Domestic Cat Angiotension Converting Enzyme 2 CDNA: Cloning, Expression, Bioinformatic Analysis And Tissue Distribution Study

Angiotensin converting enzyme 2 (ACE2), a metalloproteinase, is present in heart, lung, liver, kidney, testicle and gastrointestinal system. ACE2 plays a key role in cardio-renal functions, and it has also been implicated in the pathogenesis of diabetic complications. Recently, ACE2 was demonstrated to be the functional receptor of SARS-CoV (severe acute respiratory syndrome associated coronavirus, SARS-CoV).Spike protein of SARS-CoV mediates viral infection of their target cells by binding to the cellular receptor ACE2. Beside humans, rhesus monkeys, civets, ferrets and domestic cats have been confirmed to be susceptible to SARS-CoV. Furthermore, the ACE2 of mice, rat and civet can be utilized by SARS-CoV as a low efficiency receptor. Some evidence indicated that many coronavirus in Group I and Group III could infect target cells by using the feline aminopeptidase N as the common receptor, suggesting domestic cats may play a key role in the process of coronavirus interspecies infection and in the evolution of the novel coronavirus SARS-CoV.Because ACE2 is a novel protein identified by the year 2000, there are only limited data about human and mouse ACE2 availabe and there was no report on cat ACE2 at the beginning of this study. Therefore this study was designed to identify and characterize the cat ACE2. In this study, demestic cat ACE2 was cloned and expressed, and a series of bioinformatics analyses were performed to predict the structure and function of this protein. The tissue distribution of ACE2 prtoten in domestic cats was furhter investigated.A domestic cat was sacrified and the lung was used to extract total RNA. The cDNA was synthesized by reverse transcription. Four overlapped mRNA fragments were amplified by using the cDNA as the templates and spiced. The ACE2 mRNA coding region consists of 2418 nucleotides encoding a putative protein of 805 amino acids. Alignment analysis demonstrated that cat ACE2 has a similarity of 86%, 84% and 84% at nucleic acid level, and 85%, 81% and 81% at the amino acid level with human, mouseand rat ACE2, respectively.A series of bioinformatics analyses were performed to predict the structure and function of this protein, including prediction of physical and chemical properties, the signal peptide and transmembrane helices, the secondary structure, motif, domain and protein family. Furthermore, compared to other animal's (civet, mouse, rat) ACE2, the cat ACE2 sequences in the three functional domains of ACE2-SARS-CoVinteraction have the highest similarity with human ACE2, suggesting cats may play an important role in SARS-CoV interspecies infection.The tissue distribution of ACE2 in domestic cats detected by RT-PCR was similar to that of human ACE2.Prokaryotic expressing plasmid pET-ACE2 was constructed by inserting ACE2 into prokaryotic expressing vector pET-28(a+). ACE2 protein was expressed efficiently in E.coli with molecular weight about 85kD, and this expressed protein could cross-react with anti-human ACE2 antibody as demonstrated by westen blot analysis.Taken together, these results confirmed that the cloned sequence is the coding sequence of domestic cat ACE2. The expressed ACE2 protein shares similar antigenic characteristics of human ACE2. It is the first report of cat ACE2. Further investigation will be of significance to elucidate the role of cats in the interspecies infection of SARS-CoV and other coronaviruses.