Coevolution And Function Of FKN/CX3CR1 In Higher Primates

FKN/CX3CR1 plays a critical role in the process of trafficking white blood cells, inflammation and development of immune cells as well as allograft transplantation. In the past decades there are some progress in studying the structure and function of FKN/CX3CR1 based on the approach of biochemistry. However, few comprehensive investigations on the structure basis of the function FKN/CX3CR1 mediated have been conducted till now. In the present study, we explored the roles of possible negative selection sites, Darwin positive selection sites and deletion/insertion sites in the immunological functions by using comparative genomics study of FKN/CX3CR1 in higher primates and firstly fully elucidated the relation between the structure and function, which may provide a new strategy for studying the structure and function between chemokines and their receptors.1. The comparative genomics study of FKN/CX3CR1 in primatesAll available full-length FKN/CX3CR1 sequences of Homonid (including human chimpanzee, gorilla, orangutan and gibbon) and Old World Monkey (including macaque and leaf monkey) obtained by PCR were aligned using MegAlign. The negative selection sites were analyzed by using Datamonkey software. The phylogenetic trees were constructed using maximum evolution approach in MEGA 3. AA p-distance for each branch of FKN/CX3CR1 gene phylogeny and maximum likelihood analyses of substitution rates were estimated by codeml in PAML. Pearson's correlation coefficient values were calculated by using the statistic software SPSS. The correlation is significant when comparing pairwise distances between FKN and CX3CR1 from 7 primates at 99% level (r=0.636, p<0.01) which suggests that coevolution occurs between FKN and CX3CR1 in 7 higher primates. In addition, we discovered that 30Q, 61D, 216N are negative selection sites in FKN and 4F, 259Y in CX3CR1 in 7 primates using Datamonkey database. Besides, we deduced A33, I41, D53, K55, K60, R68 and E86 in human and chimpanzee FKN, and K95, D270 in human and chimpanzee CX3CR1 as possibleDarwinian positive selection sites by using PAML and discovered an apparent 30 bp nucleotides deletion mutation in Homonid FKN by MegAlign which possible maintain FKN/CX3CR1 gene coevolution from OWM to homonid.2. Molecular cloning and expression of FKN/CX3CR1 and their mutated counterpartsTo study the evolutionary rule in function from Old World Monkey to Homonid and the role of negative selection sites, possible Darwinian positive selection sites and deletion mutation, full-length FKN/CX3CR1 and mutant gene were obtained by overlapping extension PCR and site-directed mutagenesis. The target gene were inserted into pcDNA.3.1/myc-His(-)A eukaryotic vectors and then transfected into CHO cells via liposome after enzyme digesting and sequencing. The gene expression was analyzed with RT-PCR, SDS-PAGE and Western blot. Enzyme digestion and sequence analysis showed that the DNA fragments inserted into eukaryotic vectors were correct full-length FKN/CX3CR1 gene and the target gene could be amplified from CHO cells transfected with recombinant vectors by RT-PCR. Expressed protein in CHO cells could combine with myc antibody specifically and FKN could also be secreted into supernatant. SDS-PAGE and Western blot showed the appearance of 95kD band of FKN and 52kD band of CX3CR1. The results showed that the recombinant eukaryotic vectors with FKN/CX3CR1 and mutants were constructed successfully.3. Comparative functional study of FKN/CX3CR1 in higher primatesTo observe FKN/CX3CR1 evolution rules in immunological function among higher primates and confirm that FKN/CX3CR1 does occur gene coevolution from OWM to homonid, we compared that FKN binding activity, chemotaxis and adhesion to CX3CR1 -expressing cells among 7 primates respectively and crossly. It is showed that the function up-regulation step by step as a whole from OWM to homonid, but a temporary reduction in the gibbon, and any cross FKN/CX3CR1 interaction in different primates all caused a great reduction in function, including binding activity, chemotaxis and adhesion, which strongly suggests that the coevolution occurs between FKN and CX3CR1 in higher primates.4. Functional study of the negative selection sites, possible Darwinian positiveselection sites and deletion mutants The aim of this study was to observe the role of negative selection sites,possible Darwinian positive selection sites and deletion fragments in FKN/CX3CR1 binding affinity, chemotaxis and adhesion. Targeted mutation of the negative selection sites 30Q and 61D in FKN caused a reduction in binding affinity and chemotaxis, but no change in adhesion, and point mutation of FKN negative selection site 216N caused a great reduction in adhesion, but no binding affinity and chemotaxis. In contrast, targeted mutation of the negative selection sites 4F and 259Y in CX3CR1 caused all reduction in binding affinity, chemotaxis and in adhesion. Besides, point mutation of the possible Darwinian positive selection sites A33,141, D53, K55, K60, R68 and E86 in human FKN respectively shows that D53, K55, K60, R68 caused a reduction in binding affinity and chemotaxis, but no change in adhesion, and E86 reduction in adhesion, but no change in binding affinity and chemotaxis. In the same time, we found A33 and 141 caused no change in binding affinity, chemotaxis and adhesion. Furthermore, point mutation of the possible Darwinian positive selection sites K95 and D270 in human CX3CR1 respectively shows that they both caused a reduction in binding affinity, chemotaxis and adhesion and the deletion 10 amino acids of FKN in homonid caused no change in binding affinity and chemotaxis, but a great reduction in adhesion.In summary, FKN and CX3CR1 co-evolve in higher primates and there are some negative selection sites, a few Darwinian positive selection sites both in FKN and CX3CR1 and a deletion fragment in homonid FKN in the gene evolutionary process. And these sites play different roles in FKN/CX3CR1 binding affinity, chemotaxis and adhesion.