Chauvigné F et al. (2011), Design and characterization of genetically engi...

XB-ART-43096

BMC Biotechnol 2011 Apr 08;11:34. doi: 10.1186/1472-6750-11-34.

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Design and characterization of genetically engineered zebrafish aquaporin-3 mutants highly permeable to the cryoprotectant ethylene glycol.

Chauvigné F , Lubzens E , Cerdà J .

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Increasing cell membrane permeability to water and cryoprotectants is critical for the successful cryopreservation of cells with large volumes. Artificial expression of water-selective aquaporins or aquaglyceroporins (GLPs), such as mammalian aquaporin-3 (AQP3), enhances cell permeability to water and cryoprotectants, but it is known that AQP3-mediated water and solute permeation is limited and pH dependent. To exploit further the possibilities of using aquaporins in cryobiology, we investigated the functional properties of zebrafish (Danio rerio) GLPs. Water, glycerol, propylene glycol and ethylene glycol permeability of zebrafish Aqp3a, -3b, -7, -9a, -9b, -10a and -10b, and human AQP3, was examined. Expression in Xenopus laevis oocytes indicated that the permeability of DrAqp3a and -3b to ethylene glycol was higher than for glycerol or propylene glycol under isotonic conditions, unlike other zebrafish GLPs and human AQP3, which were more permeable to glycerol. In addition, dose-response experiments and radiolabeled ethylene glycol uptake assays suggested that oocytes expressing DrAqp3b were permeated by this cryoprotectant more efficiently than those expressing AQP3. Water and ethylene glycol transport through DrAqp3a and -3b were, however, highest at pH 8.5 and completely abolished at pH 6.0. Point mutations in the DrAqp3b amino acid sequence rendered two constructs, DrAqp3b-T85A showing higher water and ethylene glycol permeability at neutral and alkaline pH, and DrAqp3b-H53A/G54H/T85A, no longer inhibited at acidic pH but less permeable than the wild type. Finally, calculation of permeability coefficients for ethylene glycol under concentration gradients confirmed that the two DrAqp3b mutants were more permeable than wild-type DrAqp3b and/or AQP3 at neutral pH, resulting in a 2.6- to 4-fold increase in the oocyte intracellular concentration of ethylene glycol. By single or triple point mutations in the DrAqp3b amino acid sequence, we constructed one mutant with enhanced ethylene glycol permeability and another with reduced pH sensitivity. The DrAqp3b and the two mutant constructs may be useful for application in cryobiology.

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Species referenced: Xenopus laevis
Genes referenced: aqp3 mip

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	Figure 1. Effect of pH on zebrafish GLPs and Aqp1a and human AQP3 expressed in X. laevis oocytes. Oocytes were injected with 1 ng (DrAqp3b, -7, -9a, -9b, -10a, -1a or HsAQP3) or 10 ng (DrAqp3a and -10b) cRNA, or with 50 nl of water (controls). The osmotic water permeability (Pf) was measured in 10 times diluted MBS after 15 min of exposure to isotonic MBS at pH 6, 7.5 or 8.5. The swelling experiments were also performed at the corresponding pH. Data are the means ± SEM of three experiments (n = 10-12 oocytes for each aquaporin). Bars with different superscript in each panel indicate significant differences (ANOVA, p < 0.05) in Pf between aquaporin-injected oocytes. The asterisks denote significant differences with respect the control oocytes at a given pH (Student's t test, p < 0.05).
	Figure 2. PEG and ethylene glycol uptake of X. laevis oocytes expressing HsAQP3 or DrAqp3b. (A) PEG of oocytes expressing different amounts of cRNA (1-40 ng) encoding HsAQP3 or DrAqp3b. Control oocytes were injected with 50 nl of water. The PEG was measured by swelling measurements during 20 sec in isotonic MBS containing 60 mM of ethylene glycol at pH 7.5. Values are the mean ± SEM of three experiments (n = 8-10 oocytes for each aquaporin). Data with an asterisk at the same cRNA dose are significantly different (Student's t test, p < 0.01). (B) Uptake of radiolabeled ethylene glycol of oocytes injected with 50 nl of water or 5 ng of HsAQP3 or DrAqp3b cRNA. Oocytes were exposed to isotonic MBS containing 1 mM cold ethylene glycol and 5 μM radiolabelled [1,2-3H]ethylene glycol for 1 min. Values (mean ± SEM; n = 8-10 oocytes) with different superscript are significantly (ANOVA, p < 0.01).
	Figure 3. Amino acid sequence alignment of zebrafish GLPs, Aqp1a and Aqp0a with mammalian and teleost orthologs. Amino acid sequence alignment of representative GLPs and water-selective aquaporins of teleosts and mammals: Danio rerio Aqp0a (DrAqp0a; FJ666326), -0b (FJ655389), -3a (EU341833), -3b (EU341832), -7 (FJ655385), -9a (FJ655387), -9b (EU341835), -10a (FJ655388), -10b (EU341836), and -1a (AY626937), Fundulus heteroclitus Aqp0a (FhAqp0; AF191906) and -3a (ACI49539), Homo sapiens AQP3 (HsAQP3; BC013566), and Bos taurus AQP0 (BtAQP0; NM_173937). Predicted transmembrane domains (TMD1-6) of DrAqp3b are annotated by blue arrows, and external (out) and internal (in) loops are indicated. The two NPA motifs are shaded in red, whereas the four residues forming the aromatic/arginine (ar/R) constriction in zebrafish GLPs (Phe, Gly/Ser, Tyr/Ala, and Arg) [48] are pointed by red arrowheads. Potential residues involved in pH sensitivity in AQP0 and -3 orthologs are shaded in green, and mutated residues in DrAqp3b are indicated by black arrowheads.
	Figure 4. Functional characterization of DrAqp3b mutants. (A) Pf of control oocytes (water-injected) and oocytes expressing 1 ng cRNA encoding wild-type DrAqp3b (DrAqp3b-WT) or different DrAqp3b mutants at different pH. Values are the mean ± SEM of three experiments (n = 8-10 oocytes per construct). The asterisks denote significant differences between WT and mutant DrAqp3b at a given pH (Student's t test, p < 0.05). (B) Immunofluorescence microscopy of water-injected oocytes (control), or oocytes expressing DrAqp3b-WT, -H53A, -H53A/G54H, -T85A and -H53A/G54H/T85A using an affinity purified anti-DrAqp3b antiserum. The arrowhead points to the oocyte plasma membrane. (C) Immunoblot of total membrane protein extracts of control oocytes or oocytes expressing DrAqp3b-WT treated or not with N-glycosidase F (PNGase F). The arrows indicate glycosylated and deglycosylated forms of DrAqp3b-WT. (D) Representative immunoblot of plasma membrane protein extracts of oocytes expressing increasing amounts of cRNA (1-8 ng) encoding DrAqp3b-WT, -T85A and -H53A/G54H/T85A treated with PNGaseF.
	Figure 5. Ethylene glycol uptake of DrAqp3b-WT and mutants at different pH. Uptake of radiolabeled ethylene glycol by oocytes injected with water or with 5 ng of DrAqp3b-WT, -T85A or -H53A/G54H/T85A cRNA was determined as in Figure 2. Data (mean ± SEM; n = 8-10 oocytes) with an asterisk are significantly (ANOVA, p < 0.01) different from the DrAqp3b-WT.
	Figure 6. Changes in cell volume of oocytes expressing DrAqp3b-WT, DrAqp3b mutants or HsAQP3 in hypertonic solutions. (A) Oocytes expressing 1 ng of DrAqp3b-WT, -T85A, -H53A/G54H/T85A or HsAQP3 were exposed to 0.9 M sucrose in MBS for 10 min. (B-C) Oocytes expressing 1 (B) or 20 (C) ng of the same constructs were exposed to 1.3 M ethylene glycol in MBS for 10 min. Data from all panels are means ± SEM of 15-24 oocytes from 3-4 different batches.

References [+] :

Bautista, Current status of vitrification of embryos and oocytes in domestic animals: ethylene glycol as an emerging cryoprotectant of choice. 1998, Pubmed