Structure-activity Of HGlyrichin-derived Peptides And Related Studies

A novel gene mL52[1] (GeneBank number:AY028425) derived from mus musculus was obtained by using the method of suppression subtractive hybridization. According to the sequence of mL52 gene, the hL52 gene was obtained from human fetal liver cDNA library by using PCR amplification. It was designated as the hGlyrichin gene, because the protein encoded by the hL52 gene was rich in glycine. The hGlyrichin gene was composed of 240bp and had the complete ORF (open reading frame) and encoded the protein of 79 amino acids. Based on bioinformatics analysis results and antibacterial activities primarily demonstrated, the hGlyrichin was a highly coincident with the characteristic of most cationic AMPs reported. Therefore a series of peptides were synthesized and antibacterial activities were tested according to bioinformatics analysis. The results had indicated that the CM19 peptide at the position between 42th and 60th amino acids of hGlyrichin had the definite antibacterial activities[2].In the light of the results described above the CM12 peptide at the position between 42th and 53th amino acids of hGlyrichin was obtained by abscising the 7 amino acids of CM19 C-terminal GIG flexibility region according to bioinformatics analysis and the report of Yoonkyung[3]. Antibacterial activities of CM12 were detected and the results manifested that antibacterial activities of CM12 were higher than CM19. Then the antibiosis spectrum of CM12 was studied and the results confirmed that the CM12 peptide showed the antibacterial activities against not only the bacteria laboratory used but also those bacteria with pathogenesis potential, including Pseduomonas aeruginosa, S.typhi, S.aureus, AmpR S.aureus, EV76 strain of attenuation pestis. It is significant and application value that the CM12 peptide had the antibacterial activities against AmpR pET-22b+ BL-21 E.Coli and AmpR S.aureus. All the above-mentioned results revealed that the CM12 peptide had the broad-spectrum(the minimal broad spectrum including G+ bacteria, G- bacteria, coccus and bacillus, et al) antibacterial activities, and provided the valuable experimental data for the future application. After this, antibacterial activities of the CM12 peptide were quantitatively studied using 96 shadow mask method. The dose-effects curve of the CM12 peptide against E.Coli BL-21 presented broken line, and had evident deflection point that suggested that antibacterial activities could be found only when the concentration of the CM12 peptide reached the threshold dosage. And that curve was consistent with that reported by Sunkyun Kim, where dose-effects curve of the antimicrobial peptide, gaegurin 6, was studied [4]. Above-mentioned results testified that the CM12 peptide was gained and could represent the core functional domain of hGlyrichin, which was shorter and showed higher antibacterial activities than the CM19 peptide.Based on the CM12 peptide with the definite antibacterial activities, five peptides was designed and synthesized by substitution, replacement, deletion and rearrangement referring to the feature of antimicrobial peptide[5](mainly based on the feature of net positive charge) and known research reports. All the five peptides were named as hGlyrichin structure-activity peptides. The GE12 peptide was the random control, which was the random sorted peptide based on the sequence of the CM12 peptide. The SC22 peptide was the positive control, which was the P2 peptide derived from neutrophilic granulocyte [6]. Then the antibacterial activities of all above peptides were tested and the results had indicated that the first cysteine at the N-terminal of the CM19 peptide was necessary for maintaining the antibacterial activities, the GE12 peptide with rearrangement of amino acids lost the activity, and seven amino acids at C-terminal of the CM19 peptide had no antibacterial activities. Randomly increasing positive charges did not improve antibacterial activities. That would be related with the reasons that increased positive numbers should conform to the structure of peptides and pellicle interaction, including the influence of the net positive charge and positive charge distribution on the structure.According to hGlyrichin, sequences whose score was more than 50 were gained searching with BlastP tool and were multiple sequence aligned using ClustalX software. Cladogram was drawed using phylip software, then a protein assemble was obtained, which included many species with great evolution distance. And it was designated as hGlyrichin AMP family. Then the hGlyrichin AMP family members were divided into four groups and sequences of every group were multiple sequence aligned according to the evolution distance in order to choose the representative sequences. As a result, the four peptides named as hGlyrichin AMP family peptides, CM19 (CM12), GT18, AT18 and IM12, were synthesized. The results of antibacterial experiment had indicated that the antibacterial activities of the representative peptides were related with evolution of species and peptides derived from animal or plant had the different antibacterial spectrum. Of the total, the CM19 and CM12 peptide representing seven sequences of vertebrate had the greatest in-vitro antibacterial activities, the AT18 peptide representing one sequence of plant had temperate in-vitro antibacterial activities, the GT18 peptide representing four sequences of invertebrate had poor in-vitro antibacterial activities, and the IM12 peptide without representation had no antibacterial activities.In this study, the in-vitro antibacterial mechanisms of the referred peptide were preliminarily investigated. Peptides were incubated with the pTAT-EGFP E.Coli BL-21 preserved by our laboratory, and FCM analysis of the resuspension of bacteria precipitation was used to count the bacteria ratio dyed by PI and Western Blot of the supernatant was detected whether the GFP of bacteria was leaked out. Both methods co-analyzed the membrane integrity of target bacteria. All the results had indicated the CM12 and CM19 peptide might kill target bacteria by the way destroying the membrane integrity leading content of the target bacteria to leak out, which was corresponding to the mechanism of most cationic antimicrobial peptides.The hemolytic effect is a general side-effect for some of the AMPs, which is the critical obstacle to influence their development into drugs, so it is necessary to evaluate the hemolytic effect of the hGlyrichin related peptides. In this study, in-vitro hemolytic effects were evaluated by detecting the OD570 value of the supernatant after peptides were incubated with RBC. The results demonstrated hGlyrichin AMP family peptides had no in-vitro hemolytic effects to human erythrocyte, and hGlyrichin structure-activity peptides, CK17 and CT18, both had visiblely in-vitro hemolytic effects which hinted that redundant positive charges or unsuitable distribution were disadvantage to control the hemolytic effects. The CM12 peptide gained from this study had no in-vitro hemolytic effects.All the investigation indicates that:a) The CM12 peptide was discovered which was shorter and showed more great antibacterial activities than the CM19 peptide,b) Both CM19 and CM12 had the definite broad-spectrum antibacterial activities,c) Antibacterial activity could be found only when the concentration of the CM12 peptide reached the threshold dosage, 256μg/ml,d) Antibacterial activities of the hGlyrichin AMP family peptides were related with evolution of species, and peptide derived from animal or plant had the different antibacterial spectrum,e) CM12 and CM19 might kill target bacteria by the way destroying the membrane integrity and leading content of the target bacteria to leak out,f) CM12 and CM19 had not in-vitro hemolytic effects to human erythrocyteg) Redundant positive charges or unsuitable distribution are disadvantage to control the hemolytic effects,h) CM12 has greater antibacterial activities than positive control SC22 derived from BPI.