Molecular Cloning of cDNA Encoding an Aquaglyceroporin, AQP-h9, in the Japanese Tree Frog, Hyla japonica: Possible Roles of AQP-h9 in Freeze Tolerance

In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which...

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Published inZoological science Vol. 32; no. 3; p. 296
Main Authors Hirota, Atsushi, Takiya, Yu, Sakamoto, Joe, Shiojiri, Nobuyoshi, Suzuki, Masakazu, Tanaka, Shigeyasu, Okada, Reiko
Format Journal Article
LanguageEnglish
Published Japan 01.06.2015
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Abstract In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevisoocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation,confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serumand tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed afterfreezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernatingfrogs. Glycerol content in the liver did not change in the freezing experiment, whereas thatin the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a c;:ryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.
AbstractList In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence ofH. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conservedAsn-Pro-Aia motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevisoocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation,confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serumand tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed afterfreezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernatingfrogs. Glycerol content in the liver did not change in the freezing experiment, whereas thatin the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a c;:ryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.
Author Shiojiri, Nobuyoshi
Tanaka, Shigeyasu
Sakamoto, Joe
Takiya, Yu
Suzuki, Masakazu
Hirota, Atsushi
Okada, Reiko
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Snippet In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, wecloned a eDNA encoding aquaporin (AQP) 9 from its liver. The...
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StartPage 296
SubjectTerms Amino Acid Sequence
Animals
Anura - genetics
Aquaglyceroporins - genetics
Aquaglyceroporins - metabolism
Base Sequence
Cloning, Molecular
DNA, Complementary - genetics
Freezing
Gene Expression Regulation - physiology
Hibernation
Molecular Sequence Data
Phylogeny
RNA, Messenger - genetics
RNA, Messenger - metabolism
Title Molecular Cloning of cDNA Encoding an Aquaglyceroporin, AQP-h9, in the Japanese Tree Frog, Hyla japonica: Possible Roles of AQP-h9 in Freeze Tolerance
URI https://www.ncbi.nlm.nih.gov/pubmed/26402924
Volume 32
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