Study On The Interactions Of Human Chemokine CCL11-Glycosaminoglycans-CCR3

The C-C motif chemokine 11(CCL11)is the main chemokine for the recruitment and mediation of eosinophils.It is related to asthma and other allergic inflammatory reactions,and there is a balance of monomers and oligomers in the organism.CCL11 interacts with C-C motif chemokine receptor 3(CCR3)and glycosaminoglycans(GAGs)on the surface of endothelial cells to regulate immune cells to reach the site of inflammation.However,the interaction process and mechanism of chemokine CCL11-glycosaminoglycan-CCR3 are not yet clear.In this paper,to explore the interaction process of CCL11-GAGs-CCR3,we conducted the following experiments:Firstly,in order to explore the interaction between CCL11-CCR3,the chemokines CCL11 and CCR3 expression system were constructed.The fluorescent protein MKO was introduced into the C-terminus of CCL11,and the p ET28a-ccl11-mko plasmid was constructed using genetic engineering technology.After inducing expression,the CCL11-MKO protein with a purity of more than 90% was obtained by nickel affinity chromatography.To further explore the interaction between CCL11 and CCR3,we constructed a pc DNA3.1-ccr3-egfp stably transfected cell line,screened cell lines that can perform single-molecule experiments,and proved that it has normal biological functions through chemotaxis experiments.Secondly,under the condition of in vitro solution,the CCL11-MKO protein at the singlemolecule level was fixed on the surface of the glass slide through pull-down by Total Internal Reflection Fluorescence(TIRF)technology,and the single-molecule fluorescence bleaching method was used.It directly proves that CCL11-MKO protein mainly exists in monomer under low p H conditions,and the aggregation behavior of CCL11-MKO protein is regulated by p H,and the degree of aggregation increases with the increase of p H.In order to further explore the influence of GAGs on the aggregation state of CCL11-MKO,this paper selected four GAGs:heparin sodium and chondroitin sulfate(number 1-3,increasing in length),through the Isothermal Titration Calorimetry(ITC)technique,it is concluded that several glycosaminoglycans can be combined with CCL11 well,and as the chain length of chondroitin sulfate increases,the binding exotherm increases.We mixed several GAGs with CCL11-MKO protein and tested the aggregation behavior of CCL11-MKO through single-molecule fluorescence experiments.The experimental results show that Chondroitin Sulfate No.1 has almost no effect on the aggregation state of CCL11-MKO,Chondroitin Sulfate No.2 maximizes CCL11-MKO aggregates at 10:1,and Chondroitin Sulfate No.3 is at 1:1.Its aggregation behavior has reached its peak,but the degree of aggregation increase is not obvious.Finally,in order to explore the interaction between CCL11-GAGs-CCR3,this paper carried out chemotaxis experiments and single-molecule imaging at the level of living cells,showing that different types and different proportions of GAGs will affect the interaction of CCL11 and CCR3.The addition of GAGs inhibited the chemotaxis of CCL11.The chemotaxis of heparin sodium and chondroitin sulfate No.2 with similar molecular weights was significantly reduced when the addition amount was 10:1 and 1:1;as the length of chondroitin sulfate increased,The inhibition of chemotaxis is also increasing.Similarly,the results of single-molecule experiments showed that the addition of GAGs inhibited the aggregation of CCR3-EGFP caused by CCL11.When CCL11 alone was present,the oligomeric state of CCR3-EGFP reached 33%.With the addition of heparin sodium,the ratio of CCR3-EGFP oligomers decreased,from 33% to 18% at 10:1.The addition of Chondroitin Sulfate No.1 has the same effect as heparin sodium.Chondroitin sulfate No.2 and No.3 had a significant inhibitory effect on the aggregation of CCR3-EGFP when the added amount was 1:1,and chondroitin sulfate No.3 had a more obvious effect than No.2.In summary,this thesis takes the CCL11-GAGs-CCR3 system as the research object,uses TIRF technology and chemotactic experiments to explore the aggregation of chemokine CCL11 and its interaction with GAGs and chemokine receptor CCR3.It is found that the presence of GAGs can inhibit the chemotaxis induced by CCL11 and the aggregation of CCR3,and the interaction between CCL11-GAGs-CCR3 has been clarified,which provides a certain theoretical basis for the treatment of inflammatory asthma-related diseases and drug design.