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

BMC Biotechnology

Research Article

Esther Lubzens¹  François Chauvigné²  Joan Cerdಠ

[1] Department of Marine Biology, Israel Oceanographic and Limnological Research, 81080, Haifa, Israel;Laboratory of Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003, Barcelona, Spain;

关键词: Propylene Glycol;    Water Permeability;    Hypertonic Solution;    Isotonic Condition;    Control Oocyte;   

DOI  :  10.1186/1472-6750-11-34

 received in 2010-10-11, accepted in 2011-04-08,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundIncreasing 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.ResultsWater, 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.ConclusionBy 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.

【 授权许可】

Unknown   
© Chauvigné et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.