Ординатура / Офтальмология / Английские материалы / Carbonic Anhydrase Its Inhibitors and Activators_Supuran, Scozzafava, Conway_2004

TABLE 2.3

mRNA Expression of Human and Mouse CA-RPs in Vital Organs by Northern Blot Analysis

aAdapted from Akisawa, Y. et al. (2003) Virchows Archives, 442, 66–70.

bAdapted from Taniuchi, K. et al. (2003) Brain Research: Molecular Brain Research,

109, 207–215.

cOkamoto, N. et al. (2001) Biochimica et Biophysica Acta 1518, 311–316.

dAdapted from Bellingham, J. et al. (1998) Biochemical and Biophysical Research Communications 253, 364–367 and Fujikawa-Adachi, K. et al. (1999) Biochimica et Biophysica Acta 1431, 518–524.

ent, not tested.

fAs compared to a normal message size for CA-RP X (2.8 kb), a shorter transcript was seen in the placenta (~2.0 kb) on Northern blot [Okamoto, N. et al. (2001) Biochimica et Biophysica Acta 1518, 311–316 ].

gIn parentheses are shown results from mRNA dot blot hybridization [Okamoto, N. et al. (2001) Biochimica et Biophysica Acta 1518, 311–316].

2.4 EXPRESSION OF CA-RPs

2.4.1 MRNA EXPRESSION

mRNA expressions of CA-RPs VIII, X and XI have been studied in a panel of human and mouse vital organs by Northern blot analyses (Fujikawa-Adachi et al. 1999; Okamoto et al. 2001; Taniuchi et al. 2002; Akisawa et al. 2003; Bellingham et al. 1998), and the results are summarized in Table 2.3. CA-RPs X and XI show a similar expression pattern in humans and mice, with intense signals being observed in the brain. Notable differences in humans and mice are observed in the heart and kidney. Human kidney shows CA-RP X expression but mouse kidney does not, whereas mouse heart shows a positive signal for CA-RP XI but human heart does not. However, the message levels detected in these organs are much weaker than those in the brain. In this regard, mRNA expressions of CA-RPs X and XI appear to be relatively specific to the brain.

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Northern blot analysis shows single transcripts of human CA-RP X (2.8 kb; Okamoto et al. 2001), human CA-RP XI (1.5 kb; Fujikawa-Adachi et al. 1999; Bellingham et al. 1998) and mouse CA-RP XI (1.5 kb; Taniuchi et al. 2003), all of which corresponded to the apparently full-length cDNAs (Table 2.1). For murine CA-RP X mRNA expression, however, two sizes of transcripts (2.6 and 3.0 kb), possibly generated by alternative splicing, are observed (Okamoto et al. 2001). Although a number of expression sequence tags of CA-RP X from the human placenta have been deposited in GenBank (Hewett-Emmett 2000), only a shorter transcript (~2.0 kb) is detected on the Northern blot, and the molecular nature of this shorter transcript remains uncertain at present (Okamoto et al. 2001).

In contrast to CA-RPs X and XI, there is much more variety in the transcript size and distribution of CA-RP VIII mRNA expression (Taniuchi et al. 2002; Akisawa et al. 2003). Both human and mouse CA-RPs VIII show broad distribution in a panel of vital organs (Table 2.3). A distinct difference between humans and mice is observed in the lung and liver: there is no detectable signal for a CA-RP VIII message in the human liver and lung, whereas mouse lung and liver as well as the brain show the strongest signals. There is no acceptable explanation for this discrepancy other than species differences.

The blots for mRNA expression of both human and mouse CA-RPs VIII show multiple transcripts: five transcripts with different sizes (2.4, 3.0, 4.0, 4.5 and 7.0 kb) for human CA-RP VIII (Akisawa et al. 2003) and six transcripts (1.4, 1.6, 2.4, 3.4, 3.8 and 4.9 kb) for mouse CA-RP VIII (Taniuchi et al. 2002). Among these transcripts, the 2.4-kb band shows the strongest intensity and is commonly observed in organs that show positive signals. Kato has also reported multiple transcripts of mouse CA-RP VIII (Kato 1990). The different-sized RNAs might be generated by alternative splicing or by the multiple poly(A) signals found in CA-RP VIII mRNA (Akisawa et al. 2003). These findings indicate that CA-RP VIII is diversely expressed not only in the brain but also in various peripheral tissues.

2.4.2 IMMUNOHISTOCHEMICAL LOCALIZATION IN THE BRAIN

Taniuchi et al. (2000, 2002) have produced monoclonal antibodies to all three human CA-RPs that are cross-reactive with mouse homologues and have studied their regional and cellular distribution in the brain by immunohistochemical analysis. Table 2.4 summarizes their results. CA-RPs VIII and XI are consistently expressed in neural cells, astrocytes and neurites of most parts of human and mouse brains (Figure 2.3, left). Lakkis et al. (1997b) have also shown mRNA expression of mouse CA-RP VIII in cerebral neurons and Purkinje cells by an in situ hybridization method. Epithelial cells of the choroid plexus and pia arachnoid also express CA-RPs VIII and XI.

It is of great interest that CA-RP X is expressed in the myelin sheath (Figure 2.3, right). This expression has been confirmed by using brain tissue sections under two unique pathological conditions. One is acute disseminated encephalomyelitis, a disease in which there is focal demyelinization in the human brain. In a demyelinized lesion of a patient’s brain, the axons were clearly shown by lucsol fast blue stain,

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TABLE 2.4

Immunohistochemical Localization of CA-RPs in Human and Mouse Brains

Note: +, positive expression; +w, weak expression; –, no significant expression. Previous reports of CA-RP expression in the human brain [Taniuchi, K. et al. (2000) Neuroscience 112, 93–99] and the mouse brain [Taniuchi, K. et al. (2002) Brain Research: Molecular Brain Research, 109, 207–215] are summarized.

FIGURE 2.3 Immunohistochemical analysis of CA-RPs expression by using monoclonal antibodies to human CA-RPs VIII and X [Taniuchi, K. et al. (2000) Neuroscience 112, 93–99]. CA-RP VIII is expressed in the cytoplasm of neural cells (left; cerebral cortex of the frontal lobe). CA-RP X is expressed in radial myelin fibers of the basal ganglia (right).

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but CA-RP X expression was selectively lost (Taniuchi et al. 2000). The other is the shiverer demyelinated model mouse. In this mutant mouse, exons 3 to 7 of the myelin basic protein gene are deleted and thus myelinization is left incomplete (Mikoshiba et al. 1995). In the brain of the shiverer mouse, CA-RP X expression is almost eliminated from the myelin sheath (Taniuchi et al. 2002).

There is a notable difference in CA-RP X expression in humans and mice. In the human brain, neural cells in the cerebrum show no signal for CA-RP X expression, with only Purkinje cells and neurons in the olivary nuclei being weakly stained (Taniuchi et al. 2000). In contrast, in most parts of the murine brain, CA-RP X is expressed not only in the myelin sheath but also in the neural cell body (Taniuchi et al. 2002). This discrepancy might be caused by the difference in the postmortem time until fixation of the brain tissue. Mice brains were experimentally obtained by transcardial perfusion with 4% paraformaldehyde immediately after sacrifice, whereas human brains were obtained from necrosectomy cases in which postmortem degeneration had possibly occurred. Human CA-RP X probably disappeared from most neural cells during tissue fixation and partially remained in the Purkinje cells and neurons in the olivary nuclei. Taken together, these results suggest that CA-RP X is expressed in both the myelin sheath and neural cells.

2.4.3 DEVELOPMENTAL EXPRESSION

Taniuchi et al. (2003) have reported mRNA expression of all three CA-RPs in whole mouse embryos at four gestational days (Days 7, 11, 15 and 17). RT-PCR and Northern blot analyses showed that CA-RPs VIII and X appeared in the middle of gestation (Day 11 by RT-PCR and Day 15 by Northern blot) and were developmentally expressed, whereas murine CA-RP XI expression was seen at the early gestational stage (Day 7 by both RT-PCR and Northern blot) and gradually decreased with the progress of gestation. Lakkis et al. (1997a) have reported that CA-RP VIII mRNA is expressed during embryonic development in the liver, lung, heart, gut and thymus.

Developmental expression of CA-RPs has also been immunohistochemically studied in the brain. Human fetal brains at five gestational periods (Days 84, 95, 121, 141 and 222) were employed for the immunohistochemical analysis (Taniuchi et al. 2000). CA-RPs VIII and XI were expressed in the neuroprogenitor cells in the subventricular zone in the early term of gestation (as early as Day 84) and subsequently detected in the neural cells migrating to the cortex on Day 95. In the epithelial cells of the choroid plexus, CA-RPs VIII and XI appeared on Day 95. Meanwhile, CA-RP X first appeared in the neural cells in the cortex on Day 141. In the mouse fetal brain, CA-RPs were expressed in the neuroprogenitor cells in the subventricular area on Day 10 of gestation and were subsequently detected in neural cells migrating to the cortex on Day 16 (Taniuchi et al. 2002). Interestingly, in the lucher mouse, which is a mutant strain with an autosomal dominant inherited disorder causing locomotor ataxia (Caddy and Biscoe 1975), CA-RP VIII has been reported to postnatally disappear from the cerebellum (Nogradi et al. 1997). Although the exact function in the developing brain is uncertain, these findings suggest certain roles of CA-RPs in the early development or differentiation of neuroprogenitor cells.

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2.4.4 ONCOGENIC EXPRESSION

In 1994, a tumor-associated protein MN was found to be highly expressed in cervical tumors (Pastorekova et al. 1992; Zavada et al. 1993; Liao et al. 1994) and reported to have a CA domain and also CA activity (designated CA IX/MN; Pastorek et al. 1994; Opavsky et al. 1996). Since this discovery, CA IX expression has been immunohistochemically analyzed in various kinds of normal and tumor tissues (see details in Chapter 9). CA IX is expressed in a limited number of cell types in normal tissues, such as epithelial cells in the upper gastrointestinal tract, biliary tract, testis, ovary, and choroid plexus (Pastorekova et al. 1997; Ivanov et al. 2001). In contrast, markedly upregulated expression of CA IX has been reported in carcinoma cells in a number of organs, including the cervix, ovary, breast, kidney, gastrointestinal tract, biliary tract, pancreas and skin (Liao et al. 1994, 1997; Ivanov et al. 2001; Saarnio et al. 1998; Kivela et al. 2000b, 2001; Chia et al. 2001; Beasley et al. 2001; Wykoff et al. 2001). Subsequently, another transmembrane isozyme, CA XII, has also been reported to be overexpressed in certain tumors, including uterine carcinoma, breast cancer, renal cell tumors, gastrointestinal carcinoma and nonsmall cell carcinoma of the lung (Ivanov et al. 2001; Kivela et al. 2000a, 2001; Parkkila et al. 2000). It has been hypothesized that these transmembrane CA isozymes might contribute to the tumor microenvironment by maintaining extracellular acidic pH and thus help cancer cells grow and metastasize (Sly 2000).

Although CA-RPs have no CO2 hydration activity, thus invalidating the previous hypothesis as applied to them, CA-RP VIII has recently been reported to be overexpressed in certain tumors, including nonsmall cell lung cancer and colorectal cancer (Akisawa et al. 2003). In normal counterparts of these two organs, CA-RP VIII expression is limited to bronchial ciliated cells and cryptal proliferating cells of the colorectal epithelium, respectively. Among a panel of nonsmall lung cancers (24 squamous cell carcinomas, 6 adenosquamous cell carcinomas and 25 adenocarcinomas), all except one case of squamous cell carcinoma showed a positive signal for CA-RP VIII expression by immunohistochemical analysis. Interestingly, the positive signals were observed in cancer cells at the front of tumor progression. Similar findings have been obtained in colorectal carcinoma (Nishimori et al., unpublished data). Furthermore, CA-RP VIII mRNA is significantly expressed in developing human lungs, and, to a much lesser extent, in normal adult lungs. These findings suggest that CA-RP VIII plays some role in cell proliferation and carcinogenesis of lung and colorectal epithelial cells.

2.5 FUNCTIONAL PROSECUTION OF CA-RPs

Several lines of evidence suggest a pivotal role of CA-RPs in cellular function:

(1) amino acid sequences show high evolutional conservation (Table 2.2); (2) the secondary structure of CA-RP VIII is similar to that of CA II, suggesting that the binding interface is conserved (Bergenhem et al. 1998); and (3) replacement of an arginine residue at position 94 with histidine (Figure 2.1), which reconstructs the zinc-binding site, restores the CO2 hydration activity of mouse CA-RP VIII (Elleby et al. 2000). These findings suggest that CA-RPs might be involved in protein– protein

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interactions, and, in the case of RPTPs, that CA-RP domains might function as ligand-binding domains. Although informative studies on functional properties of CA-RPs are very limited, several important findings have been reported.

2.5.1LIGAND BINDING TO THE CA-RP DOMAIN OF RPTP

The most direct evidences for CA-RP function have come from the studies of RPTPs, which are type I transmembrane proteins and belong to the PTP gene family (Bixby

2001). Among the more than 30 known RPTPs, a CA-like domain has been identified in RPTPβ (= PTPξ) and RPTPγ. The extracellular part of these RPTPs possess, from

the N-termini, a CA-RP domain, a fibronectin Type III (FN III) domain and a long cysteine-free region (spacer domain; Figure 2.4A; Barnea et al. 1993b; Krueger and Saito 1992; Levy et al. 1993). The intracellular part contains two catalytic domains with tyrosine phosphatase motifs (D1 and D2). Three major isoforms of RPTPβ generated from an identical gene by alternative splicing have been reported (Barnea et al. 1993a, 1994). The two transmembrane forms differ by the absence of 860 residues from the spacer domain, and one secreted form loses the transmembrane domain and a cytoplasmic tail (Figure 2.4A). The latter soluble form has been identified in rat brain as a chondroitin sulfate proteoglycan called phosphacan (Maurel et al. 1994). In addition, an alternatively spliced miniexon that encodes a seven- amino-acid residue in the juxtamembrane domain (arrows in Figure 2.4A) has been reported in both long and short transmembrane forms (Li et al. 1998). These various structural types might result in a multifunctional protein capable of interacting with other proteins under various conditions. The expression of RPTPβ is restricted to the nervous system, where it is mainly found in a glial precursor, radial glia and astrocytes (Canoll et al. 1993) as well as in certain neurons (Snyder et al. 1996), whereas RPTPγ is expressed both in the developing nervous system and in a variety

of peripheral tissues in the adult rat (Barnea et al. 1993b; Canoll et al. 1993).

In 1995, a CA-RP domain of RPTPβ was reported to bind specifically to a 140-kDa protein, contactin, which is a glycosylphosphatidyl inositol (GPI)-anchored protein expressed on the surface of neuronal cells (Figure 2.4B; Peles et al. 1995). Contactin has been shown to form a cis complex with a 190-kDa transmembrane protein, a contactin-associated protein (Caspr; Peles et al. 1997). The cytoplasmic domain of Caspr contains a proline-rich sequence capable of binding to a subclass of SH3 domains of signaling molecules. Caspr is expressed diffusely on unmyelinated axons but becomes localized to the paranodal junctions shortly after the onset of myelination (Einheber et al. 1997), and is thus thought to promote neurite outgrowth and fasciculation and also possibly synapse formation and maintenance (Faivre-Sarrailh and Rougon 1997; Berglund et al. 1999). As expected from their molecular properties, both RPTPβ and contactin can exist as a soluble form. Along with direct glial–neuronal cell interactions, they can form a complex with each other on one side of the cell surface (Figure 2.4B). They can also transmit cytoplasmic signals and thus lead to bidirectional signals between neurons and glial cells (Peles et al. 1995). Furthermore, contactin has been shown to interact with the neuronal recognition molecules Ng-CAM and Nr-CAM (Brummendorf et al. 1993; Morales et al. 1993) and the extracellular matrix protein tenascin (Zisch et al. 1992). These

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CA-RP domain

FN III

Spacer domain

(A)

FIGURE 2.4 (A) Schematic presentation of the molecular structure of the transmembrane protein tyrosine phosphatase (RPTP). The two transmembrane forms differ by the absence of 860 residues from the spacer domain. A mini exon encoding a seven amino acid residue in the juxtamembrane domain is alternatively spliced (arrow). CA-RP, carbonic anhydraserelated protein; FN III, fibronectin type III; D1 and D2, tyrosine phosphatase domain.

(B) Proposed model of the interaction among transmembrane tyrosine phosphatase (RPTPβ), contactin and contactin-associated protein (Caspr). Along with direct glial–neuronal cell interactions, RPTPβ and contactin can form a complex with each other on one side of the cell surface. GPI, glycosylphosphatidyl inositol; PI-PLC, phosphatidyl inositol-specific phospholipase C. [Based on (A) Barnea, G. et al. (1993a,b) Cell 46, 205 and Barnea, G. et al. (1993b) Molecular and Cellular Biology 13, 1497–1506; and (B) Peles, E. et al. (1997) EMBO Journal 16, 978–988.]

findings indicate that CA-RP has ligand-binding ability and plays an important role in cell-to-cell communication between glial cells and neurons during development.

2.5.2 INTERACTION OF CA II WITH BICARBONATE TRANSPORTER

It has been reported that CA II binds to the C-terminus of a plasma membrane chloride/bicarbonate anion exchanger, AE1, and increases the rate of bicarbonate transport. This has been proved as follows: (1) the CA inhibitor acetazolamide causes

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(B)

FIGURE 2.4 (continued).

a major decrease in the bicarbonate transport rate by AE1; (2) mutant AE1 lacking the ability to bind to CA II decreases transport activity as compared with wild-type AE1; and (3) overexpression of a catalytically inactive form of CA II exerts a dominant negative effect on the wild-type CA II, resulting in a loss of anion-exchange activity by AE1 (Sterling et al. 2001). Similar to the CA II–AE1 interaction, CA II has also been shown to bind and function with another type of bicarbonate transporter, the sodium bicarbonate cotransporter (kNBC1; Gross et al. 2002). The consensus C-terminal sequence in these bicarbonate transporters, Asp–Ala/Asn–Asp–Asp, binds the basic sequence at the N-terminus of CA II (Gross et al. 2002; Vince and Reithmeier 2000; Vince et al. 2000). Furthermore, five histidine residues found in the N-terminal 17 residues of CA II (underlined, MSHHWGYGKHNGPEHWH) are thought to be important for the binding to AE1 (Vince et al. 2000). Probably because of the absence of these histidine residues, human CA I fails to bind with AE1. In an alignment of known mammalian CA II sequences, interestingly, histidine residues at Positions 3, 4 and 17 are completely conserved, suggesting that these three histidine residues are more critical than the other two. Because none of the CA-RPs have histidine residues at the equivalent positions, CA-RPs seem to have no capacity to bind to the bicarbonate transporter family. However, the evidence of the interaction between CA II and membrane proteins such as certain types of bicarbonate transporters suggests a novel function of CA-RPs that can be elucidated in the future.

2.6FUTURE DIRECTION OF FUNCTIONAL ANALYSIS OF CA-RPs

The molecular properties and expression of CA-RPs have gradually been elucidated. However, almost nothing is yet known about their biological function. The findings

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Brummendorf, T., Hubert, M., Treubert, U., Leuschner, R., Tarnok, A., and Rathjen, F.G. (1993) The axonal recognition molecule F11 is a multifunctional protein: Specific domains mediate interactions with Ng-CAM and restrictin. Neuron 10, 711–727.

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Chia, S.K., Wykoff, C.C., Watson, P.H., Han, C., Leek, R.D., Pastorek, J. et al. (2001) Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma. Journal of Clinical Oncology 19, 3660–3668.

Einheber, S., Zanazzi, G., Ching, W., Scherer, S., Milner, T.A., Peles, E., and Salzer, J.L. (1997) The axonal membrane protein Caspr, a homologue of neurexin IV, is a component of the septate-like paranodal junctions that assemble during myelination.

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