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Technical Briefs |
1 Controllo e Gestione delle Merci e del oro Impatto sull’Ambiente Department and
2
Department of Pharmacology and General Physiology, "La Sapienza" University of Rome, 00161 Rome, Italy
3 Institute of Internal Medicine, Department of Medicine, Surgery and Dentistry, University of Milan, 20142 Milan, Italy
aaddress correspondence to this author at: CGMIA Department, University of Rome "La Sapienza", Via del Castro Laurenziano 9, 00161 Rome, Italy; fax 39-06-23310228, e-mail botre{at}uniroma1.it
Carbonic anhydrase (CA; EC 4.2.1.1) is a zinc enzyme that is widely distributed in the living world and is involved in many biochemical processes that depend on the hydration/dehydration of carbon dioxide/bicarbonate [reviewed in Refs. (1)(2)(3)(4)(5)(6)].
Anti-CA antibodies have been identified, isolated, and purified from patients with a wide range of diseases, for some of which their presence can be a reliable diagnostic indicator (7). Anti-CA I and anti-CA II antibodies (aCAIab and aCAIIab) have recently been isolated from patients with systemic lupus erythematosus (8)(9), polymyositis and systemic sclerosis (9), endometriosis(10)(11), Sjögren syndrome (8)(9)(12)(13), idiopathic chronic pancreatitis (13)(14), primary biliary cirrhosis (PBC) (12)(13)(15)(16), and autoimmune cholangitis (15).
It has recently been hypothesized that all of these diseases (many of which can occur concomitantly) may have a common pathogenetic mechanism based on autoimmune reactions against a common antigen. According to this hypothesis, it seems that highly active CA isoenzymes (cytosolic CA II and membrane-bound CA IV) are particularly involved, because CA plays an important role in such biochemical processes as tissue hydration and secretory activities. In some cases, the preincubation of CA with specific inhibitors has blocked its antibody interactions, suggesting that the site of the immunologic reaction may involve the active site of the enzyme (9).
In patients with PBC, anti-CA antibodies are often associated with the presence of anti-mitochondrial antibodies, particularly anti-pyruvate dehydrogenase (17)(18)(19), which is the main diagnostic marker of the disease (20)(21). However, anti-CA antibodies have also been detected in the absence of anti-mitochondrial antibodies in patients with PBC and ascites (16).
The aim of this study was to verify whether isoenzyme-specific anti-CA antibodies, isolated from patients with PBC and ascites, can affect the catalytic activity of the different CA isoenzymes and, if so, to evaluate the isoform and species specificity of the effect.
The experiments were carried out on samples of anti-CA antibodies purified by means of affinity chromatography. The catalytic activities of the purified human CA I (hCAI), human CA II (hCAII), and bovine CA II (bCAII) isoforms were tested in the presence and absence of anti-CA antibodies by use of an electrochemical method based on measuring the rate of CO2 diffusion from a buffered NaHCO3 solution (22).
Total IgGs were obtained by means of immunoabsorption through protein G columns (HiTrap Protein G column; no. 1-7001-00; Pharmacia Biotech Italia). Pure CA I and CA II IgGs were prepared by an immunoabsorption method in which purified human CA I and CA II antigens (Sigma Aldrich) were attached to N-hydroxysuccinimide (NHS)-activated columns (HiTrap NHS-activated column; no. 71-7006-00; Pharmacia Biotech Italia), and the specific IgGs were adsorbed by passing total IgG through the antigen-bound columns. The specific IgGs were then eluted from the affinity columns with 0.1 mol/L glycine-HCl buffer at pH 2.7. The mean spectrophotometrically assessed aCAIab concentration was 30 mg/L, and the mean aCAIIab concentration was 13 mg/L.
The catalytic activities of the different CA isoforms were assessed electrochemically in the presence and absence of chemical inhibitors and anti-CA antibodies (22)(23). The measurements were made by connecting a carbon dioxide microelectrode (MI 720 Microelectrodes Inc.) to a two-channel potentiometer (Orion 940 EA Ionalyzer; Analytical Control SpA); the electrode jacket was filled with an internal filling solution of NaHCO3 (0.01 mol/L) and KCl (0.1 mol/L). All reagents were of analytical grade. The experiments were carried out in HEPES buffer, pH 7.0, in an open 200-µL measuring cell that was maintained at 25 °C by means of forced water circulation and under magnetic stirring at a constant rate. The pH of the reaction chamber was monitored throughout the assay by a commercial pH glass microelectrode (MI 410; Microelectrodes Inc.). The CA activity correlates with the rate of CO2 diffusion from the open chamber, as measured by the PCO2 microelectrode (22). The potentiometric apparatus was calibrated with samples of crystalline CA I and II, and the measurements were made under the following experimental conditions:
For system calibration with hCAI, hCAII, and bCAII, CA activity was measured in 60 µL of 0.1 mol/L HEPES, pH 7.0, at 25 °C. After a stable potential was reached, 20 µL of substrate solution (0.1 mol/L NaHCO3) was added; subsequently, at the time of maximum electromotive force (E), a known volume of the enzymatic calibration solution was added (20 µL of a 20-mg/L solution of bCAII or hCAII or 20 µL of a 200-mg/L solution of hCAI). There was a constant decrease in electromotive force after the addition of CA, which was followed for at least 20 min after the addition of NaHCO3; the 
E/t slope (in mV/min) of the final part of the curve is proportional to the rate of CO2 diffusion. Control experiments were performed by adding 20 µL of distilled water instead of CA; in this case, the slope of the final part of the curve had lower E/t values than those recorded by the PCO2 sensor during the enzymatic assays.
For studies of the interactions between CA isoenzymes and anti-CA antibodies, the assays were performed under the same conditions as those described above, except that the anti-CA antibodies were added to the reaction chamber at the beginning of the potentiometric assay. All of the chemicals were of analytical grade, and doubly distilled water was used to prepare all reagent solutions. The CA from bovine and human erythrocytes was supplied by Sigma Aldrich.
The rate of CO2 diffusion from the buffered solution of NaHCO3 was monitored by the electrochemical PCO2 sensor in the presence and absence of CA isoenzymes and anti-CA antibodies. The sensing element of the CO2 electrode, which was originally designed to measure CO2 tension in the blood (24), is a particular kind of pH glass electrode [usually a combined, semimicro, flat-tipped electrode (25)] incorporated in an outer body that has a membrane permeable to CO2 and contains a thin layer of bicarbonate solution [reviewed in Ref. (26)]. Any change in CO2 activity inside the reaction chamber causes a change in the electromotive force because of the change in the pH of the bicarbonate solution between the CO2-permeable membrane and the glass membrane. In particular, an increase in CO2 activity causes the intake of CO2 through the CO2-permeable membrane, with a consequent lowering of the pH of the inner solution and a related increase in electromotive force, whereas a decrease in CO2 activity has the opposite effect.
Shown in Fig. 1
are typical examples of the potentiometric experiments carried out in the absence of enzymes and in the presence of hCAI, hCAII, and bCAII. To evaluate the effectiveness of the method used to determine CA inhibition, a series of assays were performed under the same experimental conditions but in the presence of different concentrations of sulfonamide CA inhibitors (acetazolamide, methazolamide, and sulfanilamide) dissolved in the same starting solution (HEPES buffer) before the beginning of the assay. The calculated KI values were comparable to those reported in the literature (27); our data vs comparison values, in nmol/L, were as follows: acetazolamide, 9.0 vs 7.0; methazolamide, 13 vs 12; sulfanilamide, 680 vs 750, with good repeatability (relative SD <4%).
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The data given in Table 1
show the effect of aCAIabs and aCAIIabs on the catalytic activity of the different CA isoforms. The experiments performed in the presence of a CA isoenzyme and its specific antibody showed a reduced rate of CO2 diffusion compared with those performed in the absence of antibody: more precisely, the decrease (calculated taking into account the contribution of the uncatalyzed reaction) was 31% for the hCAI-aCAIab interaction and 16% for the hCAII-aCAIIab interaction; a smaller decrease (10%) was recorded for the bCAII-aCAIIab interaction.
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Although it is virtually impossible to obtain reliable KI values to calculate exactly the inhibitory power of each antibody in relation to the different isoenzymes, our preliminary results indicate that the interaction between CA and anti-CA antibodies is associated with a decrease in enzyme catalytic activity only in the case of specific interactions, i.e., between hCAI and aCAIabs and between hCAII (and, to a lesser extent, bCAII) and aCAIIabs. This finding suggests that the interactions of CA isoenzymes with their corresponding antibodies are specific and should therefore at least partially involve the active site of the enzyme. Furthermore, anti-CA antibodies do not affect the rate of uncatalyzed reaction, indicating that the antibody preparation is CA-free and that it is not altered during the purification procedure.
Autoantibodies to various mitochondrial enzymes (the serologic hallmark of PBC) greatly inhibit the catalytic activity of the autoantigens in vitro, and, more interestingly, it has been suggested that the antibodies may be involved in the destruction of bile duct epithelial cells (the target organ in PBC) by trafficking to mitochondria during the usual course of trans-cytosis (28). It is now well known that IgA, IgG, and IgM are transported across epithelial cells during the receptor-mediated transcytosis process (29), and it is possible that anti-CA antibodies, which are capable of inhibiting CA catalytic activity, may similarly contribute to the destruction of biliary epithelial cells during transcytosis in PBC.
Additional experiments are in progress to study the influence of other compounds (e.g., sulfonamide inhibitors) on antigen-antibody interactions and to extend our investigations to other CA isoforms and to antibodies isolated from patients with diseases other than PBC.
References
The following articles in journals at HighWire Press have cited this article:
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  P Invernizzi, C Selmi, M Zuin, and M Podda Lack of serum antibodies to membrane bound carbonic anhydrase IV in patients with primary biliary cirrhosis Gut, November 1, 2005; 54(11): 1665 - 1665. [Full Text] [PDF]
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); 4 mg/L hCAII (
). See text for details.