The Carbonic Anhydrase Gene Family
At least 11 enzymatically active CAs are recognized (referred to as CAs I, II, III, IV, VA, VB, VI, VII, IX, XII, and XIV). Several additional CA-related proteins that have complete CA domains but lack enzymatic activity because of amino acid substitutions in the active site have also been recognized. The active CAs differ considerably in tissue distribution, kinetic properties, and sensitivity to various inhibitors.
All active CAs catalyze the hydration of CO2 to form bicarbonate and a proton. Considerable evidence suggests that the reaction involves two steps,107 conversion of CO2 to HCO3 −, leaving H2O as a ligand on the zinc (ch208eq1), and transfer of proton to solvent buffer through a proton shuttle group, His64 (ch208eq2).
In the first step shown in ch208eq1, the zinc-bound OH− adds to CO2 to yield HCO3 − with a hydroxyl coordinated to the zinc. In the second step, displacement of the zinc-bound bicarbonate by a water molecule releases bicarbonate and restores zinc-bound water. The proton release reaction (ch208eq2), which is facilitated by His64, is the rate-limiting step for the high-activity isozymes like CA II.108 The direction of the reaction in a given tissue or body fluid depends on the relative concentrations of CO2, HCO3 −, and H+ ion, that is, the pH.
There is a distinctive membrane-bound CA in lung called CA IV,40 which was shown to be identical to the membrane-bound CA in the brush border lining the lumen of the proximal tubules of the kidney.41 The CA IV cDNA42 and CA IV genomic organization43 were recently reported. A distinct, secretory form of CA (CA VI) has been described in saliva of the rat,44 human,45 and sheep.46 The amino acid sequence of the ovine salivary CA was recently reported and showed 33 percent sequence identity with ovine CA II, though residues involved in the active site were more highly conserved. The structure of the CA VI gene was recently published.109 The low concentration of this isozyme in saliva is associated with prevalence of caries,110 and it is also reported to protect gastroesophageal mucosa from acid injury.111
A distinct CA was reported in mitochondria in the liver and was designated CA V.47 Since the recent report of another isozyme, CA VB, in the mitochondria of human112 and mouse,113 the isozyme previously designated CA V is renamed as CA VA. CA V (whether A or B is not yet established) has been shown to play a role in pyruvate decarboxylation in rat adipocytes114 and in the regulation of glucose-stimulated insulin secretion by isolated rat islets.115 The structure of mouse CA VA at 2.45 Å resolution116 and its catalytic properties have been reported.117 In addition, three membrane CAs, human CA IX118 and CA XII,119 as well as human120 and mouse121 CA XIV, were recently cloned and expressed. A role for CA IX and CA XII has been suggested in cancer because these two isozymes are up-regulated in certain forms of cancer, and CA XII has been shown to be regulated by von Hippel-Lindau tumor suppressor gene.122
Chromosome Localization of CA Genes
Genetic and structural evidence suggests that the CA isozymes comprise a multilocus enzyme family derived from a common ancestral gene by gene duplications.48 CAs I, II, and III are clustered at chromosome 8q22;49 CA IV is assigned to 17q23;43 and CA VI is assigned to chromosome 1p.50 CA VA (previously CA V) is assigned to chromosome 16,47 to which CA VII was previously mapped.51 Newly reported mitochondrial CA VB is located on Xp22.1.112 The chromosomal localization of CA IX is 17q21.2.122 CA XII is mapped to chromosome 15q22.119 CA XIV is located on chromosome 1q21.120
Tissue Distribution and Properties of CA Isozymes
The kinetic parameters of the different isozymes, and their sensitivities to different inhibitors, as well as their tissue distributions, can differ markedly, indicating different physiological roles for the different isozymes.52,53 The human CA II isozyme, whose turnover number for CO2 hydration (1.3 to 1.9 × 106 sec−1) is the highest known for any enzyme,54,55 is widely distributed. It has been identified in erythrocytes, brain, eye, kidney, cartilage, liver, lung, skeletal muscle, pancreas, gastric mucosa, and anterior pituitary body.39,56 The other isozymes, whose activities are lower than those of CA II, in the order CA II>CA IV>CA I>CA III>CA V, appear to have a more limited distribution.47,57 CA I is found primarily in erythrocytes. CA III is found mainly in red skeletal muscle.52,53 CA IV is expressed on the apical and basolateral surfaces of cells of the proximal tubule and thick ascending limb of the nephron,41 and on the plasma face of certain endothelial surfaces including the pulmonary microvasculature,58 the choriocapillaris,59 and microcapillaries of brain,60 heart, and skeletal muscle.57,61
CA VA is seen in liver and skeletal muscle by both western and northern blot analyses. The message for CA VA is also detectable in kidney.113 The CA VB protein is present in heart, liver, lung, kidney, testis, and muscle, with a weaker band of the same size in other tissues. The CA VB transcripts are widely distributed with strongest signal seen in RNA from the kidney.113 Both protein and RNA data show that CA VB is more widely distributed than CA VA. This observation suggests different physiological roles for the two mitochondrial CA isozymes. CA VI, the only secretory CA isozyme, is produced predominantly by serous acinar cells of submandibular and parotid glands.123 Immunohistochemical studies show the presence of CA VI in the enamel pellicle.124 In vitro studies document binding of CA VI to polished enamel. CA IX, one of the three newly discovered membrane CAs, has been found in several human carcinomas but not in corresponding normal tissues.118,122,125 Besides stomach mucosa, basolateral surfaces of the enterocytes of duodenum and jejunum, as well as crypts of ileal mucosa, are the only normal tissues found so far to express CA IX.118,126 The CA XII transcript has been reported in normal kidney and intestine.119 However, in 10 percent of patients with renal cell carcinoma (RCC), the CA XII transcript was expressed at much higher levels in RCC than in the normal surrounding kidney tissue. Ivanov et al.122 showed high expression of CA XII in colon, kidney, and prostate; moderate expression in pancreas, testis, and ovary; and very low expression in lung and brain. Recently, CA XII was shown by immunocytochemistry to be present in normal human endometrial epithelium127 and in normal human gut, as well as in colorectal tumors.128 The association of CA IX and CA XII with carcinomas points to a potential role for these CA isozymes in proliferation and cancerous cell growth. CA XIV is most abundant in kidney and heart followed by skeletal muscle, brain, lung, and liver.120,121 In situ hybridization shows that in the kidney the gene is expressed intensely in the proximal convoluted tubule, which is the major segment for bicarbonate resorption. The role of CA XIV in renal physiology remains to be established.
In 1983, the three affected sisters reported initially by Sly et al.,7 and described in detail by Whyte et al.,9 were shown to have no detectable CA II activity in their erythrocytes.24 CA I was present in near-normal levels. No immunoreactivity was detectable with specific antibody to CA II. The obligate heterozygote parents and several additional family members were found to have half-normal levels of CA II activity. These findings were subsequently extended to 110 similarly affected patients from mostly unrelated families of different geographic and ethnic origins. Every patient with osteopetrosis and renal tubular acidosis tested since has had undetectable levels of CA II activity.25,27,91-101 Thus, there has been no exception to the finding of a quantitative defect in CA II in erythrocytes of patients with this syndrome.
Mutations in the structural gene for CA II (summarized below) have been found in all patients with CA II deficiency analyzed genetically.63-65,91,95-101 Although the complete absence of CA II activity and immunoreactivity in erythrocytes are consistent findings in affected patients, it should be stressed that the residual activity in cells that continue to synthesize protein (such as osteoclasts in bone and cells in the proximal and distal tubules of the kidney) might be significantly higher than in erythrocytes. In fact, we suspect that some of the clinical heterogeneity in this syndrome may be explained by differences in residual CA II activity in bone and kidney in patients with different mutations in the structural gene for CA II.
The finding of a quantitative defect in CA II in these patients provided an unusual opportunity to assess the function of this enzyme and to understand its importance for bone, brain, and kidney metabolism.
All known forms of osteopetrosis involve the failure to resorb bone.4 Studies showing inhibition of PTH-induced release of Ca2+ from bone by CA inhibitors had suggested a role for CA in bone resorption.15-17 Also, CA had been demonstrated histochemically in chick and hen osteoclasts66 and CA II demonstrated immunohistochemically in rat67 and human15 osteoclasts. The osteopetrosis seen in patients with CA II deficiency provided genetic evidence for a role for CA in bone resorption, and specifically implicated the CA II isozyme.24
It had been suggested that CA aids the resorptive process by mediating the secretion of H+.16,67,129,130 We proposed that the role of CA II in acidifying the bone-resorbing component is an indirect one, analogous to its role in supporting the acidification of the lumen in the distal tubule of the kidney. It was recently suggested that the acidification of the bone-resorbing compartment is mediated by a proton-translocating ATPase,68 which secretes protons into the lumen. This reaction would simultaneously generate an OH− ion in the cytoplasm for each H+ translocated to the lumen. Titration of the OH− ions produced in the cytosol by CA II might be required to allow the proton-translocating ATPase to maintain the pH gradient (7.0 to 4.5) between the cytosol of the osteoclast and the bone-resorbing compartment. This could explain the pharmacologic evidence for a requirement for CA in bone resorption.15-17 Because CA II is the only CA isozyme known to be expressed in osteoclasts,67,68 it could also explain the osseous manifestations of CA II deficiency.
Three things need explanation concerning renal metabolism in these patients. First, most CA II-deficient patients have both a proximal and a distal component to the renal tubular acidosis.27 Second, some patients have predominantly proximal renal tubular acidosis, while in other patients, the distal renal tubular acidosis predominates.27 Third, CA II-deficient patients have a nearly normal bicarbonaturia following ingestion or infusion of carbonic anhydrase inhibitors.69 Some of these observations can be explained by a model in which the functions of CA II in the proximal and distal tubules are physiologically and biochemically distinct, and the major role of CA in bicarbonate reclamation is assigned not to CA II, but to CA IV, the luminal CA in the brush border of the proximal tubule.70,71 CA IV is biochemically and immunologically distinct from CA II, and appeared to be normal in CA II-deficient patients69 based on normal bicarbonaturia in response to infused acetazolamide. We demonstrated that the affected patients in the original American family with CA II deficiency have normal CA IV levels in their urinary membranes.69a
We deal first with the explanation for the proximal renal tubular acidosis. There is general agreement that renal reabsorption of bicarbonate is a major factor in acid-base homeostasis. Most of the bicarbonate reclamation takes place in the proximal tubule and is blocked by inhibitors of CA. However, two distinct CAs participate in bicarbonate reclamation by the proximal tubule, and they play separate roles in bicarbonate reclamation.
Bicarbonate reclamation depends on H+ secretion, which is mediated primarily by Na+/H+ exchange in the proximal tubule but also by the Mg2+/H+−ATPase on the apical membrane.72 The H+ secreted into the lumen of the proximal tubule is titrated by the HCO3 − in the glomerular filtrate to produce H2CO3, which is in contact with the membrane-bound CA IV. The luminal CA IV catalyzes the dehydration of H2CO3 to H2O and CO2.73,74 The bicarbonaturia seen in response to infused acetazolamide in already acidotic CA II-deficient patients is attributed to inhibition of this luminal CA IV.69
The CO2 produced by the CA IV-catalyzed reaction in the lumen diffuses freely into the cytosol of the proximal tubule. Here in the cytoplasm CO2 encounters CA II which acts to hydrate the CO2 to produce H2CO3, which dissociates spontaneously to HCO3 − and H+. The HCO3 − generated from CO2 in the cytosol is transported from the cytosol to the interstitial fluid or peritubular capillary by the Na-3HCO3 cotransporter, completing the reclamation of the filtered bicarbonate. The H+ regenerated in the cytosol by the CA II-catalyzed reaction can be secreted in exchange for Na+ to initiate another round of HCO3 − reclamation.72,74
Thus, both the luminal CA IV and the cytosolic CA II participate in the reclamation of HCO3 − in the proximal tubule. The fact that CA II-deficient patients do not spill HCO3 − when acidotic suggests that CA II is not required for HCO3 − reclamation when patients have low bicarbonate loads, that is, when acidotic.92 However, they have a lowered tubular maximum for bicarbonate and lose bicarbonate when the filtered load is increased by bicarbonate infusion or ingestion, indicating that CA II is required to regenerate H+ for bicarbonate reclamation under normal bicarbonate loads. This requirement explains the proximal component of the renal tubular acidosis in CA II-deficient patients (Fig. 208-5 A).
A, Proposed roles of carbonic anhydrases in bicarbonate reclamation in the proximal tubule. Na+ and HCO3 − enter the lumen of the proximal tubule. H+ is secreted in exchange for Na+, and H+ and HCO3 − are converted to CO2 and H2O in a reaction catalyzed by the luminal CA (CA IV). We propose that this enzyme functions normally in CA II-deficient patients, and that its inhibition explains the positive response to acetazolamide (normal bicarbonate diuresis). CO2 diffuses freely into the proximal tubular cell (and across the basement membrane [BM] and into the peritubular capillary [PC]), and is exposed to cytosolic CA II which catalyzes its rehydration to form HCO3 − and H+. 3HCO3 − is cotransported with 1Na+ by the basolateral cotransporter from the contraluminal surface of the proximal tubular cell to the peritubular capillary [PC]. The H+ generated by CA II is secreted in exchange for Na+ to initiate another cycle of HCO3 − reabsorption. Loss of CA II-mediated regeneration of H+ is suggested as the cause of HCO3 − wasting in CA II-deficient patients. B, Proposed role of CA II in distal urinary acidification. The H+ is secreted into the lumen by a proton-translocating Mg++ ATPase, as in amphibians, which produces OH− in the cytosol. CO2 can condense with OH− to form HCO3 − in a CA II-catalyzed reaction, and HCO3 − can be transported across the basement membrane and into the peritubular capillary. We suggest that failure to titrate the OH− limits the ability to secrete H+ and acidify the urine appropriately in CA II-deficient patients. (From Sly et al.69 Used by permission.)
The prominent distal component of the renal tubular acidosis in most CA II-deficient patients, evidenced by inappropriately high urine pH values when patients are acidotic, suggests a need for CA II for distal acidification as well. This is consistent with the immunohistochemical evidence showing a much more intense reaction for CA II in the distal tubule and the intercalated cells of the collecting ducts than in the proximal tubules.71 Why is there normally such an abundance of CA II in the distal tubules, when most of the HCO3 − reclamation takes place in the proximal tubule? We suggest69 that the explanation may be inferred from the analogous situation in the distal nephron and collecting system in the amphibian. In the turtle bladder, for example, the “CA-rich cells” are specialized cells that secrete H+ and are capable of generating a steep pH gradient.75,76 However, the acidification of the lumen is sensitive to inhibition by acetazolamide. It has been proposed that CA is needed in the amphibian nephron to titrate the OH− produced in the cytosol by the proton-translocating Mg2+ ATPase. We have suggested a similar role for CA II in the distal tubule of the human kidney, that is, catalyzing the conversion of OH− and CO2 to HCO3 −.69 Unless the OH− is titrated by CO2, the proton-translocating ATPase cannot generate a pH gradient and acidify the lumen. The absence of CA II for this reaction in CA II-deficient patients can explain their defect in distal tubular acidification (Fig. 208-5 B).
The third point, the basis for heterogeneity in the renal lesion in CA II deficiency, still requires explanation. Why is there variability in prominence of the proximal and distal lesions in different pedigrees? The explanation for this heterogeneity is still speculative. The different structural gene mutations producing CA II deficiency in different pedigrees may contribute to this heterogeneity in at least two ways. First, different mutations may affect the rate of enzyme turnover in proximal and distal tubular cells differentially, resulting in different levels of residual enzyme activity in the two different locations. Second, different structural gene mutations could affect the two different enzymatic activities in the two locations differentially. Thus, hydration of CO2 to produce H+ and HCO3 − in the proximal tubule and the condensation of OH− and CO2 to produce HCO3 − in the distal tubule might be differentially affected by different mutations in the CA II gene. Continued delineation of the mutations in different CA II-deficient patients and studies of the enzyme produced after expression of the cloned mutant enzymes in prokaryotic and eukaryotic cells may allow one to test this hypothesis.
Although not appreciated initially, Lien and Lai102 pointed out that the acidosis in CA II deficiency has a respiratory component. They reported that CA II-deficient mice have a high arterial blood PCO2, indicating failure of adequate compensation for metabolic acidosis. Furthermore, when the metabolic acidosis was corrected by NaHCO3 infusion, the respiratory acidosis became more profound. They suggested that CO2 retention in the animal was due to CA II deficiency in both erythrocytes and type II pneumocytes. Taki, Kato, and Yoshida131 recently studied three patients with CA II deficiency and reported that all three have non-anionic gap metabolic acidosis due to renal losses of bicarbonate, but with virtually no compensatory reduction in PCO2. Thus, impaired respiratory compensation for the metabolic acidosis due to CO2 retention is a previously unrecognized component of the CA II deficiency syndrome, although it has not been associated with symptomatic respiratory distress.
Brain Calcification and Cerebral Function
The mechanism of the cerebral calcification is unclear. CA II is primarily a glial enzyme that occurs predominantly in oligodendrocytes.77 It is the only soluble carbonic anhydrase in brain homogenates. As much as 50 percent of the total CA II activity occurs in a membrane-bound or myelin-associated form.78 The function of CA II in brain is not known. Morphologic changes in oligodendrocytes were reported in brains of CA II-deficient mice.132 Whether the cerebral calcification in carbonic anhydrase II deficiency is a direct effect of the deficiency of CA II in the brain or an indirect effect—for example, of carbonic anhydrase deficiency in erythrocytes, or of chronic systemic acidosis—is not clear.
While brain development and central nervous system function are not profoundly deranged in patients with this syndrome, psychomotor delay, learning disabilities, and even mental retardation are evident in most affected patients.27 The mental retardation was not so obvious in the initial reports of patients with CA II deficiency syndrome, but it is now clear that over 90 percent of the reported patients have mental retardation severe enough to prevent school attendance. Whether this is a direct consequence of the CA II deficiency, or an indirect effect, is not yet clear.
Although CA II is the only soluble CA expressed in brain, CA IV is expressed on the plasma face of cerebral capillaries and anchored to the capillary membrane by a glycosylphosphoinositol linkage.60
Growth failure appears to result from the combined effects of the osteopetrosis on bone elongation, and of the chronic metabolic acidosis on general health. Correction of the acidosis has been followed by a growth spurt in one patient,27 but the dramatic reduction in final height achieved by this patient makes it clear that the growth retardation is not due to the acidosis alone.