First published online February 20, 2004
Journal of Experimental Biology 207, 1217-1227 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00867
Isolation of a novel aquaglyceroporin from a marine teleost (Sparus auratus): function and tissue distribution
C. R. A. Santos1,
M. D. Estêvão1,
J. Fuentes1,
J. C. R. Cardoso1,
M. Fabra2,
A. L. Passos1,
F. J. Detmers3,
P. M. T. Deen3,
J. Cerdà2 and
D. M. Power1,*
1 Centre of Marine Sciences (CCMAR), Universidade do Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
2 Center of Aquaculture-IRTA, 43540-San Carlos de la Rapita, Tarragona,
Spain
3 Department of Cell Physiology, University Medical Center St Radboud,
6500HB Nijmegen, The Netherlands
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Fig. 1. Sea bream AQP cDNA and deduced amino acid sequence. Two MIP family
signatures (NPA) were identified and are indicated in bold at positions 76 and
208. The six transmembrane domains predicted using the Kite and Doolittle
method are underlined in italics.
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Fig. 3. Northern blot of sbAQP expression. (Ai) sbAQP transcripts were expressed in
the gastrointestinal tract, where it was most abundant in the hind-gut and
rectum, with lower levels detected in the mid-gut. Two transcripts of 2 kb and
1.6 kb were identified in the gastrointestinal tract. A single 2 kb transcript
was detected in the kidney. (Aii) The results of northern blot hybridisation
with ß-actin. (B) From the semi-quantitative analysis of sbAQP expression
(Ai) relative to that of ß-actin (Aii), the hind-gut and kidney were the
tissues with the highest expression of sbAQP followed by the rectum and
mid-gut. Hatched bars, 2 kb transcript; open bars, 1.6 kb transcript.
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Fig. 4. In situ hybridisation localises sbAQP expression in the kidney,
gills and gastrointestinal tract. sbAQP was highly expressed in some of the
kidney tubules (A) and (C). In the gill, expression was detected along the
length of the lamellae with the most intense signal present in the apical
cellular region of lamellae (D,F) where the chloride cells (arrows) were
localised (F). An intense expression was found in the hind-gut in cells
localised in the lamina propria (G) and in cells (arrows) at the
interface between the two muscle layers of the gut (I). No sbAQP expression
was observed in the liver (J) or in tubules (arrowheads) in control sections
of the kidney (B), gills (E) or gut (H). Scale bars, 1 µm. Asterisks
indicate haem deposits in the kidney.
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Fig. 5. Multiple sequence alignment of sbAQP, putative Fugu GLPs extracted
in silico from the genome and GLP proteins selected to cover the
principal groups. Species identification is indicated in
Table 1. Fugu
sequences (M00...) are identified by the scaffolds from which they were
obtained. Identical sequences are highlighted in black. Dark and light tints
indicate partial identity.
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Fig. 6. Consensus tree with corresponding bootstrap values (from sampling 1000
trees) obtained by Parsimony analysis of vertebrate GLP proteins using the
sequence of Allium cepa (Ac) AQP1 as outgroup. sbAQP fails to cluster
with any of the previously isolated GLPs and instead cluster with a gene
predicted from the Fugu genome. Species identification is indicated
in Table 1. Fugu
sequences (M00...) are identified by the scaffolds from which they were
obtained.
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© The Company of Biologists Ltd 2004
) and
(C) urea uptake of Xenopus oocytes expressing sbAQP. Oocytes were
injected with 50 nl of water containing 10 ng cRNA of sbAQP or 50 nl of
distilled water only (control oocytes) 48 h prior to the experiments.
Pf and