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Physiol Rep
2017 Aug 01;515:. doi: 10.14814/phy2.13331.
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Expression of the aquaglyceroporin HC-9 in a freeze-tolerant amphibian that accumulates glycerol seasonally.
Stogsdill B
,
Frisbie J
,
Krane CM
,
Goldstein DL
.
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As ambient temperatures fall in the autumn, freeze-tolerant Cope's gray treefrogs, Dryophytes chrysoscelis (formerly Hyla chrysoscelis), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP9 and that AQP9 expression is upregulated in the cold to facilitate glycerol transport. We sequenced 1790 bp from cloned cDNA that codes for a 315 amino acid protein, HC-9, containing the predicted six transmembrane spanning domains, two Asn-Pro-Ala (NPA) motifs, and five amino acid residues characteristic of aquaglyceroporins. Functional characterization after heterologous expression of HC-9 cRNA in Xenopus laevis oocytes indicated that HC-9 facilitates glycerol and water permeability and is partially inhibited by 0.5 mmol/L phloretin or 0.3 mmol/L HgCl2 HC-9 mRNA (qPCR) and protein (immunoblot) were expressed in most treefrog tissues analyzed (muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin) except the protein fraction of red blood cells. Contrary to our prediction, both mRNA and protein expression were either unchanged or downregulated in most tissues in response to cold, freezing, and thawing. A notable exception to that pattern occurred in liver, where protein expression was significantly elevated in frozen (~4-fold over warm) and thawed (~6-fold over warm) conditions. Immunofluorescence labeling of HC-9 protein revealed a signal that appeared to be localized to the plasma membrane of hepatocytes. Our results indicate that gray treefrogs express an AQP9-like protein that facilitates glycerol permeability. Both the transcriptional and translational levels of HC-9 change in response to thermal challenges, with a unique increase in liver during freezing and thawing.
Figure 1. Nucleotide and Protein Sequence of Dryophytes chrysoscelis HC‐9: (A) The nucleotide sequence of HC‐9 from D. chrysoscelis is shown with the translated (boxed) and untranslated (unboxed) regions displayed. The predicted amino acid sequence is shown above the nucleotide sequence. (B) The protein secondary structure topography was predicted using Phobius and generated using Protter (http://wlab.ethz.ch/protter/). Includes the predicted location of the NPA motifs, the N‐linked glycosylation site, and five conserved aquaglyceroporin amino acids.
Figure 2. Comparison of AQP1, AQP2, AQP3, and AQP9 in Dryophytes chrysoscelis Based on the Amino Acid Sequence: An unrooted tree of aquaporins and aquaglyceroporins identifies all aquaporins currently published from the D. chrysoscelis (HC‐1, HC‐2, HC‐3, and HC‐9). The protein sequences from Dryophytes chrysoscelis are compared to the protein sequence of Dryophytes japonicus (Japanese Treefrog), Xenopus laevis (African Clawed Frog), Xenopus tropicalis (Western Clawed Frog), Mus musculus (house mouse), and Homo sapiens (humans). The scale represents genetic change as amino acid substitutions per site. The sequence is aligned and generated using Geneious version 10.0.9 (http://www.geneious.com, Kearse et al. 2012).
Figure 3. Comparative Real‐time Expression of HC‐9 mRNA in the Dryophytes chrysoscelis: HC‐9 mRNA expression is shown for muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin of the D. chrysoscelis. Real‐time PCR of selected tissues from warm‐acclimated, cold‐acclimated, frozen, and thawed treefrog tissue were measured (n = 5). The standard mean of 2−ΔΔCt is graphed with ±SEM. * indicates a condition with a P < 0.05 compared by one‐way ANOVA to data from the warm‐acclimated condition.
Figure 4. Protein Expression by Immunoblot for Various Tissues of Dryophytes chrysoscelis: (A) Immunoblots from muscle, liver, bladder, stomach, kidney, dorsal skin, ventral skin, and red blood cells are shown. A band just under the 34 kDa marker is observed in all tissues except the red blood cells. Other bands appear in various tissues, including a 55–95 kDa band that shows strongly in the liver and ventral skin. (B) Densitometry of immunoblot including all immunoreactive bands normalized to βactin and taken as a ratio of arbitrary units over the average warm‐acclimated tissue (mean fold change). Error bars are ±SEM. * indicates a condition with a P < 0.05 compared by one‐way ANOVA to data from the warm‐acclimated condition.
Figure 5. Detailed Liver Protein Expression from Immunoblots of Dryophytes chrysoscelis Including Treatment with PNGase F: (A) The mean measurement of the individual bands in the liver are shown by densitometry (n = 3). Liver showed three distinct bands. While the 33.69 kDa band did not change between warm‐acclimated, cold‐acclimated, frozen, and thawed, the other bands increase by 4‐ to 6‐fold. The error bar is ±SEM. (B) An immunoblot of the liver before and after treatment with PNGase F, a deglycosylating enzyme, across warm‐acclimated, cold‐acclimated, frozen, and thawed treefrog tissue. A band that appears around 72 kDa is reduced in intensity while a band around 45 kDa increases after treatment. * indicates a condition with a P < 0.05 compared by one‐way ANOVA to the band from the warm‐acclimated condition.
Figure 6. Immunofluorescence in the Liver of Warm‐acclimated and Cold‐acclimated Dryophytes chrysoscelis: Liver was fixed in 10 μmol/L sections. Sections were stained with DAPI (purple) and HC‐9 (green) and images were taken on a confocal microscope. The DAPI stains the nucleus, whereas the HC‐9 appears in the plasma membrane of the hepatocytes in both (A) warm‐acclimated and (C) cold‐acclimated treefrog tissue. This signal is blocked after treatment with peptide in both (B) warm‐acclimated and (D) cold‐acclimated treefrog tissue.
Figure 7. Water and Glycerol Permeability of cRNA‐injected Oocytes from Cloned HC‐9: (A) A measure of the mean volume/initial volume and (B) the mean water permeability (P
f) of various injection groups (n = 10) given after 5 min of exposure to a hypoosmotic water solution (~67 mOsM). (C) A measure of the mean volume/initial volume over time and (D) the mean solute permeability (P
s) of various injection groups (n = 10) after 5 min of exposure to a high glycerol (~130 mmol/L) solution. Phloretin and HgCl2 conditions included a 5‐min preincubation with 0.5 mmol/L phloretin or 0.3 mmol/L HgCl2 prior to the swelling assay. The error bar is ±SEM. (E) An immunoblot from HC‐9‐, hAQP1‐, and water‐injected oocytes incubated with HC‐9 antibody. The expected 33.69 kDa band shows only in the HC‐9‐injected oocytes, as expected. * indicates a condition with a P < 0.05 when compared by Student's t‐test to the P
f or P
s of the water‐injected control. ** indicates a condition with a P < 0.05 when compared by Student's t‐test to the Pf or Ps of HC‐9‐injected oocytes.
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