Kabutomori J et al. (2018), Lithobates catesbeianus (American Bullfrog) ooc...

XB-ART-54669

Biol Open 2018 Mar 29;74:. doi: 10.1242/bio.031880.

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Lithobates catesbeianus (American Bullfrog) oocytes: a novel heterologous expression system for aquaporins.

Kabutomori J , Beloto-Silva O , Geyer RR , Musa-Aziz R .

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Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys - hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 - were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp3 aqp7 aqp8 aqp9

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	Fig. 1. Process of oocyte isolation. (A) 1. Enzymatic and mechanical dissociation of Lithobates oocytes. First, the ovary fragments were cut into small pieces and treated with Type VII Collagenase (0.25 mg/ml) for 5 min. 2. After this first enzymatic dissociation, healthy stage V-VI oocytes were mechanically isolated with tweezers. (B) Comparison between a defolliculated isolated Lithobates oocyte and a defolliculated isolated Xenopus oocyte. Note: the image of the Xenopus oocyte was adapted from Batra (2016).
	Fig. 2. Sequence alignment of hAQP1, rAQP3, hAQP7, hAQP8 and rAQP9. The sequence alignment shows the two conserved NPA (asparagine-proline-alanine) motifs (bold), as well as the mercury-sensitive cysteine residues in AQP1 (Cys 189), AQP3 (Cys 11), AQP8 (Cys 202) and AQP9 (Cys 213) (highlighted in gray). Each Cys is located in the mouth of the aquapore, with the exception of the Cys in AQP3 (Cys 11), which is located on the intracellular side of the protein. AQP7 does not have a mercury-sensitive Cys residue, and is not inhibited by mercury.
	Fig. 3. Surface expression of hAQP1, hAQP1FLAG, hAQP1C189S, rAQP3, hAQP7, hAQP8 and rAQP9 versus H2O-injected control oocytes. The surface expression of hAQP1, hAQP1C189S mutant, rAQP3, hAQP7, hAQP8 and hAQP9 monomers (∼28 kDa) is shown by immunoreactive bands detected at a molecular weight (MW) between 25 and 37 kDa, using polyclonal antibodies (anti-AQP1, anti-AQP3, anti-AQP7, anti-AQP8 and anti-AQP9, respectively). The surface expression of hAQP1FLAG monomer is shown by a band at a MW between 25 and 37 kDa, using a monoclonal antibody anti-FLAG. All the western blots show the absence of this immunoreactive bands in the H2O-injected control oocytes. Oocytes from 8–18 different frogs (i.e. batches of oocytes) were analyzed for surface expression, depending on the AQP construct used.
	Fig. 4. Time course of cell swelling for hAQP1-expressing oocytes and H2O-injected controls. (A) The pictures in the upper panel show three H2O-injected oocytes exposed to a hypotonic ND96 variant solution (∼70 mOsm) over the time course of 5 min, in which they do not show any significant cell swelling. (B) The pictures in the lower panel show three hAQP1-expressing oocytes exposed to the same hypotonic ND96 solution over the time course of 5 min, in which they show a significant cell swelling, as well as cell explosion. Values are means±s.e., with numbers of oocytes in parentheses.
	Fig. 5. Osmotic water permeability (Pf) values of oocytes expressing hAQP1, hAQP1C189S, hAQP1FLAG, rAQP3, hAQP7, hAQP8 and rAQP9 versus their day-matched H2O-injected control oocytes, before and after the treatment with pCMBS (1 mM for 30 min). The Pf values of AQP-expressing oocytes (black bars) are significantly greater than those of the Pf of day-matched H2O-injected controls (gray bars) (P<0.0001, t-test). Treatment with pCMBS significantly reduced the Pf of oocytes expressing AQP1 (P=0.002, t-test), AQP1FLAG (P=0.01, t-test), AQP8 (P=0.007, t-test), and AQP9 (P=0.005, t-test) versus their day-matched H2O controls, but had no effect on oocytes expressing hAQP1C189S, rAQP3 and hAQP7 versus their day-matched H2O controls (P<0.0001, t-test). In addition, a comparison of hAQP1C189S mutant, rAQP3 and hAQP7 before and after pCMBS treatment was not significant different based on a t-test (P values: 0.86 for hAQP1C189S, 0.33 for rAQP3 and 0.59 for hAQP7). Oocytes from 8–18 different frogs (i.e. batches of oocytes) were used for calculating the Pf of each AQP.
	Fig. 6. Channel-dependent osmotic water permeability (Pf) of oocytes expressing hAQP1, hAQP1FLAG hAQP1C189S, rAQP3, hAQP7, hAQP8 and rAQP9 before and after treatment with pCMBS (1 mM for 30 min). Subtracting the Pf value for day-matched H2O-injected control oocytes from the Pf of each AQP-expressing oocyte, before and after pCMBS treatment yields the channel-dependent osmotic water permeability (Pf). Treatment with pCMBS reduces the Pf* of hAQP1 (P=0.01, t-test), hAQP1FLAG (P=0.03, t-test), hAQP8 (P=0.03, t-test) and rAQP9 (P=0.02, t-test), but has no effect on Pf* of hAQPC189S mutant (P=0.85, t-test), AQP3 (P=0.10, t-test), and AQP7 (P=0.54, t-test). A one-way ANOVA, followed by an SNK post hoc analysis to compare Pf* before and after treatment with pCMBS was also performed. Overall P values: P=0.27 before pCBMS treatment and P=0.0001 after pCMBS treatment).

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