Phylogeny and expression of carbonic anhydrase-related proteins

Abstract

Background: Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate alpha-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR.

Results: We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye.

Conclusions: CARP sequences have been strongly conserved between different species, and all three CARPs show high expression in the mouse brain and CARP VIII is also expressed in several other tissues. These findings suggest an important functional role for these proteins in mammals.

Figure 1

Multiple sequence alignment of CARP VIII sequences. Comparison of 33 CARP VIII sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right side of the figure. Details of the sequences and full species names are provided in Table 1. The sequences of CAH8_Rnor and CAH8_Spur are trimmed in the end by 21 and 89 residues, respectively.

Figure 2

Multiple sequence alignment of CARP X sequences. Comparison of 32 CARP X sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right. Details of the sequences and full species names are provided in Table 1.

Figure 3

Multiple sequence alignment of CARP XI sequences. Comparison of 19 CARP XI sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right. Details of the sequences and full species names are provided in Table 1.

Figure 4

Evolutionary relationships of CARP sequences. The phylogenetic tree of 84 CARP sequences used in the study was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated sequences clustered together in the bootstrap test is shown above the branches.

Figure 5

Expression pattern of Car8 mRNA in different murine tissues.

Figure 6

Expression profile of Car10 mRNA in murine tissues.

Figure 7

Distribution of Car11 mRNA in murine tissues.

Figure 8

Immunohistochemical staining of CARP VIII, CARP X, and CARP XI proteins in mouse cerebellum (A, C, E) and cerebrum (B, D, F). Arrows indicate the location of the Purkinje cells, which are strongly positive for CARP VIII and moderately positive for CARP XI. The molecular layer (m) of the cerebellum is also intensely labeled with CARP VIII, whereas the granular cell layer is negative (g). Panel B shows strong punctuate immunostaining for CARP VIII in the cerebrum. The arrowheads in panel D indicate CARP X-positive microcapillaries in the cerebrum. Immunostaining reactions for CARP XI remained very weak in the cerebrum (F). Original magnifications are at × 20.

Figure 9

Immunohistochemical staining of CARP VIII proteins in mouse tissues. CARP VIII was observed moderately in the liver hepatocytes (A), ducts (*) and acini (arrows) of the submandibular gland (C), gastric glands (arrows in D), respiratory epithelium (arrowhead), and rounded alveolar cells (arrows) of the lung (G). Extremely low expression was observed in the pancreatic Langerhans islets (*in B), colonic glands (arrows in E), and occasionally in the tubule cells of the kidney (arrow in F indicates positive macula densa cells). No staining was present in the skeletal muscle (H) and testis (I). Original magnifications are at × 20.

Figure 10

Immunohistochemical staining of CARP X protein in mouse tissues. Moderate CARP X expression was observed in the respiratory epithelium (arrowhead) of the lung (G). Moderate expression was also present in the gastric glands (arrows in panel D). The heart muscle cells occasionally showed extremely weak signals (I). The other tissues including the liver (A), pancreas (B), submandibular gland (C), colon (E), small intestine (F), skeletal muscle (H) remained negative. Original magnifications are at × 20.

Figure 11

Immunohistochemical staining of CARP XI protein in mouse tissues. Weak immunoreactions for CARP XI were observed in the crypts of Lieberkuhn (arrows in D), gastric glands (arrows in C), and renal tubule cells (F). Extremely weak signals can also be observed in the liver (A), colon (E), and testis (I). The pancreas (B), lung (G), and skeletal muscle (H) were all negative. Original magnifications are at × 20.