the gram-positive (G+) bacteria, but it was not for the gram-negative (G-) bacteria. Different from these, the reconstructed polypeptide P26 had the most weak bactericidal activity on the gram-positive(G+) bacteria, and none of bactericidal activity on the gram-negative (G-) bacteria.CONCLUSIONS:The linear and cyclic polypeptide P26 which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2possesses potent bactericidal activity against G+ bacteria. It is speculated that the more potent action of the cyclic peptide on gram-positive(G+) bacteria related to its cylic structure;when changed four basic amino acid residues of the linear polypeptide P26 into non-basic amino acid residues, bactericidal activity of the reconstructed polypeptide P26 against G+ bacteria would be decreased obviously.