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First published online April 18, 2006
Journal of Experimental Biology 209, 1696-1708 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02198

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COX2 in a euryhaline teleost, Fundulus heteroclitus: primary sequence, distribution, localization, and potential function in gills during salinity acclimation

Keith P. Choe^1,2,*, Justin Havird^1,2, Rachel Rose^1,2, Kelly Hyndman^1,2, Peter Piermarini³ and David H. Evans^1,2

¹ Department of Zoology, University of Florida, Gainesville, FL 32611, USA
² Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672, USA
³ Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA

View larger version (122K): [in a new window]   Fig. 1. Amino acid alignment of chordate cyclooxygenases (labeled by genus). Gaps (dashes) were introduced to maintain alignment. All amino acids that are identical or similar to those of the mouse COX2 are shaded (Blosum 62 scoring matrix with the following amino acid groups considered similar: DN, EQ, ST, KR, FYW and LIVM). The dimerization domains were well conserved in all the vertebrates, but not in the urochordate (Ciona). The membrane-binding domain was well conserved in all species. The catalytic domain begins just after the second dimerization domain and constitutes about 80% of the protein. When folded properly, the catalytic domain is further divided into cyclo-oxygenase and peroxidase active sites. Within the cyclooxygenase site, active site tyrosine and serine (ASA-acetylated) residues are conserved in all species (Simmons et al., 2004). Importantly, the substitution of an isoleucine for a valine within the cyclooxygenase active site of COX1 only occurs in mammalian COX1. This substitution allows some drugs to be COX2 specific (Gierse et al., 1996a), and therefore these drugs would not be expected to discriminate between COX1 and COX2 of non-mammalian vertebrates. Within the peroxidase active site, the proximal histidine that binds heme is conserved in all species. 13–15 amino acids of mouse COX2 that were used as an antigen to generate antibody 126 are conserved in killifish COX2. GenBank accession numbers are, from top to bottom: COX2 – Mus (Q05769), Gallus (P27607), Fundulus (AAS21313), and Onchorynchus (CAB46017); COX1 – Mus (NP_032995), Gallus (XP_425326), Onchorynchus (CAC10360) and Squalus (AAL37727); COXa – Ciona (scaffold 118); COXb – Ciona (scaffold 207).

View larger version (22K): [in a new window]   Fig. 2. Phylogenetic tree of chordate COX homologues. Database searches were used to find COX homologues in Fugu and Ciona. The tree was constructed using the neighbor-joining method with Poisson-correction, and numbers indicate bootstrap values for 1000 replicates. All homologues are labeled by genus. GenBank accession numbers are, from top to bottom: COX1 – Homo (AAL33601), Mus (NP_032995), Gallus (XP_425326), Danio (NP_705942), Onchorhynchus (CAC10360), Salvelinus (AAF14529), Fugu 1a (FuguGenscan_21183), Fugu 1b (FuguGenscan_4741); COX2 – Homo (BAA05698), Mus (Q05769), Gallus (P27607), Danio (NP_705943), Onchorhynchus (CAB46017), Salvelinus (AAD45896), Fundulus (AAS21313), Fugu (FuguGenscan_3088); COXa – Ciona (scaffold 118), COXb – Ciona (scaffold 207). Scale bar represents 20% amino acid replacement per site.

View larger version (22K): [in a new window]   Fig. 3. Multiple tissue semi-quantitative PCR. Multiplex PCRs with primer pairs for COX2 and a 315 bp fragment of the 18S ribosome were conducted with randomly primed killifish cDNA as a template. The 479 base pair COX2 fragment was amplified, in decreasing order, from gill, opercular epithelium, kidney, heart, stomach, intestine and brain. 18S was amplified approximately equally from the cDNA of all tissues. 100 bp ladder molecular mass markers were run in the lanes to the left and right of samples.

View larger version (119K): [in a new window]   Fig. 4. Representative light micrographs of gill sections from killifish that demonstrate the localization of COX2 protein. Gill sections were incubated with antibody 160126 (A,C), antibody 160126 and antigen (B), or antibody 5 (D). Peroxidase substrates used to label antibodies were brown (160126=COX2) and blue (Na+/K+-ATPase). No immunolabeling was observed in negative control sections that were incubated with antibody 160126 and excess antigen followed by multilink (anti-mouse, rabbit and donkey) secondary antibodies (B); however, strong immunolabeling occurred in a population of epithelial cells with antibody 160126 (A). In serial sections, immunolabeling with antibody 160126 was always in cells with Na+/K+-ATPase (C,D); arrows mark cells that stained with both antibodies. Lamellae are numbered for clarity. Scale bars, 50 µm.

View larger version (18K): [in a new window]   Fig. 5. Relative expression of (A) COX2, (B) CFTR, (C) NKCC1 and (D) NKA1 mRNA was measured by quantitative real-time PCR in gills following chronic acclimation to seawater (SW; white bars) or freshwater (FW; black bars). Values are means ± s.e.m., N=6, *P<0.05.

View larger version (18K): [in a new window]   Fig. 6. Relative expression of (A) COX2, (B) CFTR, (C) NKCC1 and (D) NKA1 mRNA was measured by quantitative real-time PCR in gills following acute transfer from freshwater (FW) to seawater (SW; solid lines) or from seawater to freshwater (broken lines). To adjust for different starting expression levels between the two salinities, the relative expression values of the freshwater to seawater series was standardized to the relative chronic salinity means of Fig. 5. Values are means ± s.e.m., N=5 or 6, *P<0.05 for FW to SW, P<0.05 for SW to FW.