Function Of CXCL8 Homolog And Verification Of Relationship Between Receptor And The Ligand In Lamprey

Lamprey is considered to be one of the most ancient jawless vertebrates remaining,which connects invertebrates and vertebrates.Research on its immune system is significant in theory.Sea lamprey(Petromyzon marinus)is one of the representatives,whose complete geneome has been sequenced.Northeast Chinese lamprey(Lethenteron morii)distributes in northeast of China,which is closed related to P.marinus.CXCL8,also known as interleukin 8(IL-8),was reported to possess the ability to promote the processes of inflammatory reactions,stimulate the occurrence of blood vessels,promote mitosis,adjust the host's immune functions,etc.,by binding to its receptor CXCR1 in innate immunity.In this study,we identified CXCL8 from sea lamprey and Northeast Chinese lamprey,which were highly homologous to other animals utilizing homology cloning PCR approaches,named by PmCXCL8 and LmCXCL8 separately,and its receptor CXCR1 named by PmCXCR1 and LmCXCR1 separately.On this basis,we found the distribution of LmCXCL8 and LmCXCR1 in tissues of Northeast Chinese lamprey,then explored the factors of LmCXCL8 prokaryotic expression,and finally validated the chemotaxis of the recombinant protein.After the calcium fluxing experiments,we confirmed the relationship between LmCXCL8 and LmCXCR1.Firstly,we cloned the PmCXCL8,our results showed that the opening reading frame of pmcxcl8 gene was 309 nt,encoded 102 amino acids.The molecular weight of PmCXCL8 was 11.23 kDa,and the isoelectric point was 9.37.The hydropathy index of it ranges from-4.500 to 4.500.From the structure analysis of amino acid sequence,PmCXCL8 belonged to CXC chemokine subfamily,and the ELR motif(Gly-Gly-Arg)was replace by GGR(Gly-Gly-Arg)at its N-terminus.Recombinant PmCXCL8 showed a better expression in E.coli Rosetta(DE3)than in BL21 strain,was purified by Ni2+affinity chromatography column.SDS-PAGE and Western blot detections showed our recombinant PmCXCL8 was purely and correctly expressed in prokaryotic expression system.This study provides the opinions of function study and antibody preparation of PmCXCL8,and the research basis of sea lamprey innate immunity system.Then,we cloned the LmCXCL8,whose full-length cDNA consists of 1055 nucleotides(nt),including a 5'-untranslated region(UTR)of 142 nucleotides and a 3'-UTR of 604 nucleotides,and an open reading frame(ORF)of 309 nt encoding a polypeptide of 102 amino acid residues with a predicted molecular weight of 11.23 kDa.Molecular structure analysis showed that the LmCXCL8 possessed GGR(Gly-Gly-Arg)motif instead of the typical ELR(Glu-Leu-Arg)motif.Q-PCR results showed that the LmCXCL8 constitutively expressed in tissues,and that it also could be induced to express after Pseudomonas aeruginosa stimulation,indicating that the LmCXCL8 involved in the immune defense of L.morii.Chemotaxis assays showed that the the recombinant LmCXCL8 protein do have the activity to attract PBLs of lamprey,which first discovered the chemotactic activity caused by chemokine in Agnatha.Then we cloned the PmCXCR1 and LmCXCR1,their ORF were composed of 1088 nucleotides encoding a polypeptide of 360 amino acid residues.The identity of LmCXCR1 and PmCXCR1 amino acid sequences was 95.83%.Q-PCR results showed that the LmCXCR1 constitutively expressed in tissues and also can be induced to express after P.aeruginosa stimulation like LmCXCL8,which validated that LmCXCR1 involved in the innate immune defense as well.LmCXCR1 was successfully expressed in HEK293 T cells.Calcium fluxing experiments preliminarily confirmed that CXCR1 was the receptor of CXCL8 in Korean lamprey.This research helped us understand the function and evolution of CXCL8 in the lamprey immune system.It also provided some ideas for further research of innate immunity.