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First published online December 3, 2004
Journal of Experimental Biology 207, 4559-4572 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01287

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A GPI-linked carbonic anhydrase expressed in the larval mosquito midgut

Terri J. Seron^1,2, Jennifer Hill¹ and Paul J. Linser^1,2,*

¹ The Whitney Laboratory, University of Florida, Saint Augustine, FL 32080, USA
² Department of Fisheries and Aquatic Sciences, University of Florida, Saint Augustine, FL 32080, USA

View larger version (98K): [in a new window]   Fig. 1. Alignment of several mammalian CA IV enzymes with two mosquito CA IV-like isoforms. The leucine-rich signal sequences are displayed in all aligned isoforms (red), along with the 3 essential zinc-binding histidines (blue), and cysteine residues (green) that form disulfide bonds. The reduced activity in rodent CA IVs is caused by the glycine-69 mutation to glutamine (orange; Tamai et al., 1996a,b), which the mosquitoes do not display. Important conserved residues are boxed. The position of mammalian signal sequence cleavage is shown (vertical line) and therefore the following amino acid is residue #1 in the functional protein. The peptide sequence used for antibody generation is also displayed (violet horizontal box). Asterisks, identical residues; dots, conserved residues. Broken orange lines mark the shortened active site region within the two mosquito sequences when compared to mammalian CA IV enzymes.

View larger version (101K): [in a new window]   Fig. 2. Localization of CA mRNA in a whole-mount preparation of early fourth instar Ae. aegypti midgut. (A) The whole-mount gut preparation localizes CA message to specific cells of the gastric caeca (GC) and posterior midgut (PMG). The anterior midgut (AMG) epithelial cells and the malpighian tubules (MT) showed little or no labeling. (B) A subset of cardia (arrows) and GC cells display the CA message. The distal lobes of the GC, called Cap cells, display no staining (asterisks). (C) There is a distinctive labeling pattern of CA message within a specific band of PMG epithelial cells. In addition, numerous trachea are heavily labeled along the length of the midgut (arrows). Scale bars, 300 µm (A), 150 µm (B), 75 µm (C).

View larger version (66K): [in a new window]   Fig. 3. Ae. aegypti anterior midgut in situ hybridization CA labeling. While the GC and PMG display heavy epithelial labeling for the CA message (A), there is also specific labeling seen in muscle (B,C, large arrowheads) and nerve cells (B, small arrows). (A) A representative whole-mount larva displaying the strong epithelial label in GC and PMG along with muscle fiber staining in the AMG that can be overlooked at low magnification. (B) The beginning of the anterior midgut displays both muscle and nerve fiber labeling. The labeled fibers reveal striated muscle running longitudinally down the length of the anterior gut and circularly around the girth of the gut (B,C, large arrowheads). The nerve fibers can be distinguished by their non-striated wavy appearance (B,C, small arrows). (C) The AMG (left) displayed strong labeling in muscle and nerve fibers while displaying no epithelial cell labeling. The PMG (right, asterisk) shows fiber labeling as well as intense epithelial cell labeling. Scale bars, 300 µm (A), 25 µm (B), 50 µm (C). Abbreviations as in Fig. 2.

View larger version (103K): [in a new window]   Fig. 4. Localization of CA message within Ae. aegypti CNS tissue. In situ hybridization localized the CA message within all ventral ganglia CNS clusters (A, arrows) as well as hair sensory cells (A, asterisk) and longitudinal nerve fibers (A, arrowheads). (B) Sense control showed no labeling in ganglia (arrows) or other neural structures. Scale bars, 300 µm.

View larger version (27K): [in a new window]   Fig. 5. Real-time PCR analysis of relative concentrations of CA message in Ae. aegypti larvae. The GC tissue displays the greatest amount of CA message. This value was arbitrarily set to 1 so that the other tissue sections could be relatively compared. The AMG and PMG along with the MT display very little CA message. The head section displays roughly half the amount of message found in the gastric caeca. All samples were normalized to 18S RNA. Values are means ± 1 S.E.M. Abbreviations as in Fig. 2.

View larger version (65K): [in a new window]   Fig. 6. Immunoblot analysis of bacterial expressed Aedes CA. (A) Protein staining (Fast Green) of an electroblot from an SDS-PAGE analysis of extracts from cultures of bacteria that had been transformed with the CA expression vector. Labels at the bottom of the panel show the molecular mass marker lane (M) and extracts from 3 or 24 h of culture time. Labels on the left indicate molecular masses of the markers. A protein of approximately 35 kDa displays the highest level of expression. In (B) the same blot was subsequently immunostained with the XPress epitope antibody (Invitrogen, lane XP) preimmune serum from rabbit 33 (left lane labeled PI) or rabbit 34 (right lane labeled PI) or the antisera from the rabbits (lanes 33 and 34). Note intense labeling of the same band identified by the XPress antibody as detected by the two rabbit antisera to the CA peptide at approximately 35 kDa, the expected mass of the expressed recombinant CA protein (arrow).

View larger version (80K): [in a new window]   Fig. 7. Immunofluorescence and confocal imaging of CA antibody labeling. (A–C) An isolated midgut from a fourth instar Ae. aegypti larva immunostained for CA (A, green) and labeled with TRITC-conjugated Phalloidin (B, red) to visualize actin. (C) Merge of the two colored images. Note the specific checkerboard arrangement of CA labeling in AMG (A, arrows), which corresponds to a subset of the phalloidin labeled muscles seen in B and C. (D–F) A similar subset of the gut musculature at higher magnification, labeled for CA from a fourth instar larva of An. gambiae. Scale bars 200 µm (A–C); 80 µm (D–F).

View larger version (66K): [in a new window]   Fig. 8. The Ae. aegypti CNS ganglia express this CA IV-like isoform. (A) Pre-immune serum does not show any immunoreactivity for the CNS tissue. (B) Strong immunolabeling for the mosquito CA is displayed in the ventral ganglion clusters, as displayed by the fluorescent green coloring as compared to the yellow control (pre-immune) ganglia. Scale bars, 100 µm.

View larger version (83K): [in a new window]   Fig. 9. High magnification confocal imaging of CA (green) actin (red) and DNA (blue) in the AMG of a fourth instar Ae. albopictus larva. (A–D) A maximum projection of a z-stack of images showing muscle fibers which are clearly labeled for CA (green in A and yellow in the overlay D) and ones that do not label for CA (only red in D). (E,F) Selected planes of focus from the same z-stack in three-color overlay. Note CA labeling (green) outlines the CA-positive muscles which are internally red, supporting a cell surface CA localization. All images are the same magnification so the magnification bars shown in A–D (20 µm) apply to all images.

View larger version (55K): [in a new window]   Fig. 10. Clustal alignment of CA protein sequences. All characterized human CA isoforms are presented along with predicted GPI-linked CA isoforms from Aedes aegypti, Anopheles gambiae and Drosophila melanogaster. Histidine residues that are required for the essential binding of zinc are shaded in blue. Note that one or more of these histidine residues are missing from the inactive human CA-related proteins VIII, X and XI, while all three histidines are present within the Dipteran sequences. These three species of Dipteran CAs contain a shortened active site region (marked by red dashes) when compared to any of the human (or other mammalian) CA sequences. This difference may provide a potential target for mosquito-specific CA inhibitors. Asterisks, identical residues; dots, conserved residues.