Research On Novel Biological Function Discovery Of Scorpion And Human Defensins And Their Interaction Mechanism

As an ancient molecular,defensins play an important role in innate and adaptive immunity system.In recent years,new progresses is being beyond the understanding of their biological functions.Such as,our group first reported defensins from fungi and human inhibited Kv1.3 channel current,and regulate the secretion of cytokines in T cells(Feng,Yang et al.2015,Xiang,Xie et al.2015,Xie,Feng et al.2015,Yang,Feng et al.2015).In view of defensins widely distributing in fungi,plant,invertebrate and vertebrate,this thesis focuses on new function and mechanism of the scorpion and human defensins.Scorpion,as the known "living fossil",completed the natural evolution from the sharp metasoma in sea scorpion to extant scorpion-like telson before more than 400 million years and further evolved into terrestrial animal.As the well known venomous animal,scorpion neurotoxin evolution always remains fascinating.In view of the venom gland evolutionary process and amino acid residues sequence similarity between scorpion defensin and classical scorpion neurotoxin,we infer that scorpion haemolymph and defensin ingredients likely play an important role in the molecular evolution of scorpion neurotoxin.Before investigating the relationship between scorpion defensin and scorpion neurotoxin,we first studied the antimicrobial function of scorpion defensin.Based on the six defensin genes from Buthus martensii,which were previously identified from our research team(Cao,Yu et al.2013),we successfully obtained three scorpion defensin BmKDfsin3,BmKDfsin4 and BmKDfsin5 through the prokaryotic expression and chemical synthesis technology.Assays were performed to detect their antimicrobial activity,and they were found to be antimicrobial peptides against several species of Gram-positive bacteria,such as the MICs of BmKDfsin3,BmKDfsin4 and BmKDfsin5 for S.sureus ATCC25923 were 2.5 ?M,0.08 ?M and 2.5?M,respectively.Scorpion defensins also showed the effective antibacterial activity against resistant strains,such as the MICs of BmKDfsin3,BmKDfsin4 and BmKDfsin5 for S.epidermis PRSE P1389 were 1.25 ?M,0.31 ?M and 1.25 ?M.In order to explore the antibacterial mechanism of scorpion defensin,we selected the peptide BmKDfsin4 against S.sureus AB94004 as the model.It was found that BmKDfsin4 could destory the cell structure integrity through plasmolysis,and eventually lead to cell death.These work showed that scorpion defensins BmKDfsin3,BmKDfsin4 and BmKDfsin5 had the typical function characteristics of antibacterial defensins,indicating that scorpion defensin play their inherent basic function in the survival and evolution of scorpion for hundreds of millions of years.Scorpion venom gland evolutionary process showed that neurotoxins were evolved after the emergence of defensins.In order to survive,the sharp metasoma of sea scorpion slowly evolved into telson of aquatic scorpion through continuous use.There is an obvious artery and vein in the front of extant scorpion telson.These evolutionary processes imply that scorpion haemolymph and defensin ingredients play an important role in the evolution of scorpion neurotoxin.In order to verify this hypothesis,we first investigated the inhibitory activity of scorpion haemolymph on potassium channel currents in mammalian cell.In line with our inference,electrophysiological experiments found that 1:10 diluted scorpion haemolymph could inhibit 45.7%,82.8%,86.1%and 52.8%of Kv1.1,Kv1.2,Kvl.3 and SK3 channel currents,respectively.These results showed that scorpion haemolymph contained potassium channel inhibitors.In view of the amino acid residue sequence similarity between scorpion defensins and classical scorpion neurotoxins,their structure and function correlations were investigated.on the one hand,the structure of BmKDfsin3 was analyzed by NMR technology,which was found to be similar to those of scorpion neurotoxin.Their structures composed of a helix and anti-parallel ? sheet and were stabilized by three pairs of disulfide bonds with the same pattern as toxins.On the other hand,the patch clamp experiments showed that scorpion defensin BmKDfsin3,BmKDfsin4 and BmKDfsin5 were blockers with differential potencies on the different potassium channels.Scorpion defensin BmKDfsin3 had different inhibitory activity on Kv1.1,Kv1.2,Kv1.3 and SK3.Concentration-dependent experiments indicated that BmKDfsin3 blocked Kv1.3 channel currents with IC50 value of 23.4 nM.Scorpion defensin BmKDfsin4 had all inhibitory activities on Kv 1.1,Kv 1.2 and Kv1.3.The IC50 value of BmKDfsin4 on Kv1.3 channel was 510.2 nM.BmKDfsin5 was only much potent on SK3 channel.1 ?M BmKDfsin5 can inhibit 46.3%of SK3 channel current.These results first showed that scorpion defensins can inhibit the neurotoxin-sensitive potassium channel current.Apart from the functional similarity between scorpion defensin and classical neurotoxin,we further investigated the mechanism of interactions between scorpion defensins and classical neurotoxin.Chimeric potassium channel showed that BmKDfsin3 inhibited Kv1.3 channel current through recognizing the pore region.Mutagenesis of potassium channel further showed that BmKDfsin4 also adopted the same mechanism of recognizing the pore region for interacting with Kvl.3.We found that the K13,R19 and R21 played an important role in inhibiting Kv1.3 channel current through alanine scanning of basic amino acid residues of BmKDfsin4.K13 was predicted to be the channel pore-blocking residue.These results showed that scorpion defensins adpoted the neurotoxin-identical model to block potassium channel current.Based on the analysis of the activity of scorpion haemolymph on potassium channel and the similarity of structure-function between scorpion defensins and neurotoxins,which provided the powerful experimental evidence for the viewpoint that defensins evolved into neurotoxins.In recent years,we found that human defensins interacted with Kvl.3 channel in T cell membrane(Xie,Feng et al.2015,Yang,Feng et al.2015),which indicated that defensins had more biological function than what we had known.In order to further explore the function of human defensins,we selected the chronic wound of diabetic foot as the model for primary research.We found that the expression of human endogenous human ? and ? defensins showed characteristic distribution in wound by investigating the clinical samples of patients with the diabetic foot.The expression of a defensins in the center of wound is higher than those in the border of wound,however,the expression of ? defensins in the center of wound is lower than those in the border of wound.Diabetic patients accumulate a lot of advanced glycosylation end products(AGEs)due to long-term high hyperglycemia in the body.Based on these phenomena,we found that HNP1 and HNP4,which highly expressed in the center wound of diabetic foot,with AGE-BSA synergistically promoted the secretion of proinflammatory cytokines IL8 in fibroblast,but only HNP1 and HNP4 did not.This phenomenon suggested that human a defensins worked against bacteria and exacerbated the inflammatory response through interaction with the fibroblast in diabetic foot wound under the pathological condition.And excessive inflammation further leaded to difficult wound healing.In order to explore the mechanism of human a defensins promoting the IL8 secretion of fibroblast,we found that HNP1 and AGE-BSA regulated the secretion of inflammationary cytokine IL8 by promoting NF-?B-p65(Ser536)phosphorylation.These work not only found the specific distribution and pathological function of human defensin in diabetic foot wound,but also improved the understanding of the immune function of defensins in pathological conditions.In summary,this paper carried out the research on the new biological function and mechanism of scorpion and human defensins,on the one hand,it provided the experimental evidence for the molecular evolution of scorpion neurotoxins,on the other hand,it further revealed the mechanism of chronic wound difficult to heal in patients with diabetic foot.These work laid the foundation for further understanding and application of the biological functions of different types of animal defensins.