Design and characterization of genetically engineered zebrafish aquaporin-3 mutants highly permeable to the cryoprotectant ethylene glycol
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Publication date
2011-04-08ISSN
1472-6750
Abstract
Background: 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.
Results: 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, DrAqp3bT85A 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.
Conclusion: 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.
Document Type
Article
Document version
Published version
Language
English
Subject (CDU)
639 - Hunting. Fishing. Fish breeding
Pages
16
Publisher
BMC
Is part of
BMC Biotechnology
Citation
Chauvigné, François, Esther Lubzens, and Joan J. Cerdà. 2011. “Design and Characterization of Genetically Engineered Zebrafish Aquaporin-3 Mutants Highly Permeable to the Cryoprotectant Ethylene Glycol.” BMC Biotechnology 11 (1): 34. doi: 10.1186/1472-6750-11-34
Grant agreement number
MEC/ /AGL2007-60262/ES/MECANISMOS MOLECULARES IMPLICADOS EN LA REGULACION DE AQUAPORINAS EN EL OOCITO DE PECES MARINOS Y APLICACIONES PARA LA CRIOPRESERVACION DE GAMETOS FEMENINOS EN ACUICULTURA/ACU
EC/FP6/35995/EU/Integrated analyses of aquaporin structure and function/AQUA(GLYCERO)PORINS
Program
Aqüicultura
This item appears in the following Collection(s)
- ARTICLES CIENTÍFICS [2838]
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by/4.0/