MOLECULAR CLONING AND CHARACTERIZATION OF FAS-ASSOCIATED DEATH DOMAIN (FADD) FROM BRANCHIOSTOMA BELCHERI

FADD(Fas-associated death domain protein),also termed MORT1,plays a critical role in the apoptotic signaling pathways of both CD95(Fas/APO-1) and certain members of the tumor necrosis factor receptor(TNFR) superfamily,and is well conserved in vertebrates,especially in mammals.To reveal the features of FADD in lower animals,we have characterized FADD from amphioxus,Branchiostoma belcheri(Chordata,Cephalochordata),which is a model animal,and have an insight into the origin and evolution of apoptotic signaling system of the vertebrate.Branchiostoma belcheri FADD(bbFADD) cDNA and genomic DNA sequences were obtained by using RACE-PCR and Genomic Walking,respectively.The full-length cDNA of bbFADD consisted of 1239 base pairs(bp) encoding 217 amino acid residues(aa) with a death effector domain(DED)(12— 95th;84 aa) near the N-terminal and a death domain(DD)(129— 211st;83 aa) by the C-terminal.bbFADD genomic sequence,the length of which was 2840 bp,consisted of three exons and two introns in the gene coding region.The structure of bbFADD gene was different from that of its vertebrate counterparts,with two exons.The result of multiple alignments among FADDs from various species supported that DED and DD regions were more conservative than other regions.Furthermore,the 33rd phenylalanine residue(F33) of bbFADD amino acid sequence,which was equivalent to the 25th phenylalanine residue(F25) in human FADD.This site,which was crucial to mediate FADD self-association,was well conserved through sea urchin and human.Homology analysis showed that the percent identity and the percent similarity between bbFADD and vertebrate FADDs were 27.5%— 30.0% and 44.7%— 55.3%,while 35.1%— 36.9% and 51.1%— 52.0% between bbFADD and sea urchin FADD,respectively.In NJ phylogenetic tree based on amino acid sequences of FADDs,B.belcheri was grouped with sea urchin with the suggestion that comparing with lower vertebrate,fish,amphioxus was closely relative to sea urchin although amphioxus.However,generally amphioxus was regarded as the evolutional intermediate stage between invertebrate and vertebrate.In order to examine whether bbFADD was functionally conserved,bbFADD was transfected into HeLa cells by using the expression vector pEGFP-N3-bbFADD,which was constructed in the present study.24 hours post-transfection,over-expression of bbFADD resulted in cell shrinkage and DNA fragmentation.In addition,DNA content of the transfected cells were detected by flow cytometry.12.6% cells in pEGFP-N3-bbFADD transfected HeLa cells underwent apoptosis while 0.35% in the control.All data suggested that bbFADD might participate in the human apoptotic signaling pathway and induced the apoptosis of human HeLa cells.Therefore,it was speculated that the apoptosis signaling system was conserved during the evolution course through amphioxus to mammal.