Journal of Data Mining in Genomics & Proteomics

Comparative Studies of Vertebrate Mitochondrial Carbonic Anhydrase (CA5) Genes and Proteins: Evidence for Gene Duplication in Mammals with CA5A Being Liver Specific and CA5B Broadly Expressed and Located on the X-Chromosome

Roger S Holmes^* Griffith Research Institute for Drug Discovery (GRIDD) and School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia
^*Correspondence: Roger S Holmes, Griffith Research Institute for Drug Discovery (GRIDD) and School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia, Email:

Received: 04-Jun-2020 Published: 25-Jun-2020

Introduction

At least fifteen families of mammalian carbonic anhydrases (CA) (E.C. 4.2.1.2) catalyse the hydration of carbon dioxide and related functions, and are involved in a range of biological functions, including respiration, bodily fluid formation, calcification, regulating acid base balance and bone reabsorption [1-5]. CA genes are differentially expressed in the body and have diverse tissue and subcellular distribution profiles [2,4,5]. These include the CA5A and CA5B genes, which encode distinct mitochondrial enzymes and perform essential biochemical roles, including ammonia detoxification and glucose metabolism, and have similar 3D structures with the other CA isozymes [6-20]. Targeted mutagenesis studies have shown that both enzymes play important metabolic roles although Ca5a ‘null’ mice showed more significant deleterious effects than CA5B ‘null’ mice, with Ca5A/Ca5b double knockouts showing more substantial effects [7]. Genetic analyses of human CA5A and CA5B have shown that these enzymes are encoded by distinct genes, with CA5A and CA5B localized on separate chromosomes, chromosome 16 and the X-chromosome, respectively [8,20]. Moreover, genetic variants of CA5A in human populations have caused hyperammonia in early childhood [20] and mutagenesis studies of zebrafish CA5 suggested that this enzyme regulates acid-base homeostasis in this organism [14].

This paper reports the predicted amino acid sequences, gene locations and exon structures for CA5-like vertebrate genes and proteins, including primates (human (Homo sapiens) and baboon (Papio anubis), other eutherian mammals (mouse (Mus musculus), rat (Rattus norvegicus) and cow (Bos Taurus)), a marsupial mammal (opossum) (Monodelphis domestica), a monotreme mammal (platypus) (Ornithorhynchus anatinus)), and representatives of birds (chicken (Gallus gallus)), reptiles [alligator (Alligator mississippiensis)), frogs (Xenopus tropicalis and Xenopus laevis), fish [zebra fish (Danio rerio), medaka (Oryzias latipes) and coelacanth (Latimeria chalumnae)) and elephant shark (Callorhinchus millii). The phylogenetic and evolutionary relationships of these genes and enzymes are described with a hypothesis for a gene duplication event for an ancestral mammalian CA5 gene, generating CA5A and CA5B genes, which are separately localized on mammalian genomes, including the X-chromosome for CA5B, and are differentially expressed in tissues of the body.

Methods

Vertebrate CA5 gene and protein identification

BLAST (Basic Local Alignment Search Tool) studies were undertaken using web tools from the National Center for Biotechnology Information (NCBI) [21]. Protein BLAST analyses used the human and mouse CA5A and CA5B amino acid sequences deduced from reported sequences for these genes [7,8,15]. Nonredundant protein sequence databases for several mammalian and other vertebrate genomes were obtained using the blastp algorithm for the following genome sequences: human (Homo sapiens) [22]; baboon (Papio anubis) [23], cow (Bos Taurus) [24]; mouse (Mus musculus); [25] rat (Rattus norvegicus); [26] opossum (Monodelphis domestica); [27] platypus (Ornithyrinchus anatinus); [28] chicken (Gallus gallus); [29] alligator (Alligator mississippiensis); frog (Xenopus tropicalis); [30] and zebrafish (Danio rerio) [31].

BLAT analyses were subsequently undertaken for each of the predicted CA5-like amino acid sequences using the UC Santa Cruz web browser [32] to obtain the predicted locations for each of the vertebrate CA5-like genes, including exon boundary locations and gene sizes . Structures for human CA5A and CA5B isoforms were obtained using the AceView website to examine predicted gene and protein structures to interrogate this database of human mRNA sequence [33] (Table 1).

Table 1: Mammalian CA5A and CA5B and other vertebrate CA5 genes and proteins

Predicted structures and properties of vertebrate CA5 subunits

Alignments of predicted CA5-like amino acid sequences and estimates of sequence identities were undertaken using a ClustalW method [34]. Predicted secondary structures for vertebrate CA5 subunits were obtained using alignments with the reported tertiary structures for mouse CA5A9 and CA5B [10]. Predictions of the CA5A, CA5B and CA5 protein N-terminal sequences serving as mitochondrial targeting peptides, and the cleavage site for this peptide, were undertaken using MITOPROT [35].

Human CA5A and CA5B gene expression and predicted gene regulation sites

The human genome browser was used to examine predicted CpG islands [36] and transcription factor binding sites (TFBS) (ORegAnno IDs: Open Regulatory Annotations) [37] for human CA5A and CA5B using the UC Santa Cruz Genome Browser [32]. The GTEx web browser was used to examine the human tissue expression profiles for CA5A, CA5Aps and CA5B [38].

Phylogenetic studies and sequence divergence

Mammalian CA5A and CA5B and other vertebrate CA5 sequences were subjected to phylogenetic analysis using the http://www. phylogeny.fr/ portal to enable alignment (MUSCLE), curation (Gblocks), phylogeny (PhyML) and tree rendering (TreeDyn) to reconstruct phylogenetic relationships [39]. Mammalian sequences were identified as members of the CA5A or CA5B (mitochondrial) groups, whereas non-mammalian vertebrate sequences were identified as members of the CA5 group.

Results and Discussion

Alignments and biochemical features of vertebrate CA5 amino acid sequences

Amino acid sequence alignments for opossum (marsupial) CA5A and CA5B, chicken and zebrafish CA5 amino acid sequences are shown in Figure 1, together with the previously reported sequences for human and mouse CA5A and CA5B [7,8,15]. The vertebrate CA5-like sequences exhibited >50% identities, suggesting that these protein subunits are products of the same gene family (Table 2).

Figure 1. Amino acid sequence alignments for vertebrate CA5A, CA5B and CA5 sequences
See Table 1 for sources of CA5A, CA5B and CA5 sequences; * identical residues; : 1 or 2 conservative substitutions; . 1 or 2 non-conservative substitutions; active site His residues for binding Zn2+; catalytic active site residues; helices H1, H2 etc; sheets B1 B2 etc; conserved serine and threonine residues are involved in substrate binding; bold underlined font shows predicted exons (numbered) junctions; and mitochondrial transit peptides are shown.

Table 2: Percentage identities for mammalian CA5A and CA5B and other vertebrate CA5 amino acid sequences. Numbers show the percentage of amino acid sequence identities. Numbers in bold show higher sequence identities for more closely related CA5 family members.

Amino acid sequences for the mammalian CA5-like proteins examined contained 299-310 (CA5A) and 313-317 (CA6B) residues, whereas the lower vertebrate CA5 sequences examined contained 290-314 residues. The elephant shark CA5 was the smallest among the vertebrate CA5-like proteins examined with 290 amino acid residues (Table 1). The N-termini showed the lowest levels of sequence identity among the sequences examined perhaps due to the presence of the transit peptide for facilitating mitochondrial localization (Figure 1).

X-ray crystallographic studies for mouse CA5A and CA5B have enabled the identification of key structural and catalytic residues among those aligned for vertebrate CA-like sequences examined (Figure 1) [7,15]. These included mouse Tyr94, Tyr158 and Tyr161 which were identified as catalytic residues; His124, His126 and His149, which were responsible for chelating the Zinc residue at the active site; and 229Thr-230Thr, involved in substrate binding. Tyr 94 is conserved for all of the vertebrate CA5 sequences examined, whereas Tyr158 underwent a substitution with phenylalanine for human and mouse CA5B; and Tyr161 underwent a similar phenylalanine substitution in the mammalian CA5B and lower vertebrate CA5 sequences examined. Genetic substitution of mouse CA5A Ser233 with Pro 233 resulted in markedly reduced activity, [20] which suggests a significant role in catalysis for this conserved amino acid.

Predicted gene locations, exon structures and tissue expression for vertebrate CA5 genes

Table 1 and Figure 1 summarize the predicted locations and exon structures for vertebrate CA5-like genes based upon BLAT interrogations of several vertebrate genomes using the sequences for human [6,8] and mouse [7] CA5A and CA5B, and the predicted sequences for other vertebrate CA5 subunits (Table 1) and the UC Santa Cruz Web Browser [32]. Vertebrate CA5 genes contained 7 coding exons with the predicted exon start sites in identical or similar positions. Figure 2 describes the tissue expression profiles for CA5A and CA5B, as well as a CA5A pseudogene (CA5Aps) [38] (Figure 2).

Figure 2. Comparative tissue expression levels for human CA5A, CA5Aps and CA5B
RNA-seq gene expression profiles across 53 selected tissues (or tissue segments) were examined from the public database for human CA5, CA5Aps and CA5B based on expression levels for 175 individuals³⁸ (http://www.gtex.org). Tissues: 1. Adipose-Subcutaneous; 2. Adipose-Visceral (Omentum); 3. Adrenal gland; 4. Artery-Aorta; 5. Artery-Coronary; 6. Artery- Tibial; 7. Bladder; 8. Brain-Amygdala; 9. Brain-Anterior cingulate Cortex (BA24); 10. Brain-Caudate (basal ganglia); 11. Brain-Cerebellar Hemisphere; 12. Brain- Cerebellum; 13. Brain-Cortex; 14. Brain-Frontal Cortex; 15. Brain-Hippocampus; 16. Brain- Hypothalamus; 17. Brain-Nucleus accumbens (basal ganglia); 18. Brain- Putamen (basal ganglia); 19. Brain-Spinal Cord (cervical c-1); 20. Substantia nigra; 21. Breast-Mammary Tissue; 22. Cells-EBV-transformed lymphocytes; 23. Cells- Transformed fibroblasts; 24. Cervix-Ectocervix; 25. Cervix-Endocervix; 26. Colon-Sigmoid; 27. Colon-Transverse; 28. Esophagus-GastroesophagealBrain-Junction; 29. Esophagus-Mucosa; 30. Esophagus-Muscularis; 31. Fallopian Tube; 32. Heart-Atrial Appendage; 33. Heart-Left Ventricle; 34. Kidney-Cortex; 35. Liver; 36. Lung; 37. Minor Salivary Gland; 38. Muscle-Skeletal; 39. Nerve-Tibial; 40. Ovary; 41. Pancreas; 42. Pituitary; 43. Prostate; 44. Skin-Not Sun Exposed (Suprapubic); 45. Skin-Sun Exposed (Lower leg); 46. Small Intestine-Terminal Ileum; 47. Spleen; 48. Stomach; 49. Testis; 50. Thyroid; 51. Uterus; 52. Vagina; 53. Whole Blood. TPM, Transcripts per million, calculated from a gene model with isoforms collapsed to a single gene. Box plots show a median and 25th and 75th percentiles; points are shown as the outliers if they are above or below 1.5 times the interquartile range.

Human CA5A was predominantly expressed in liver, as compared with CA5Aps which was detected at low levels only in human testis, with both genes located on chromosome 16: CA5A covering 48.5kb from 87,970,135-87,921,623 on the reverse strand, while CA5Aps covered 17.5kb from 29,618,785-29,636,328, also on the reverse strand of chromosome 16. The human CA5B gene comprised two consecutive components (CA5BP1 and CA5B) located on the plus strand of the X-chromosome (15,693,048-15,806,528) (Figure 3) and was expressed with a broad tissue distribution profile, with highest levels of expression observed in testis and arteries (Figure 2). Comparative levels of total median expression for these genes showed nearly 6 times higher levels for human CA5B as compared with CA5A, but with human liver showing > 2 times the liver specific expression of the CA5A gene as compared with tissue specific expressions of the CA5B gene.

Figure 3. Gene structures for human CA5A and CA5B
From AceView website33 http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/ The major isoforms are shown for the CA5A and CA5B transcripts; note that the CA5B transcript was split into 2 components with CA5B encoding the CA5B enzyme and CA5BP1 encoding a pseudogene; capped 5’- and 3’- ends for the predicted mRNA sequences are identified; a predicted CpG island (CpG47), a gene enhancer regulatory element (GERE)⁴³; and transcription factor binding sites (EGR1,⁴⁰ HNF4A,⁴¹ CEBPA,⁴² STAT3,⁴⁴ FOXH1,⁴⁵ ZEB1⁴⁶) are shown. The numbers of nucleotides separating exons are also shown.

Figure 3 presents the predicted structures of human CA5A and CA5B gene transcripts [33] (Figure 3).

There were 7 coding exons for the CA5A isoform 2 precursor mRNA (RefSeq:NM_001367225.1) sequence which contained several transcription factor binding sites in the 5’ region: an early growth response gene (EGR1), involved in regulating synaptic plasticity [40]; hepatocyte nuclear factor 4A (HNF4A), a nuclear transcription factor which controls the expression of several hepatic genes [41]; and CCAAT enhancer binding protein alpha (CEBPA), a transcription factor that coordinates proliferation arrest and the differentiation of hepatocytes [42]. There were also 7 coding exons for the human CA5B gene on the X-chromosome, which included a gene enhancer regulatory element (GERE) at the 5’end of the gene [43], located near CpG47 and several transcription factor binding sites: STAT3 (a tyrosine phosphorylated transcription factor) [44]; EGR140 (shared with CA5A); FOXH1 (a fork head DNA binding transcription factor which is essential for development of the anterior heart field) [45]; and ZEB1 (Zinc finger E-box-binding homeobox 1, which is required for neural differentiation of human embryonic stem cells [46].

Phylogeny of primate CA5A and CA5B and vertebrate CA5 sequences

A phylogenetic tree (Figure 4) was constructed from alignments of mammalian CA5A and CA5B amino acid sequences with other lower vertebrate CA5 sequences, with representatives of bird (chicken), reptile (alligator), amphibian (frogs), fish and a shark species. The phylogram showed clustering into 2 major groups of the mammalian CA5 sequences consistent with previous reports for mammalian CA5A and CA5B genes and enzymes [7-11], [15-20]. In contrast, evidence was obtained for single copies of CA5 genes and enzymes among lower vertebrates, including the Xenopus laevis (clawed toad) species, for which multiple copies of genes have been reported due to the genome undergoing tetraploidization [47]. The phylogram suggested an ancestral relationship between lower vertebrate CA5 and mammalian CA5, with the latter gene undergoing a gene duplication event resulting in the appearance of CA5A and CA5B genes, in the ancestor leading to the appearance of monotremes, and subsequently marsupial and eutherian mammals. Monotremes have been described as arising from primitive birds which diverged from marsupials and eutherians about 163 to 186 Ma (million years ago) [48]. Platypus and opossum genomic sequences have been reported [27,28] and incorporated into the genome browser, enabling identification of CA5A and CA5B-like genes and enzymes sequences in these species. Moreover, this study of other vertebrate CA5-like genes and enzymes is consistent with CA5 being an ancient gene present throughout vertebrate evolution, including sharks, fish, frogs, reptiles and birds, which has undergone a major gene duplication event during the emergence of mammals, generating the separate CA5A and CA5B evolutionary pathways (Figure 4).

Figure 4. Phylogenetic tree of mammalian CA5A and CA5B sequences and other vertebrate CA5 sequences
The tree is labeled with the CA5 gene name and the name of the vertebrate; note the major clusters include the lower vertebrate CA5 group and two groups for the mammalian CA5A and CA56B enzymes; a gene duplication event generating the mammalian CA5A and CA5B gene families is proposed to have occurred in a mammalian CA5 ancestral gene leading to the formation of the monotreme, marsupial and eutherian mammal groups. A genetic distance scale is shown. The number of times a clade (sequences common to a node or branch) occurred in the bootstrap replicates are shown. Only replicate values of 0.9 or more which are highly significant are shown. 100 bootstrap replicates were performed in each case. Note the higher level of sequence conservation observed for the eutherian mammalian CA5B sequence. Sequences were derived from those reported in Table 1.

It may be noted however that the CA5B gene is consistently located on the X-chromosome in eutherian mammalian genomes but is located on chromosome 7 (an autosome) in the opossum genome (Monodelphis domestica) (Table 1). This may reflect on the evolution of the mammalian X-chromosome which is highly conserved among eutherians due to suppression of recombination between X and Y chromosomes [49,50].

Phylogenetic relationships among primate CA5A (Figure 5 and Table 3) and CA5B (Figure 5 and Table 4) were examined using known and predicted genomic and enzyme sequences for 15 primates which are representative of species separated by > 40 million years of primate evolution [51]. Both phylograms separated into 3 distinct groups, with species representative of Hominidae (great apes, including humans and related species); old world monkeys (including rhesus, baboons and related species); and Cebidae (marmosets and squirrel monkeys).

Figure 5. Phylogenetic trees of primate CA5A and CA5B sequences
The trees are labeled with the CA5 gene name (CA5A for upper phylogram; and CA5B for lower phylogram) and the name of the primate; note 3 major clusters in each case for primates which are closely related phylogenetically; genetic distance scales are shown for each enzyme, with CA5A showing ~3 times larger genetic distances than for CA5B. The number of times a clade (sequences common to a node or branch) occurred in the bootstrap replicates are shown. Only replicate values of 0.9 or more which are highly significant are shown. 100 bootstrap replicates were performed in each case. Sequences were derived from those reported in Tables 3 and 4.

Table 3: Primate CA5A genes and proteins

Table 4: Primate CA5B genes and proteins

Conclusion

Phylogeny studies examined several vertebrate CA5 subunits and demonstrated that this is an ancient gene in vertebrate evolution which appears to have undergone a gene duplication event in a mammalian ancestral gene prior to the appearance of monotreme, marsupial and eutherian genomes, generating 2 distinct related mitochondrial CA5 genes and enzymes, CA5A and CA5B. These enzymes have been shown to play key roles in ammonia detoxification and glucose metabolism, with similar 3D structures to other CA isozymes.

Acknowledgement

The advice of Dr Laura Cox of the Centre for Precision Medicine, Wake Forest School of Medicine, Winston Salem NC USA is gratefully acknowledged.

Conflict of Interest

The author reports no conflicts of interest.

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