Evolution And Functional Analysis Of CXC Chemokines And Receptors In Lamprey

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense.They interact with chemokine receptors and are indispensable for coordination of cell migration in diverse physiological processes.The CXC subfamily is one of the largest groups of chemokine family and consists of multiple members.Lamprey is one of the extant jawless vertebrates and serves as an excellent model for study on the evolution of immune system.In this study,we identified homologues of three chemokine ligands(CXCL8,CXC_F5 and CXCL12)and two CXCRs(CXCR_L1 and CXCR_L2)in Lamprey.Using bioinformatics methods to analyze their molecular structure,phylogenetic relationship and gene synteny,etc.Real-time fluorescent quantitative PCR(q PCR)was used to analyze their expression in various tissues of healthy Northeast China lamprey(Lampetra morii).Expression of CXCLs and CXCRs in Northeast China lamprey after infection with Vibrio anguillarum and Staphylococcus aureus.Expression analysis of CXCLs and CXCRs in Japanese lamprey(Lampetra japonica)leukocyte after in vitro stimulation with Lipopolysaccharide(LPS),Polyinosinic acid-polycytidylic acid(poly I:C)and pokeweed mitogen(PWM).Construction of eukaryotic expression vectors to obtain recombinant CXCLs proteins and CXCRs cells for chemotaxis assay.(1)The three chemokine ligands(CXCL8,CXC_F5 and CXCL12)full length CDS of the Northeast China lamprey and the full length c DNA of the Japanese lamprey were cloned.Sequence analysis revealed that lamprey CXCL8,CXCL_F5 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues and contains multiple cationic aa residues in the extended C terminal region.Phylogenetic trees were constructed to infer the homologous relationships of lamprey CXCLs with members of CXCL family.CXCL8,CXCL_F5 and CXCL12 were clustered together with homologous proteins from other fish.Genomic organization and synteny analysis found they share the same genomic organizations with their counterparts in jawed vertebrates but no conservation in synteny.The three CXCLs displayed a helical turn at the N terminus,followed by three ?-sheets.The overall topology of the N-terminal helix of CXCL8 and CXCL_F5 is more similar to that of human CXCL8 than that of human CXCL9,but differ significantly with CXCL12 where notably the N terminal helical turn has a large distance with the three ?-sheets(2)The two CXCRs(CXCR_L1 and CXCR_L2)full length CDS of the Northeast China lamprey and the full length c DNA of the Japanese lamprey were cloned.Sequence analysis revealed that the two CXCRs possess seven transmembrane domains,a conserved DRY domain in the second intracellular region and multiple acidic aa residues in the N terminal region.In the tree rooted with the RXFP3 sequences,CXCR_L1 and CXCR_L2 were clustered together and formed a large clade consisting of CXCR1-5 and ACKR4.Genomic organization and synteny analysis found they share the same genomic organizations with their counterparts in jawed vertebrates but no conservation in synteny.The two CXCRs contain 7 well defined helices forming hydrophobic transmembrane domains.The predicted structure of CXCR_L1 is remarkably similar to that of human CXCR1.Curiously,the CXCR_L2 is structurally more similar to human CXCR3 than CXCR1.(3)The CXC chemokines and receptors were constitutively expressed in tissues including liver,kidney,intestine,heart,gills,supraneural body and primary leukocytes but exhibited distinct expression patterns.The highest levels of expression of CXCL8,CXCL_F5 and CXCR_L1 were found in gills whilst the highest levels of expression of CXCL12 and CXCR_L2 observed in supraneural body.The lowest expression of CXCL8,CXCL_F5 and Lm CXCR_L2 was found in the liver,and in contrast heart exhibited the lowest expression for CXCL12 and CXCR_L1.In blood leukocytes,the expression of CXCL8 was the highest,followed by CXCR_L1,CXCR_L2,CXCL12 and CXCL_F5.(4)The modulation of CXCLs and CXCRs expression of Northeast China lamprey in response to V.anguillarum and S.aureus infection were investigated.The CXCL8 and CXCL_F5 were significantly up-regulated in the kidney and intestine after each stimulants,but the degree of change was weak in the intestine.The expression of CXCL12,CXCR_L1 and CXCR_L2 was significantly up-regulated only after stimulation by V.anguillarum,and the degree of change was weak.The expression of CXCLs and CXCRs was investigated in primary leukocytes of Japanese lamprey in response to PAMPs such as LPS,poly I:C and pokeweed.The CXCL8 and CXCL_F5 were induced after stimulation.The CXCL12 expression was upregulated by LPS and PWM at 12 h whilst remained unchanged at other stimulation conditions.The CXCR_L1 and CXCR_L2 expression were increased in cells treated with LPS and PWM.Treatment of poly I:C resulted in inhibition of expression of both CXCR_L1 and CXCR_L2.(5)Chemotaxis assay analysis found,CXCL8 enhanced migration of HEK293 T cells expressing CXCR_L1 but had no effect on the migration of cells expressing CXCR_L2.Conversely,CXCL_F5 was stimulatory on the migration of cells transfected with CXCR_L2 but not CXCR_L1.Taken together,the results demonstrate that CXCL8 and CXCL_F5 specifically interact with CXCR_L1 and CXCR_L2,respectively.In conclusion,we identified three CXCLs and two CXCRs in lamprey,suggesting that multiple CXC chemokines have diverged in jawless fish.Sequence analysis revealed that they share the same genomic organizations with their counterparts in jawed vertebrates but no conservation in synteny.This may be due to the significant revamping of the genome caused by the second round WGD event occurred.The CXC chemokines and receptors respond differently to stimulation of PAMPs and bacterial infection.PAMPs are ligands of pattern recognition receptors,indicating that they can be activated via PRR mediated signal pathways.In vivo,they were regulated after infection with bacteria,confirming their involvement in inflammation.The CXCL8 and CXCL_F5 specifically interact with CXCR_L1 and CXCR_L2,respectively.The C terminal region of chemokines is critical for the specificity of interactions with the N terminal region of their receptors.Low sequence homology and differences in aa charge are apparent between these regions in CXCL8 and CXCL_F5 and between CXCR_L1 and CXCR_L2,which may contribute to binding specificity.