东北林蛙抗菌肽dybowskin-1ST的结构预测及生物学活性分析

Structure prediction and biological activity analysis of dybowskin-1ST antimicrobial peptide in Rana dybowskii

运用生物信息学方法进行东北林蛙抗菌肽dybowskin-1ST的进化、结构及抗原表位预测,分析其抑菌机理及结构性质,应用小鼠伤口愈合实验及体外抑菌实验进行活性验证。同时为改良亲本肽、进行新型衍生肽的研发提供理论基础。使用软件MEGAX对dybowskin-1ST及其他蛙类抗菌肽进行同源性比对并绘制系统进化树;通过在线软件ProtParam、ProtScale、PeptideCutter、SignalP、TMHMM Server分别预测抗菌肽dybowskin-1ST的理化参数、亲/疏水性、剪切位点、信号肽及跨膜区域;分别应用SOPMA、Jpred4及DNAstar Protean软件多重分析预测dybowskin-1ST的二级结构,利用SWISS-MODEL和I-TASSER软件进行三级结构预测。通过在线软件ABCpred和SYFPEITHI进行T/B抗原表位预测。构建小鼠伤口模型,观察dybowskin-1ST促进伤口愈合活性。应用纸片法及96孔板法,确定dybowskin-1ST的抑菌活性。抗菌肽dybowskin-1ST含有59个氨基酸,其中亮氨酸占16.9%,分子式为C_(318)H_(510)N_(80)O_(93)S_(2),理论等电点为5.10,电荷量为-2。抗菌肽dybowskin-1ST与东北林蛙抗菌肽dybowskin-1CDYa亲缘较近。三种方法二级结构预测结果相似,dybowskin-1ST中α-螺旋、延伸链、β-转角、无规卷曲,所占比例分别为44.07%、16.95%、3.39%、35.39%。三级结构预测中显示该抗菌肽大部分为α-螺旋结构,抗菌肽dybowskin-1ST总体预测为亲水性蛋白,具有信号肽序列。亚细胞定位分析显示,其分泌线粒体靶向肽的可能性为0.944。该蛋白属于膜外蛋白,无跨膜结构区,有7个磷酸化位点,3个T细胞抗原表位和8个B细胞抗原表位。dybowskin-1ST具有促进伤口愈合的作用,能够有效抑制大肠杆菌和金黄色葡萄球菌活性,但对真菌及耐药菌的抑菌活性有限。dybowskin-1ST结构中虽富含α-螺旋,但验证实验表明其抑菌能力仍有待加强,原因可能是由于其带负电荷且为亲水性蛋白,以提高正电荷数及改变疏水性为基本思路进行氨基酸改造可获得活性升级的衍生肽。

The evolution,structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1 ST were carried out using bioinformatics software available online.Its antibacterial mechanism and structural properties were analyzed,and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro.This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides.The software MEGAX were used to conduct homology alignment and to construct a phylogenetic tree.The online software ProtParam,ProtScale,PeptideCutter,signal,TMHMM Server were respectively used to predict the physicochemical parameters,hydrophilia/hydrophobicity,shear sites,signal peptides,and transmembrane domains of dybowskin-1 ST.The online software SOPMA,Jpred4,DNAstar Protean were used to predict the secondary structure of dybowskin-1 ST,and SWISS-MODEL,I-TASSER were used to predict the tertiary structure.ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes.The effect of dybowskin-1 ST on the wound healing was observed on experimental mice.Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1 ST.The dybowskin-1 ST consists of 59 amino acid residues,of which leucine accounts for 16.9%,with a molecular formula of C_(318)H_(510)N_(80)O_(93)S_(2).Its theoretical isoelectric point is 5.10 and the charge is-2.The dybowskin-1 ST and dybowskin-1 CDYa are closely related phylogenetically.The secondary structure of dybowskin-1 ST predicted by the three methods were similar,which consisted ofα-helix(44.07%),extended strand(16.95%),β-turns(3.39%),and random coil(35.39%).The prediction of tertiary structure showed that dybowskin-1 ST was mainly composed ofα-helix,and it was regarded as a hydrophilic protein with signal peptide sequence.Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944.Dybowskin-1 ST is an extracellular protein with no transmembrane structure region,but contains seven phosphorylation sites,three T-cell epitopes and eight B-cell epitopes.The dybowskin-1 ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus.However,it had limited antibacterial activity against fungi and drug-resistant bacteria.Although the structure of dybowskin-1 ST is rich inα-helix,the verification experiments showed that its antibacterial ability needs to be enhanced.The reason may be that it is a negatively charged and hydrophilic protein,and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.