The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Immunochemical evidence for a unique GPI-anchored carbonic anhydrase isozyme in human cardiomyocytes

To clarify the controversial question of cell-specific distribution of carbonic anhydrase (CA) in the heart, endothelial cells and cardiomyocytes were isolated from porcine and human hearts and were characterized with cell-specific markers. CA activity was found in the microsomal fraction of both cell types. It was shown by Triton X-114 phase separation that both cell types possess a membrane-bound form of CA. These CAs share the same mechanism of membrane-anchoring via glycosylphosphatidylinositol (GPI), which excludes identity with transmembrane isoforms CA IX or CA XII. Western blotting analysis of human microsomes with anti-human CA IV antibodies revealed a marked difference in immunoreactivity. Endothelial CA activity resulted in 11-fold stronger CA IV bands compared with identical amounts of myocytic CA activity, indicating that cardiac endothelium and cardiomyocytes possess immunologically distinct forms of CA. We conclude that in human hearts CA IV is associated with the endothelium, whereas most of the CA in myocytes is not identical with one of the known CA isozymes. This suggests that cardiomyocytic CA is a novel isozyme.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

Occurrence and Structure of Iron Inclusions in Adipocytes of Larval Lampreys

The occurrence and structure of adipocytes in the larvae of two lamprey species, Geotria australis and Petromyzon marinus, were examined by electron microscopy. Adipocytes from both species possessed large electron-dense inclusions which histochemical and energy dispersal X-ray analyses show as containing iron. The greatest concentration of inclusions in adipocytes was found in the nephric fold of G. australis. While some iron is present in the cytoplasmic matrix as ferritin, the majority is seen in large ammocoetes in membrane-bound dense aggregations of haemosiderin. The wide variety of inclusion types seen in smaller larvae may reflect on the method of formation of these inclusions within the cell. Because of the high level of iron loading in the larval lamprey nephric fold, this readily accessible tissue may provide a valuable model for studies of iron metabolism in vertebrates.     

A quantitative structure–activity relationship study on some aromatic/heterocyclic sulfonamides and their charged derivatives acting as carbonic anhydrase inhibitors

A quantitative structure–activity relationship (QSAR) study is made on a series of aromatic/heterocyclic sulfonamides and their charged derivatives acting as carbonic anhydrase (CA) inhibitors. These compounds were studied by Scozzafava et al. (J. Med. Chem. 2000; 43: 292) for the selective inhibition of CAs—sulfonamides generally do not discriminate between different CA isozymes and hence exhibit many undesirable side effects when used as drugs against a particular disease. In this communication, an attempt has been made to investigate the physicochemical and structural properties that can make them selective for a given CA isozyme. Based on in vitro data reported by Scozzafava et al. against two cytosolic isozymes and one membrane-bound isozyme, the QSAR study has shown that uncharged compounds cannot be made selective for cytosolic or membrane-bound isozyme since in both the cases the compounds appear to follow the same mechanism of inhibition. However, for the charged compounds the polarizability of the molecule seems to greatly favor the inhibition of the membrane-bound enzyme, and hence they can be made selective for this enzyme by enhancing their polarizability, which is found to play no role in the inhibition of cytosolic enzymes.     

Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum

SUMMARY Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum . We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium ( LjTbx20, LjTbx4/5 , and LjIsl1/2A ), establishment of left–right heart asymmetry ( LjPitxA ), and partitioning of the heart tube ( LjTbx2/3A ), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A -positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.     

On the vagal cardiac nerves,with special reference to the early evolution of the head–trunk interface

The vagus nerve, or the tenth cranial nerve, innervates the heart in addition to other visceral organs, including the posterior visceral arches. In amniotes, the anterior and posterior cardiac branches arise from the branchial and intestinal portions of the vagus nerve to innervate the arterial and venous poles of the heart, respectively. The evolution of this innervation pattern has yet to be elucidated, due mainly to the lack of morphological data on the vagus in basal vertebrates. To investigate this topic, we observed the vagus nerves of the lamprey (Lethenteron japonicum), elephant shark (Callorhinchus milii), and mouse (Mus musculus), focusing on the embryonic patterns of the vagal branches in the venous pole. In the lamprey, no vagus branch was found in the venous pole throughout development, whereas the arterial pole was innervated by a branch from the branchial portion. In contrast, the vagus innervated the arterial and venous poles in the mouse and elephant shark. Based on the morphological patterns of these branches, the venous vagal branches of the mouse and elephant shark appear to belong to the intestinal part of the vagus, implying that the cardiac nerve pattern is conserved among crown gnathostomes. Furthermore, we found a topographical shift of the structures adjacent to the venous pole (i.e., the hypoglossal nerve and pronephros) between the extant gnathostomes and lamprey. Phylogenetically, the lamprey morphology is likely to be the ancestral condition for vertebrates, suggesting that the evolution of the venous branch occurred early in the gnathostome lineage, in parallel with the remodeling of the head–trunk interfacial domain during the acquisition of the neck. J. Morphol. 277:1146–1158, 2016. © 2016 Wiley Periodicals, Inc.     

Subcellular distribution and characterization of gill carbonic anhydrase and evidence for a plasma carbonic anhydrase inhibitor in Antarctic fish

This main purpose of this study was to examine the subcellular distribution and isozyme characteristics of branchial carbonic anhydrase (CA) in Chaenocephalus aceratus, an Antarctic icefish that lacks erythrocytes. The Antarctic fish, Notothenia coriiceps, which possesses erythrocytes, was also studied for comparative purposes. The gills of both species were found to have measurable activity of CA. N. coriiceps also had normal levels of blood CA activity. In contrast, the icefish, C. aceratus, lacked blood CA activity, but was found to possess an endogenous plasma CA inhibitor. The large majority of branchial CA in the gills of these species was located in the cytoplasmic fraction whereas less than 3% was associated with the membrane fraction. In both species, CA from the cytoplasmic gill fraction and membrane fraction differed markedly in terms of their sensitivity to the plasma CA inhibitor from C. aceratus. In addition, treatment with the cleaving enzyme phosphatidylinositol-specific phospholipase C indicated that CA from the branchial membrane fraction of both species is anchored to the membrane via a phosphatidylinositol-glycan linkage. Taken together, these results provide evidence for a CA IV-like isozyme in the gills of Antarctic fish. At present, the functional significance of this membrane-bound CA is unknown, but the relative amount of this isozyme appeared to be greater in the gills of C aceratus, the species that lacked erythrocytes.     

Unexpected expression of carbonic anhydrase I and selenium-binding protein as the only major non-heme proteins in erythrocytes of the subterranean mole rat (Spalax ehrenbergi)

Chromatographic separation of the non-heme proteins from the erythrocytes of the subterranean mole rat belonging to the superspecies Spalax ehrenbergi from Israel revealed two major peaks. On sequence analyses, the larger peak corresponded to a 56 kDa selenium-binding protein (SeBP) previously characterized from mouse and human liver, and the second peak to the low-activity carbonic anhydrase (CA) isozyme, CA I. There was no evidence of the high-activity CA II isozyme normally found in the red cells of all amniotes tested to date. Thus, the mole rat appears to be the first mammalian species to express both a SeBP and the low-activity CA I isozyme, as the major non-heme proteins in its red blood cells. It is possible that the absence of the high-activity CA II isozyme may be advantageous to the mole rat in adapting to the low O₂ and high CO₂ environment of its underground burrows. It is also likely that the 56 kDa SeBP may play an important adaptive role in the physiology of the red cell.     

Carbonic Anhydrase Inhibitors. Arylsulfonylureido-and Arylureido-Substituted Aromatic and Heterocyclic Sulfonamides: Towards Selective Inhibitors of Carbonic Anhydrase Isozyme I

Abstract

Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydra-zino or hydroxyl group, with tosyl isocyanate or 3,4-dichlorophenyl isocyanate afforded two series of derivatives containing arylsulfonylureido or diarylureido moieties in their molecule respectively. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Potent inhibition was observed against all three isozymes but especially against CA I, which is generally 10-75 times less susceptible to inhibition by the classical sulfonamides in clinical use as compared to the other major red cell isozyme, CA II, or the membrane-bound one, CA IV. The derivatives obtained from tosyl isocyanate were generally more potent than the corresponding ones obtained from 3,4-dichlorophenyl isocyanate. This is the first reported example of selective inhibition of CA I and might lead to more selective drugs/diagnostic agents from this class of pharmacologically relevant compounds.     

Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.     

A proteomic study of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>AAA+ protease FtsH

Background  

The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis.     

Parathyroid hormone-related protein production in the lamprey <Emphasis Type="Italic">Geotria australis</Emphasis>: developmental and evolutionary perspectives

This study explored the distribution of parathyroid hormone-related protein (PTHrP) and its mRNA in tissues of the lamprey Geotria australis, a representative of one of the two surviving groups of an early and jawless stage in vertebrate evolution. For this purpose, antibodies to N-terminal and mid-molecule human PTHrP were used to determine the locations of the antigen. Sites of mRNA production were demonstrated by in situ hybridisation with a digoxigenin-labelled riboprobe to exon VI of the human PTHrP gene. The results revealed that antigen and its mRNA were widely distributed among similar sites of tissue localisation to those described for mammalian and avian species. However, some novel sites of localisation, such as in the gill and notochord, were also found. Some differences in PTHrP localisation were noted among individuals at different intervals of the life cycle, indicating that the distributions of PTHrP, and possibly its roles, change with the stage of development in this species. The widespread tissue distribution in G. australis implies diverse physiological roles for this protein. The presence of PTHrP in the lamprey, a representative of a group of vertebrates, which apparently evolved over 540 million years ago, strongly suggests that it is a protein of ancient origin. In addition, the successful use of antibodies and probes based on the human sequence in the lamprey also provides evidence that the PTHrP molecule may have been conserved from lampreys through to humans.     

Lamprey <Emphasis Type="Italic">snail</Emphasis> highlights conserved and novel patterning roles in vertebrate embryos

snail genes mark presumptive mesoderm across bilaterian animals. In gnathostome vertebrates, snail genes are a multimember family that are also markers of premigratory neural crest (pnc) and some postmigratory neural crest derivatives in the pharyngeal arches. Previous studies of nonvertebrate chordates indicate that they have single snail genes that retain ancestral functions in mesoderm development and perhaps in specification of a pnc-like cell population. Lampreys are the most basal extant vertebrates, with well-defined developmental morphology. Here, we identify a single snail gene from the lamprey Petromyzon marinus that is the phylogenetic outgroup of all gnathostome snail genes. This single lamprey snail gene retains ancestral snail patterning domains present in nonvertebrate chordates. Lamprey snail is also expressed in tissues that are broadly equivalent to the combined sites of expression of all three gnathostome snail paralogy groups, excepting in embryonic tissues that are unique to gnathostomes. Importantly, while snail does not appear to demarcate an early neural crest population in lampreys as it does in gnathostomes, it may be involved in later neural crest development. Together, our results indicate that significant cis-regulatory innovation occurred in a single snail gene before the vertebrate radiation, and significant subfunctionalization occurred after snail gene duplications in the gnathostome lineages.     

The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO₂) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO₂ excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O₂)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.

     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Carbonic Anhydrase in Mouse Testis and Epididymis; Transfer of Isozyme IV to Spermatozoa During Passage

Spermatozoa are subjected to major changes as they pass through the epididymal duct. The aim of the present study was to describe the distribution of carbonic anhydrase (CA) in the mouse testis and epididymis using a histochemical technique showing total catalytic activity, in combination with immunohistochemistry for the two important isoforms CAs II and IV. By comparing normal mice with CA II-deficient mice, we were able to study membrane-bound CA without influence from the ubiquitous cytoplasmic CA II. Spermatozoa, when studied in both the scanning electron and light microscope, were found to pickup membrane-bound CA IV during their passage through the epididymal duct. The transfer appeared to take place in the proximal part of the corpus, where the apical membrane and vesicles of principal cells were richly supplied with CA IV. In addition to CA IV, another membrane-bound isozyme was located in basolateral membranes of principal cells. Cytoplasmic CA II was found in varying amounts in apical/narrow cells and principal cells of the corpus in control animals. The significance of CA for pH-regulating processes vital for sperm storage and motility is discussed. A function in HCO3- transport during sperm capacitation at fertilization is suggested for the CA IV found in spermatozoa.     

ATP-dependent fructose uptake system in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA–fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.