Analysis On Rab-GAP Activity And Evolution Characteristics Of TBC1D15

TBC1D15(TBC domain family,member 15)protein has a highly conserved TBC domain among different species and the key arginine residues that influence the activity of TBC1D15 are also highly conserved.Previous studies have indicated that TBC1D15 in mice and human have Rab-GAP activity,which is similar to other members of the TBC domain protein.However,TBC1D15 studies in Sharks were rarely reported.What's more,there are no related research on the evolution of Rab-GAP specificity of TBC1D15 and effects on Rab-GAP activity by changing some key amino acid residues.In this study,the TBC1D15 proteins of several different species(Shark,Sus,Mus,Homo)were selected to carry out prokaryotic expression and purification.The results showed that the expression level of TBC1D15 was low.Shark TBC1D15 protein in particular,was easy to break.Stable full-length protein was hard to obtain.Different GAP(GTPase activating protein)were acquired by truncating TBC1D15 protein sequences of four different species,which were the core sequence that decided Rab-GAP activity of TBC1D15.Sequence alignment software was used to analyze the conservation of amino acid sequence of TBC1D15-GAP among Shark,Sus,Mus and Homo.It was found that five sites of amino acid residues were arginine residues conserved in species Sus,Mus,Homo.However,they were replaced by the G28,K45,K119,K122,K221 residues in Shark-TBC1D15-GAP.The five different residues in Shark-TBC1D15-GAP were all mutated into arginine residues.Simultaneously,the arginine residues in GAP of other species were all mutated into G28,K45,K119,K122,K221 amino acid residues corresponding to the Shark.The relationship between the amino acid residues and TBC1D15 evolution was analyzed by studying the variation of Rab-GAP specificity before and after TBC1D15-GAP mutation in different species.We established the phylogenetic tree of the GAP sequences and the full length of TBC1D15 for four species.Results showed that the phylogenetic relationship of species Mus and Homo was closely related,while the genetic relationship between Shark and Sus,Sus and Homo were relatively far regardless of the GAP or the full length of TBC1D15.Therefore,TBC1D15-GAP of the three representative species Shark,Homo and Sus were selected to carry out the amino acid mutation and the analysis of and Rab-GAP specificity.High concentration and purity of proteins were obtained by prokaryotic expression and purification of pETduet-his-sumo-Shark/Sus/ Homo-TBC1B15-GAP and their mutants.Meanwhile,substrate Rab4/5/7/11 proteins were also expressed and purified.Dynamic experiments were carried out at first.The Rab-GAP activity of different species of TBC1D15-GAP were analyzed by calculating the rate of hydrolysis of GTP of Rab4/5/7/11 that catalyzed by different GAP proteins.It was shown that the Rab-GAP activity of different species of TBC1D15-GAP changed after the mutation of specific amino acid sites.Shark-TBC1D15-GAP is similar to wild type of Sus-TBC1D15-GAP after mutating the G28,K45,K119,K122,K221 residues into arginine residues.At the same time,Rab-GAP activity of Homo-TBC1D15-GAP after the specific amino acid mutation is also similar to wild type of Sus-TBC1D15-GAP.These results suggest that these specific amino acid sites play a certain role in the Rab-GAP activity of TBC1D15.Rab-GAP specificity of the six GAP proteins was further analyzed.the catalytic efficiency was compared to determine the effects of the mutated amino acid site on the activity of GAP protein by culculating the kcat/Km of the hydrolysis process.It was found that the catalytic efficiency of wild type of TBC1D15-GAP of Sus and Homo was similar in catalyzing Rab4/5/7/11.And both of them had the greatest substrate preference for Rab11.Nevertheless,wild type of Shark-TBC1D15-GAP had a large substrate preference for Rab5.It had a higher catalytic efficiency for Rab11 after the mutation,which was similar to wide type of Sus-TBC1D15-GAP,Homo-TBC1D15-GAP.All these results further showed that these amino acid sites might be related to the evolution of TBC1D15.This study preliminarily validated that the five specific amino acid sites might be the key amino acid residues during TBC1D15 evolution by Rab-GAP dynamics and specificity analysis of TBC1D15-GAPs and their mutants.With the continuous evolution of species,the substrate specificity of TBC1D15-GAP gradually preferred to Rab11.It might be related to the improvement of environmental adaptability in the process of species evolution.