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At least 19 recordsLinked to original sources
PubMed1998

Different susceptibility of mice to immune-mediated cholangitis induced by immunization with carbonic anhydrase II.

Carbonic anhydrase II (CA-II), an enzyme that catalyzes hydration of carbon dioxide to bicarbonate and hydrogen ions, is located exclusively in cholangiocytes in the liver. Recently, patients with autoimmune cholangitis have been reported to have serum antibodies to CA-II. Moreover, active immunization of susceptible mice with CA-II results in inflammation of submandibular glands, where CA-II is also expressed. In the present study, we attempted to produce cholangitis by immunization with CA-II using two strains of mice with different potential susceptibilities. Balb/c and DBA/1J mice were immunized with a dose of human CA-II (100 microg) intraperitoneally every other week on three occasions. One week after the final immunization, mice were killed and blood and tissue samples harvested. Light and electron microscopic evaluation for inflammation was performed under coded identification. After immunization of Balb/c mice, numerous mononuclear cells, mostly CD4-positive T cells, appeared around bile ducts; lymphocyte invasion between cholangiocytes was also seen. Inflammation was not observed outside the liver. Morphologic evidence of cholangitis was observed in 8 (53.3%) of 15 Balb/c mice and in 3 (20%) of 15 DBA/1 J mice. In the control mice immunized with bovine serum albumin (BSA), cholangitis was observed in only 1 (6.7%) of 15 Balb/c mice and none of 15 DBA/1J mice. Balb/c mice immunized with CA-II had statistically significant cholangitis compared with those immunized with BSA (p < 0.01), whereas DBA/1J did not show a significant difference from controls. Balb/c mice immunized with CA-II showed specific antibody production after immunization, whereas DBA/1J mice immunized with CA-II had anti-CA-II antibody even in preimmune sera. Adoptive transfer of splenocytes from CA-II-immunized Balb/c mice resulted in cholangitis in two (66.7%) of three Balb/c recipients. These data strongly suggest that the cholangitis can be induced by CA-II immunization in susceptible strains of mice.

Adoptive Transfer↗
PubMed1989

The role of the metal ion in the refolding of denatured bovine Co(II)-carbonic anhydrase II.

Conditions for reactivation of guanidine-HCl-denatured bovine Co(II)-carbonic anhydrase II are given. The renaturation is accompanied by recovery of the native Co(II)-spectrum of the enzyme. After studying the kinetics of the renaturation process, the metal ion involvement in the refolding pathway can be summarized as follows: (1) Formation of an inactive Co(II)-intermediate with the metal ion firmly bound. No native Co(II)-spectrum is observed in this state, probably due to octahedral coordination of the metal ion in this intermediate. (2) Formation of an inactive Co(II)-intermediate with a native Co(II)-spectrum. The final tetrahedral coordination of the metal ion seems to have been formed in this state. (3) Formation of the active conformation of the enzyme. A functioning active-site is formed after some rearrangements of the polypeptide chain. This isomerisation step does not need to be preceded by formation of the intermediate with a native Co(II)-spectrum. Coordination of Co2+ in a native-like manner is, however, a prerequisite for enzymic activity. It is tentatively suggested that the metal ion is involved in stabilizing a nucleation structure formed at the bottom of the active centre. This probably occurs through binding of Co2+ to some or all of its histidyl ligands in this region after an early structuration of the metal ion binding site. The mechanisms of Co2+ appear to be similar for the refolding enzyme and the native apoenzyme, inferring that the binding site formed as a result of the nucleation process probably has the same structure as in the native conformation.

Animals↗
PubMed1994

Up-regulation of carbonic anhydrase isozyme IV in CNS myelin of mice genetically deficient in carbonic anhydrase II.

Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.

Animals↗
PubMed1997

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice.

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level unit the Purkinje cells population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

Animals↗
PubMed1985

Catalysis by cobalt(II)-substituted carbonic anhydrase II of the exchange of oxygen-18 between CO2 and H2O.

We have measured the catalysis by Co(II)-substituted bovine carbonic anhydrase II from red cells of the exchange of 18O between CO2 and H2O using membrane-inlet mass spectrometry. We chose Co(II)-substituted carbonic anhydrase II because the apparent equilibrium dissociation constant of HCO3- and enzyme at pH 7.4, KHCO3-eff approximately equal to 55 mM, was within a practicable range of substrate concentrations for the 18O method. For the native, zinc-containing enzyme KHCO3-eff is close to 500 mM at this pH. The rate constant for the release from the active site of water bearing substrate oxygen kH2O was dependent on the fraction of enzyme that was free, not bound by substrate HCO3- or anions. The pH dependence of kH2O in the pH range 6.0-9.0 can be explained entirely by a rate-limiting, intramolecular proton transfer between cobalt-bound hydroxide and a nearby group, probably His-64. The rate constant for this proton transfer was found to be 7 X 10(5) S-1 for the Co(II)-substituted enzyme and 2 X 10(6) S-1 for the native enzyme. These results are applied to models derived from proton-relaxation enhancement of water exchanging from the inner coordination shell of the cobalt in carbonic anhydrase. The anions iodide, cyanate, and thiocyanate inhibited catalysis of 18O exchange by Co(II)-substituted carbonic anhydrase II in a manner competitive with total substrate (CO2 and HCO3-) at chemical equilibrium and pH 7.4. These results are discussed in terms of observed steady-state inhibition patterns and suggest that there is no significant contribution of a ternary complex between substrate, inhibitor, and enzyme.

Animals↗
PubMed2005

Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II.

Human carbonic anhydrase II (HCA II) is a zinc metalloenzyme that catalyzes the reversible hydration and dehydration of carbon dioxide and bicarbonate, respectively. The rate-limiting step in catalysis is the intramolecular transfer of a proton between the zinc-bound solvent (H2O/OH-) and the proton-shuttling residue His64. This distance (approximately 7.5 A) is spanned by a well defined active-site solvent network stabilized by amino-acid side chains (Tyr7, Asn62, Asn67, Thr199 and Thr200). Despite the availability of high-resolution (approximately 1.0 A) X-ray crystal structures of HCA II, there is currently no definitive information available on the positions and orientations of the H atoms of the solvent network or active-site amino acids and their ionization states. In preparation for neutron diffraction studies to elucidate this hydrogen-bonding network, perdeuterated HCA II has been expressed, purified, crystallized and its X-ray structure determined to 1.5 A resolution. The refined structure is highly isomorphous with hydrogenated HCA II, especially with regard to the active-site architecture and solvent network. This work demonstrates the suitability of these crystals for neutron macromolecular crystallography.

Binding Sites↗
PubMed2005

Proton transfer in a Thr200His mutant of human carbonic anhydrase II.

Human carbonic anhydrase II (HCA II) has a histidine at position 64 (His64) that donates a proton to the zinc-bound hydroxide in catalysis of the dehydration of bicarbonate. To examine the effect of the histidine location on proton shuttling, His64 was replaced with Ala and Thr200 replaced with histidine (H64A-T200H HCAII), effectively relocating the proton shuttle residue 2 A closer to the zinc-bound hydroxide compared to wild type HCA II. The crystal structure of H64A-T200H HCA II at 1.8 A resolution shows the side chain of His200 directly hydrogen-bonded with the zinc-bound solvent. Different proton transfer processes were observed at pH 6 and at pH 8 during the catalytic hydration-dehydration cycle, measured by mass spectrometry as the depletion of 18O from C18O2 by H64A-T200H HCA II. The process at pH 6.0 is attributed to proton transfer between the side chain of His200 and the zinc-bound hydroxide, in analogy with proton transfer involving His64 in wild-type HCA II. At pH 8.0 it is attributed to proton transfer between bicarbonate and the zinc-bound hydroxide, as supported by the dependence of the rate of proton transfer on bicarbonate concentration and on solvent hydrogen isotope effects. This study establishes that a histidine directly hydrogen-bonded to the zinc-bound hydroxide, can adopt the correct distance geometry to support proton transfer

Amino Acid Substitution↗
PubMed1985

Positive renal response to intravenous acetazolamide in patients with carbonic anhydrase II deficiency.

Carbonic anhydrase II (CA II) is the only soluble isozyme of CA which is known to be expressed in kidney. We recently identified a deficiency of this enzyme as the basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. In order to explore the physiological importance of CA II in the kidney, we studied the renal response to intravenously infused acetazolamide in two CA II-deficient patients and two control subjects. Following acetazolamide infusion, the CA II-deficient patients exhibited a prompt rise in urinary pH and HCO3- excretion similar to the response seen in control subjects. These findings indicate that CA II-deficient patients, who lack detectable CA II in their erythrocytes, still expressed an acetazolamide-inhibitable CA activity in their kidneys. These results can be explained in three ways: 1) the CA II deficiency which is profound in the erythrocytes of these patients may not be expressed in their kidney. 2) An acetazolamide-sensitive CA other than CA II, such as CA I and CA III, which is not normally expressed in kidney, is expressed in kidneys of CA II-deficient patients. 3) The CA II deficiency is expressed in kidney in these patients but the acetazolamide response is due to inhibition of the luminal, membrane-bound CA which is the product of a different gene and unaffected by the CA II deficiency mutation. We favor the third possibility.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed2000

Localization of the Cl-/HCO3- anion exchanger binding site to the amino-terminal region of carbonic anhydrase II.

Human carbonic anhydrase II (CAII) possesses a binding site for an acidic motif (D887ADD) within the carboxyl-terminal region (Ct) of the human erythrocyte chloride/bicarbonate anion exchanger, AE1. In this study, the amino acid sequence comprising this AE1 binding site was localized to the first 17 residues of CAII, which form a basic patch on the surface of the protein. Truncation of the amino terminal of CAII by five residues resulted in a 3-fold reduction in the apparent affinity of the interaction with a GST fusion protein of the Ct of AE1 (GST-Ct) measured by a sensitive microtiter plate binding assay. Further amino-terminal truncation of CAII by 17 or 24 residues caused a loss of binding. The homologous isoform CAI does not bind AE1, despite having 60% sequence identity to CAII. One major difference between the two CA isoforms, within the amino-terminal region, is a high content of histidine residues in CAII (His3, -4, -10, -15, -17) not found in CAI. Mutation of pairs of these histidines (and one lysine) in CAII to the analogous residues in CAI (H3P/H4D or K9D/H10K or H15Q/H17S), or combinations of these various double mutants, did not greatly affect binding between GST-Ct and the mutant CAII. However, when all six of the targeted CAII residues were mutated to the corresponding sequence in CAI, binding of GST-Ct was lost. These results indicate that the AE1 binding site is located within the first 17 residues of CAII, and that the interaction is mediated by electrostatic interactions involving histidine and/or lysine residues. Further specificity for the interaction of AE1 and CAII is provided by a conserved leucine residue (L886) in AE1 that, when mutated to alanine, resulted in loss of GST-Ct binding to immobilized CAII. The binding of the basic amino-terminal region of CAII to an acidic Ct in AE1 provides a structural basis for linking bicarbonate transport across the cell membrane to intracellular bicarbonate metabolism.

Amino Acid Sequence↗
PubMed2003

Influence of backbone conformations of human carbonic anhydrase II on carbon dioxide hydration: hydration pathways and binding of bicarbonate.

In this study, the hydration of carbon dioxide and the formation of bicarbonate in human carbonic anhydrase II have been examined. From semiempirical QM/MM molecular dynamics studies, dominant conformations of the protein backbone, possibly contributing to the catalytic activity, have been isolated and further examined by means of density functional QM/MM methods. In agreement with experimental observations, a binding site for cyanate, which acts as an inhibitor, has been located, whereas for carbon dioxide, depending on the conformation of the protein environment, either a different binding site or no binding site has been found. In the latter case, carbon dioxide diffuses barrierless to the zinc-bound oxygen, and then a weakly bound bicarbonate complex is formed. The formed complex is characterized by a long C-O bond to the zinc-bound hydroxide. The nature of the calculated stationary points was verified by determination of vibrational frequencies. Finally, the dissociation of the formed bicarbonate from zinc has been considered. Therefore, a water molecule was included in the QM zone of the QM/MM hybrid potential, and minimization yielded a pentacoordinated intermediate. From a potential energy scan, an activation energy of 6.2 kcal/mol for dissociation of bicarbonate from Zn has been found.

Bicarbonates↗
PubMed2002

Structural aspects of isozyme selectivity in the binding of inhibitors to carbonic anhydrases II and IV.

Carbonic anhydrase inhibitors are effective in lowering intraocular pressure, the primary indication of glaucoma. Human carbonic anhydrase II, and possibly carbonic anhydrase IV (CAII and CAIV, respectively), help regulate fluid secretion into the anterior chamber of the eye. Because inhibitors currently formulated as drugs to treat glaucoma were designed to target CAII, an understanding of the structural basis of CAII-CAIV discrimination by inhibitors would be useful for probing the role of each isozyme in the etiology of the disease. Here, we report the X-ray crystal structures of three novel thieno[3,2-e]-1,2-thiazine-6-sulfonamides complexed with CAII and the computationally predicted structures of the same compounds complexed with CAIV. All three compounds bind with similar affinity to CAII, but they bind with up to 100-fold lower affinities to CAIV. Comparisons of experimentally determined structures of CAII-inhibitor complexes and computationally predicted structures of CAIV-inhibitor complexes allow us to rationalize these affinity trends and outline molecular features that may contribute to high-affinity inhibitor binding to CAIV. This study demonstrates how experimental structure determination methods and computational structure prediction methods can be used together to answer questions that cannot be answered by either method alone.

Carbonic Anhydrase II↗
PubMed1993

Carbonic anhydrase II deficiency.

Carbonic anhydrase (CA) isoenzyme II deficiency--formerly called the syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification--is an autosomal recessive "inborn error of metabolism" that has disclosed important insight concerning osteoclast function. Nearly 50 cases have been described, predominantly from the Middle East and Mediterranean region. It is discovered late in infancy or early in childhood through developmental delay, short stature, fracture, weakness, cranial nerve compression, dental malocclusion, and/or mental subnormality. Typical radiographic features of osteopetrosis are present, and histopathologic study of the iliac crest reveals unresorbed calcified primary spongiosa. The radiographic findings are unusual, however, in that cerebral calcification appears by early childhood and the osteosclerosis and skeletal modeling defects may gradually resolve by adulthood. Patients are usually not anemic. A hyperchloremic metabolic acidosis, sometimes with hypokalemia, is caused by renal tubular acidosis that may be a proximal, distal, or combined type. Several different mutations within the CA II gene have been identified. There is no established medical therapy, and the long-term outcome remains to be characterized. Prenatal diagnosis has not been reported. Delineation of CA II deficiency establishes an important role in humans for CA II. The pathogenesis of the mental subnormality and cerebral calcification is poorly understood; however, CA II deficiency provides significant insight concerning CA II in renal regulation of acid/base homeostasis and osteoclast-mediated bone resorption.

Acidosis, Renal Tubular↗
PubMed1992

Molecular basis of human carbonic anhydrase II deficiency.

Deficiency of carbonic anhydrase II (carbonate hydro-lyase, EC 4.2.1.1) is the primary defect in the syndrome of osteopetrosis, renal tubular acidosis, and cerebral calcification. In this report we describe the molecular basis for carbonic anhydrase II deficiency in the American family in which the association of carbonic anhydrase II deficiency with this syndrome was first recognized. The three affected siblings from this family are compound heterozygotes, each having inherited two different mutations in the structural gene for carbonic anhydrase II. The paternal mutation is a splice acceptor site mutation at the 3' end of intron 5. The maternal mutation is a missense mutation in exon 3 that substitutes a tyrosine for histidine-107. We show that the mutant enzyme expressed in bacteria from the cDNA containing the His-107----Tyr mutation has detectable, though greatly reduced, activity. We suggest that residual activity of the His-107----Tyr mutant enzyme may explain the absence of mental retardation and the relatively mild phenotype of carbonic anhydrase II deficiency in affected members of this family.

Base Sequence↗
PubMed1998

Identification of a vitamin D response element in the proximal promoter of the chicken carbonic anhydrase II gene.

The carbonic anhydrase II gene, whose transcription is enhanced by 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), encodes an important enzyme in bone-resorbing cells derived from the fusion of monocytic progenitors. We analyzed the 1,25-(OH)2D3-mediated activation of the avian gene by transient transfection assays with promoter/reporter constructs into HD11 chicken macrophages and by DNA mobility shift assays. Deletion and mobility shift analyses indicated that the -62/-29 region confers 1,25-(OH)2D3 responsiveness and forms DNA-protein complexes. The addition of an anti-vitamin D receptor (VDR) antibody inhibited binding to this sequence, whereas anti-retinoid X receptor (RXR) antibody generated a lower mobility complex. Therefore, we concluded that this element binds a VDR.RXR heterodimer, but the addition of extra 1,25-(OH)2D3 had no effect on the formation of this complex. Moreover, the use of nuclear extracts from 1,25-(OH)2D3-treated macrophages led to the formation of an additional high mobility complex also composed of VDR.RXR heterodimer. Mutations provided evidence that the 1, 25-(OH)2D3-mediated activation of the carbonic anhydrase II gene is mediated by VDR.RXR heterodimers bound to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8 osteoblasts.

Animals↗
PubMed1994

c-ErbA, but not v-ErbA, competes with a putative erythroid repressor for binding to the carbonic anhydrase II promoter.

The carbonic anhydrase II (CAII) gene is the only known gene identified as direct target for v-ErbA-mediated repression in avian erythroleukemic cells transformed by Avian Erythroblastosis Virus (AEV). This gene is transcriptionally activated by thyroid hormone (T3) in normal erythrocytic cells. In this work we have analysed the molecular basis of the transcriptional control of the CAII gene by c-ErbA and v-ErbA. We show that several domains in the promoter control hormonal regulation of transcription. One domain proximal to the TATA box mediates T3 response but contains no identified binding site for c-ErbA. An other domain termed PAL2 is approximately 600 bp upstream the transcription initiation site and contains a c-ErbA binding site. We show that when it is associated to a heterologous promoter this site mediates transcriptional repression in erythrocytic cells but not in HeLa cells. Moreover, this site binds a nuclear erythrocyte-specific factor that we called NFX, which is different from c-ErbA. heterodimers between c-ErbA and the 9-cis retinoic acid receptor (RXR) compete with NFX for binding to PAL2. In contrast, v-ErbA alone or in association with RXR is a very poor competitor and is unable to chase NFX out of the PAL2 site. We propose that NFX is a transcription repressor whose activity is inhibited by c-ErbA but not v-ErbA. This mechanism might contribute to the overall regulation of the carbonic anhydrase II promoter. These data illustrate another possible mechanism through which v-ErbA might antagonize the function of c-ErbA in controlling gene expression.

Animals↗
PubMed2005

Clinical and molecular findings in a family with the carbonic anhydrase II deficiency syndrome.

Carbonic anhydrase II (CA2) deficiency syndrome is an autosomal recessive disorder leading to osteopetrosis, renal tubular acidosis, and cerebral calcifications. Affected members of an Arab family with the CA2 deficiency syndrome carried the "Egyptian mutation" in CA2, i.e., c.191 del A, H64fsX90. One affected member, homozygote for the mutation, developed primary pulmonary hypertension. Primary pulmonary hypertension was never described before in patients with this unique syndrome. The likelihood of both occurring randomly in a single individual is very low. We therefore speculate that there might be a possibility of an etiologic link between these entities.

Acidosis, Renal Tubular↗
PubMed2001

Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome.

Carbonic anhydrase II (CAII), found in renal tubules, brain, and osteoclasts, is critical in acid-base homeostasis and bone remodeling. Deficiency of CAII gives rise to a syndrome of osteopetrosis, renal tubular acidosis (RTA), and cerebral calcification with associated developmental delay. It is inherited in an autosomal recessive fashion and found most frequently in the Mediterranean region and the Middle East. We report 2 related Irish families with clinically severe CAII deficiency in whom the gene mutation has been fully elucidated. Two children, one from each family, have undergone allogeneic bone marrow transplantation because of severe progressive visual and hearing loss. The older 2 children had already developed cerebral calcification and marked visual loss at the time of diagnosis and were treated symptomatically. Post-transplantation evaluation at 2 and 3 years demonstrates histologic and radiologic resolution of their osteopetrosis with stabilization of hearing and vision. Both children remain developmentally delayed and continue to have RTA, and the older child has now developed cerebral calcification. Allogeneic bone marrow stem cell replacement cures the osteoclast component of CAII deficiency and retards the development of cerebral calcification, but it appears to have little or no effect on the renal lesions. (Blood. 2001;97:1947-1950)

Acidosis, Renal Tubular↗