| Pepsins, with responsibility for the preliminary digestion of proteins in the stomach, are important gastric digestive proteinases belonging to a family of aspartic proteinases. They are synthesized in the gastric mucosa of stomach as inactive pepsinogens, which are converted to mature pepsins in the acidic environment of stomach created by chlorhydric acid. Gastric proton pump, a key enzyme involved in the secretion of chlorhydric acid, also known as gastric H~+/K~+-ATPase, is a heterodimer composed ofαandβsubunits. Theαsubunit is responsible for the catalysis and transportation of H~+ and K~+, while theβsubunit stabilizes theαsubunit and plays an auxiliary role in gastric acid secretion. So the pepsins and hydrochloric acid secreted by the gastric proton pump together constitute the acidic digestion of proteins in the stomach. During the early ontogeny of fish, the differentiation of gastric glands and the secretion of pepsinogens and chlorhydric acid are considered as events of great significance. The former indicates the completion of morphological development of the stomach, while the latter signifies the functional maturation of the stomach. Therefore, the expression of pepsinogen and proton pump genes could be used as a molecular indicator of stomach differentiation and functionality.In this study, the full-length cDNA of bothαandβsubunits of the gastric proton pump were isolated and cloned by RT-PCR (Reverse transcription-polymerase chain reaction) and RACE (Rapid amplification of cDNA ends) from mandarin fish firstly. Theαsubunit of gastric proton pump was 3581 bp in length, which comprised a 178 bp 5'UTR, a 3069 bp ORF encoding 1022 amino acid residues and a 334 bp 3'UTR. The full-length ofβsubunit was 1669 bp, including a 5'UTR of 220 bp, a 3'UTR of 573 bp, and an ORF of 876 bp encoding a polypeptide of 291 amino acid residues. Amino acids sequences analysis showed that theαsubunit of gastric proton pump possessed 10 transmembrane domains, a E1-E2 type ATPase phosphorylation site (DKTGTLT) at the position of 374 - 380 amino acid residues, as well as a lysine/glycine cluster structure (KKKKKKKMKKK) at the position of 18 - 28 amino acid residues, which was the conservative domain ofαsubunit with high homology among different species. While theβsubunit formed only one transmembrane domain, and contained seven potential glycosylation sites and three disulfide bonds. Sequences alignment and similarity analysis revealed high sequence conservation ofαsubunits and moderate variability ofβsubunits among vertebrates.To further explore the temporal and spatial expression pattern of pepsinogens and gastric proton pump, as well as their mutual synergistic action during early ontogeny of mandarin fish, specific primers and specific digoxin-labelled RNA probes was designed based on sequences of three pepsinogens (PG A1, PG A2, PG C) of mandarin fish obtained in our laboratory before andα,βsubunits of the gastric proton pump identified in this study for RT-PCR and in situ hybridization (ISH) analysis in mandarin fish. The RT-PCR analysis revealed that the expression of pepsinogen and proton pump genes were sequential instead of simultaneous along the development of the stomach (0 - 40 dph, days post-hatch). The weak expression of pepsinogen A1 was detected as early as 4 dph, before the appearance of gastric glands; whereas transcripts of pepsinogen A2 andα,βsubunits of gastric proton pump were not detected until 12 dph; transcripts of pepsinogen C arose at 14 dph finally, coinciding with the appearance of gastric gland cells. The ISH study located the gene expression of all three pepsinogens andαsubunit of gastric proton pump in the same gastric gland cells in gastric mucous membrane of adult fish and larvae or juveniles of every stage. These proved that the gastric gland cells of mandarin fish were oxynticopeptic cells, which could secret both pepsinogens and chlorhydric acid simultaneously. The simultaneously significant expression of three pepsinogen and gastric proton pump genes at 12 - 14 dph, together with their expression in the same oxynticopeptic cells, showed the temporally and spatially synergistic action between pepsinogens and gastric proton pump. In addition, no oxynticopeptic cell was found in the gastric mucous membrane of larvae before 10 dph, the oxynticopeptic cells appeared in the gastric mucous membrane and their number begun to increase from 14 dph to 40 dph gradually. The HE (Hematoxylin and Eosin) staining and ISH also identified two other kinds of cells except oxynticopeptic cells, namely surface mucous cells and mucous neck cells in the gastric mucous membrane of mandarin fish, but neither surface mucous cells nor mucous neck cells contained transcripts of pepsinogens or gastric proton pump.In conclusion, this study obtained the full-length cDNAs of bothαandβsubunits of gastric proton pump from mandarin fish, the sequences characters were analyzed; sequential expression of three pepsinogens and gastric proton pump, as well as their mutual synergistic action during early ontogeny of mandarin fish were revealed; moreover, the cellular expression localization of pepsinogen and gastric proton pump genes was identified and the gastric gland cells of mandarin fish were determined to be oxynticopeptic cells. These results have enriched the knowledge of fish digestion physiology and may provide information for a better understanding of the development process of larval digestive system and establishing preferable rearing methods to improve larval culture. |
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