Full-length cDNA cloning and cellular expression of the pepsinogen A and gastric proton pump genes of mandarin fish(Siniperca chuatsi)

Pepsinogens(PGs)are inactive precursors of pepsins,which convert to mature pepsins in gastric acidic environment.In this study,the complete cDNA sequence of PG A2 was obtained from the stomach of mandarin fish(Siniperca chuatsi)by Rapid Amplification of cDNA Ends(RACE).The full-length of PG A2 cDNA was 1 322 bp,which contained an open reading frame(ORF)of 1 131 bp encoding a peptide of 319 amino acids.Three regions were identified in PG A2 amino acid sequence:the signal peptide,the activation segment and the pepsin moiety.The pepsin moiety had two aspartates functioned as catalytic residues and three disulfide bonds.There were significant differences in sequence composition,physical and chemical properties,functional sites and spatial structure between pepsin A2 and pepsin A1 of mandarin fish,suggesting that they may have divergent functions.Gastric proton pump(gastric H⁺/K⁺-ATPase)is a key enzyme involved in the secretion of gastric acid.Both cDNAs of α and β subunits of the gastric proton pump were also isolated and cloned,with a length of 3 581 bp and 1 669 bp,respectively.High sequence conservation was revealed in α subunits which had multiple functional sites,while moderate variability was in β subunits.mRNA expressions of PG A1,PG A2 and α subunit of the gastric proton pump were detected in the same gastric gland cells of gastric mucosa using in situ hybridization,which implies these cells not only synthesized pepsinogens but also secreted hydrochloric acid,thus,they belonged to the oxynticopeptic cells.