Arginase, member of ureohydrolase family, catalyses arginine into ornithine and urea. As a classical molecule, arginase involved in nitrogen detoxification as it is majorly reported in the hepatic tissue of ureotelic organisms (Clementi, 1937), later on, arginase reported in mitochondria of extra hepatic tissues of fungi, plants, invertebrates and vertebrates where it appears to be involved in ornithine metabolism (reviewed by Jenkinson et al., 1996).
Arginine, a positively charged amino acid, important for the protein synthesis and serving as substrate for synthesis of ornithine, another positively charged amino acid that acts as substrate pool for glutamate, GABA, glutamine and proline. The arginine regulation is important to various pathological conditions. The regulation of arginine impacts on the progression and cure of pathological conditions and its deficiency leads to poor wound healing, delayed immune response, poor growth, mental retardation (Crenn and Cynober, 2010) and argininemia (Morris, 2007). However, deficiency of arginine could be regulated by arginine supplementation which is found useful to reduce risk of vascular and cardiovascular dyfunction, in improvement of immune response and collagen synthesis (Morris, 2007).
Arginase serves as an important regulator of arginine homeostasis and it was suggested as central molecule involved in multiple metabolic functions, such as cellular proliferation and differentiation, ornithine, proline and glutamate synthesis, protein synthesis, immune response, anti-inflammatory response, ion channel regulation, neuroprotection etc. along with removal of nitrogenous waste in the form of urea (reviewed by Caldwell et al., 2015). In past decades, arginase impacts as molecular marker observed more significantly due to its role in regulation of Nitric Oxide Synthetase (NOS) that also acted upon arginine and produces citrulline and nitric oxide (NO). In pathological conditions, the increased activity of NOS causes production of superoxide and peroxynitrate further leads to oxidative and nitrosative stress. Other than this, NOS acts as proinflammatory marker. Elevated NOS level caused vascular and cardiovascular dysfunction by production of NO. So, arginase activity is required to regulate the hyperactivity of NOS by removal of arginine substrate, as arginase has more affinity for arginine than NOS. Reports suggests arginase could serve as biomarker in different pathological conditions, such as in carcinomas (Dzik, 2014), diabetes (Romero et al., 2008), vascular dysfunction (Lucas et al., 2014), cardiovascular diseases (Zimmerman and Rothenberg, 2006) and also associated with neurological complications (Ljubisavljevic et al., 2014). The versatility of arginase induces interest in this molecule as it alone regulates multiple metabolic functions and associated disorders.
Isoforms of arginase
Two major isoforms of arginase is reported in various organisms throughout the evolutionary spectrum from bacteria to mammal, first is cytosolic arginase-I and second is mitochondrial arginase-II (Jenkinson et al., 1996). Both isoforms are different gene products and they are proposed to evolve from a common ancestor (Dzik, 2015). Mommsen and Walsh, (1989) proposed that replacement of mitochondrial arginase-II by a cytosolic arginase-I is the key change observed that converted an arginine biosynthetic pathway of bacteria into arginine degradation pathway of vertebrates. The pattern of secondary structure organisation and the trimeric three dimensional crystal structures of the both proteins confirmed that both have conserved pattern of protein folding and organisation (Cama et al., 2003; D’Costanzo et al., 2007). Not only at protein folding, but pattern of exons and introns are also conserved in both arginase-I and arginase-II (Iyer et al., 1998; Shi et al., 2001). Despite all these similarities, both isoforms were different in their molecular weight, physicochemical properties, regulation, tissue and sub-cellular distribution (Cederbaum et al., 2004).
Mitochondrial membrane-bound arginase
Besides these two isoforms, other forms of arginase are also reported that were differ in ionic charge and immunological properties (Gasiorowska et al., 1970; Kaysen and Strecker, 1973; Tarrab et al., 1974; Herzfeld and Raper, 1976; Porembska and Zamecka, 1985; Spolarics and Bond, 1988; Venkatakrishnan et al., 2003; Venkatakrishnan and Reddy, 2010). The mitochondrial membrane-bound arginase was proposed on the basis of their sub-cellular localization (Cheung and Raijman, 1981; Nissim et al., 2005; Srivastava and Ratha, 2013). The mitochondrial membrane-bound arginase is associated to other organelle, like nucleus, endoplasmic reticulum but enriched in mitochondrial fraction most (Skrzypek-Osiecka et al., 1980). Cheung and Raijman, (1981) first demonstrated the association of mitochondrial membrane-bound arginase and show the mitochondrial membrane-bound arginase activity that is 60% of the total mitochondrial arginase activity. Later on, Nissim et al., (2005) studied role of mitochondrial membrane-bound arginase and suggested that 90% of the total mitochondrial arginase activity associated to the outer mitochondrial membrane. They proposed that this significant activity of arginase may have role in regulation of ureogenesis by efficient channeling of arginine and ornithine. Srivastava and Ratha, (2013) reported the release of cytosolic arginase-I from mitochondrial fraction in KCl dependent manner.
Statement of Problem
Distribution of arginase-I and arginase-II isoforms were completely different at organisms, tissue and cellular level. The reports regarding the mitochondrial membrane-bound arginase are confined to the rat liver (Cheung and Raijman, 1981; Nissim et al., 2005) and from fish Heteropneustes fossilis liver (Srivastava and Ratha, 2013). They reported the solubilization of mitochondrial membrane-bound arginase in cytosol and simultaneous decrease in mitochondrial arginase-II with increasing KCl concentration. Rat is a ureotelic organisms and the Heteropneustes fossilis is an ureogenic fish (Saha and Ratha, 1987) that has intermediate position in reference to nitrogen metabolism. Other than rat and fish liver, there is no report on the KCl dependent solubilization of mitochondrial membrane-bound arginase is known. Mitochondrial membrane-bound arginase is hypothesized as cytosolic arginase adsorbed at the surface of sub-cellular organelles (Skrzypek-Osiecka et al., 1980). As literature suggested, arginase isoforms are result of enzyme duplication event that evolved to make them more efficient for regulation of arginine homeostasis (Dowling et al., 2008). The presence of mitochondrial arginase-II is known as the ancestor enzyme, from which the cytosolic arginase-I is evolved. The several hypotheses are proposed regarding the evolution of arginase enzyme (Srivastava and Ratha, 2013). The isoform are evolved due to physiological force towards the different metabolic pathway. Localization of arginase isoforms are the most important criteria to determine its physiological function as by catalyzing the same reaction they are having different metabolic function. Mitochondrial membrane-bound localization is reported by Cheung and Raijman, (1981) which is further supported by Nissim et al., (2005) who proposed its role in ureogenesis. The channeling of substrate between the two compartments could be a possibility of origin of mitochondrial membrane-bound arginase. By doing so, it enhances the rate of the reaction and regulated the level of arginine in both cytosolic and mitochondrial compartment. Other than evolutionary pressure, the modification at transcript and protein level could be a cause of mitochondrial membrane-bound arginase. As arginase-I proposed to evolve from ancestral arginase-II and during this evolution mitochondrial targeting sequence of this arginase is lost, that results as its cytosolic localization (Srivastava and Ratha, 2013). There is also probability of alterations in mitochondrial targeting sequence of mitochondrial arginase-II that results as its association with mitochondrial membrane. Post translational modification in arginase-I and arginase-II also raised possibility of alterations of charge and detection of multiple charge variants (Gasiorowska et al., 1970; Kaysen and Strecker, 1973; Tarrab et al., 1974; Herzfeld and Raper, 1976; Cheung and Raijman, 1981; Porembska and Zamecka, 1985; Spolarics and Bond, 1988; Venkatakrishnan et al., 2003; Nissim et al., 2005; Venkatakrishnan and Reddy, 2010; Srivastava and Ratha, 2013).
The mitochondrial membrane-bound isoform is really an isoform or it is under the influence of misunderstandings or misinterpretation of an existing isoform of arginase. The assumptions related to the mitochondrial membrane bound arginase is triggered in this work and tried to overcome the misunderstandings of this enzyme, like if membrane-bound arginase is an isoform of arginase it may associate with mitochondrial membrane of other organisms also. What is the importance of membrane-bound arginase? Why it is associated to mitochondrial membrane? It is really a new isoform of arginase or it is similar to any one of the known isoform of arginase.
To study the aspects of mitochondrial membrane-bound arginase following objectives were designed-
1) To study distribution of mitochondrial membrane-bound arginase
2) To do biochemical and molecular characterization of mitochondrial membrane-bound arginase in fish
3) To do biochemical and molecular characterization of mitochondrial membrane-bound arginase in mice
4) In silico and molecular analysis of arginase isoforms
Review of literature
The evolution of metabolic processes would be expected as a result of selective pressure, on the other hand, could be deviated from the ideal in particular species. In the course of evolution subtle changes may occur in the metabolic context of a biochemical process, and some of its characteristics, even though still suitable, may no longer serve their original function as well. To understand a biochemical process, it is also necessary to examine it not only in its current metabolic context, but it is also necessary to examine its evolutionary history (Paulus, 1983).
The ureohydrolase superfamily includes arginase (EC:3.5.3.1), agmatinase (EC:3.5.3.11), formiminoglutaminase (EC:3.5.3.8) and proclavaminate amidinohydrolase (EC:3.5.3.22). These enzymes share a 3-layer alpha-beta-alpha structure, and play important roles in arginine/agmatine metabolism, the urea cycle, histidine degradation, and other pathways.
Agmatinase hydrolyses agmatine to putrescine, the precursor for the biosynthesis of higher polyamines, spermidine and spermine. Formiminoglutamase catalyses the fourth step in histidine degradation, acting to hydrolyse N-formimidoyl-L-glutamate to L-glutamate and formamide. Proclavaminate amidinohydrolase is involved in clavulanic acid biosynthesis. Clavulanic acid acts as an inhibitor of a wide range of beta-lactamase enzymes that are used by various microorganisms to resist beta-lactam antibiotics. As a result, this enzyme improves the effectiveness of beta-lactamase antibiotics.
Arginase [3.5.3.1], a manganese metallo-enzyme, catalyzes the metal dependent hydrolytic cleavage of the guanidine-group of arginine into ornithine and urea, and is one of the five members of the urea cycle enzymes.
Figure 1 Reaction mechanism of arginase
Presence of arginase was first observed by Kossel and Dakin, (1904) in liver. After that it’s, involvement in production of urea in ureotelic vertebrates (Hunter and Dauphini, 1925), and distribution of arginase in different organisms (Clementi, 1937) were described. Clementi suggest arginase was distributed to ureotelic mammals only. But no cycle of this kind which is known as “metabolic cycle” had been known before the Krebs and Hensleit, (1932) demonstrated its role in Ornithine-Urea cycle in which low molecular intermediate are formed cyclically. Krebs and Hensleit, (1932) conducted a series of experiments using liver slices and manometric assays to show that in presence of arginase ornithine and urea. Hellerman and Perkins, (1935) first showed its activation by bivalent metal ions including cobalt, nickel, manganese and iron. In 1940, Richards and Hellerman showed that the activity of pH inactivated arginase could be restored by manganese and iron ions. In 1956, partial purification was further improved by Robbins and Shield. They demonstrated that the activity of arginase was dependent on manganese ion and found the optimal pH at 9.2. Much work was done investigating inhibitors in 1970-1980. Bedino, (1977) studied the effect of the product and the inhibitor ornithine and proposed allosteric model for the enzyme activity. Much work has been done after investigating the inhibitors of arginase (Rosenfeld et al., 1975; Bedino, 1977; Pace and Landers, 1981). Later on, extensive study on the purification, distribution, and different physiochemical properties had done in different organisms (reviewed by Jenkinson et al., 1996).
Reaction Mechanism
The reaction of arginase is simply the hydrolysis of arginine into ornithine and urea. The nucleophilic attack leads to the formation of metastable tetrahedral intermediate. The metal ions are essential in transition state stabilization by keeping the metal-bridging hydroxide in position. The subsequent addition of a water molecule to the binuclear manganese cluster facilitates urea departure, which may trigger the ionization of the metal-bridging water molecule to regenerate the nucleophilic metal-bridging hydroxide ion (Ash, 2004).
The amino acid side chains that coordinate to the manganese ion in the active site are located on the edge of the central β-sheet in loop segments immediately adjacent to strands 8, 7, and 4. The binuclear cluster in the unliganded enzyme exhibits a MnA2+ – MnB2+ separation of 3.3Å in agreement with the separation measured in an electron paramagnetic resonance spectroscopic study (Sossong et al., 1997). The crystal structure of unliganded rat arginase-I reveals that MnA2+ is coordinated by H101, D128, D124, D232, and the metal bridging hydroxide ion with square pyramidal geometry (Kanyo et al., 1996). However, the crystal structure of the unliganded arginase-I reveal and additional water molecule coordinated with MnA2+, which completes a distorted octahedral metal coordination polyhedron. MnB2+ ion is coordinated by H126, D234, D124, D232, and a hydroxide ion with distorted octahedral geometry in rat arginase-I and human arginase-I. The structure of unliganded human arginase-I show that the metal bridging hydroxide ion and H141 interact with each other through hydrogen bonded water molecule. These interactions are consistent with the proposed role of H141 as a proton shuttle in the regeneration of the nucleophilic metal bridging hydroxide ion in catalysis.
The reaction mechanism suggested that this enzyme has a high degree of specificity with respect to chain length, stereo arrangement, and substituent’s of the α-carbon, and the guanidino ends of arginine (Ash, 2004).
Evolution of arginase
In the course of evolution, however, the single enzymatic step catalyzed by arginase may have been added to a preexisting multi-enzyme biosynthetic pathway for arginine (Paulus, 1983). It has been postulated that arginase got derived from an universal common ancestor before its divergence into archaea, eubacteria, and eukarya is complemented by the evolutionary analysis of these two pathways and helpful to understand the diverse physiological role and distribution of arginase (Ouzounis and Kyrpides, 1994).
Figure 2 Arginine biosynthetic pathway in E. coli
In E. coli arginine biosynthetic pathway is found for the production of arginine an essential amino acid, later on requirement of ammonia in Bacillus subtilis forced the hydrolysis of arginine into ornithine and urea. The urea produced here is utilized by the urease for the production of ammonia. By comparison the arginine metabolism in both organisms it was found that the additional step of arginase is to provide ornithine for the synthesis of glutamate by an intermediate glutamic semi aldehyde.
Figure 3 Arginine biosynthetic pathway in S. cerevisiae
The basic difference between the eukaryotes and prokaryotes is compartmentalization, thus by the comparison arginine biosynthetic pathway of simplest eukaryote Saccharomyces cerevisiae and Bacillus subtilis give an excellent example of regulation of biosynthetic pathway. In Bacillus subtilis the formation of ornithine occurs by the hydrolysis of N-acetylornithine, in Saccharomyces cerevisiae N-acetyl ornithine transfers its acetyl group to glutamate to yield ornithine and N-acetylglutamate. The transacetylation pathway to ornithine is more economical since it recycles the N-acetyl group and therefore does not consume a stoichiometric amount of acetyl-CoA. Under these conditions, the synthesis of N-acetylglutamate from acetyl-CoA serves as regulatory point of N-acetylglutamate synthetase. Like the enzyme of E. coli N-acetylglutamate synthetase of Saccharomyces cerevisiae is subject to the feedback inhibition by arginine but unlike the former, its sensitivity to inhibition by arginine is enhanced by N-acetylglutamate assuring that this product is synthesized only when its intracellular concentration declines.
Another lower eukaryote Neurospora crassa the arginine biosynthesis and degradation is similar to the Saccharomyces cerevisiae but Neurospora crassa has taken further opportunities for compartmentalization provided by the mitochondria and has confined all but the last two enzymes of arginine biosynthesis this organelle that separate the arginine catabolism to cytosol and the flux of catabolic ornithine cannot pass the mitochondrial membrane, hence confined to cytosol only whereas the biosynthetic ornithine converted into citrulline inside the mitochondria. The separation of anabolic and catabolic pool was an adaptation to the selective pressure toward the minimization of the energy cost to produced arginine and ornithine.
Arginase-I is one of the two major isoforms of arginase found in cytosol of liver of ureotelic organisms (Dkhar et al., 1991; Jenkinson et al., 1996). Arginase-II is the second major isoform of arginase reported in mitochondria of hepatic and extra hepatic tissues of various organisms (Glass and Knox, 1973; Spector et al., 1983; Vockley et al., 1996; Joerink et al., 2006). The distribution of arginase in plants is to provide proline and acts as reservoir of nitrogen source. The polyamine production is also an important factor for presence of arginase in developing tissue of plant or high activity is found in developing tissue. In variety of invertebrates, like as Bipalium kewense (Reddy and Campbell, 1970), Helix aspersa (Porembska, 1973) and Hyalophora gloveri (Reddy and Campbell, 1969), Lumbrichus terrestris (Reddy and Campbell, 1968), and Pheretima communissima (Reddy and Campbell, 1968) arginase is involved in the production of proline and glutamate for the muscle formation.
Figure 4 Arginine metabolic pathways in plants, invertebrates, fish, bird and reptiles
Fishes have variability in distribution of arginase-I in tissue as well as sub-cellular level. Clarias batrachus comprises two differently charged variants after bidirectional electrophoresis. Such charge variants may be due to the presence of different arginase isozymes in the same tissue and have been repeatedly observed in a variety of mammalian tissues. The ureoosmotic fishes have mitochondrial arginase-II in all tissues (Casey and Anderson, 1985) whereas air breathing cat fishes have arginase-I in cytosol and arginase-II in mitochondria of hepatic and extra hepatic tissues (Dkhar et al.,1991).
Amphibians have cytosolic arginase-I and mitochondrial arginase-II in their hepatic and extra hepatic tissues (Xu et al.,1993; Patterton and Shi, 1994). However, amphibians could be a good model to understand the evolution of ureotelism from ammoniatelism. The evident molecular masses of arginase from the liver and kidney of the frog Rana tigerina varied with the protein concentration of extracts, due to protein aggregation. It was also proposed that the smaller form was an active subunit of a heteromeric adult enzyme. Not only charge difference but, variable extra hepatic arginases have been cloned from X. laevis, having sub- unit molecular masses of about 40 kDa, slightly larger than the hepatic enzyme, and are induced at metamorphosis and also by thyroxine in a tissue and temporal pattern distinct from the liver enzyme (Venkatakrishnan and Reddy, 1993).
In birds and reptiles, arginase-I is not present whereas, arginase-II is present in mitochondria of all tissues (Grazi and Magri, 1975; Baby and Reddy, 1980; Baby et al.,1976). Till now limited information is present related to the presence of arginase-I, in birds and reptiles. In some birds ARG I is found at transcript level, but at proteomic level distribution and characterization of arginase-I is required.
Primarily arginase-I was identified in liver of rat (Gassiorowska et al., 1970; Tarrab et al.,1974; Skrzypek-Osiecka et al., 1983; Gotoh et al., 1996), mouse (Spolarics and Bond, 1988) and human (Bruter and Colombo, 1978; Takiguchi et al., 1987).
The arginase has been crystallized from liver of rat and human (Kanyo et al., 1996; D’Costanzo et al., 2005). The crystal structure of rat liver arginase reveals that it is member of α/β protein class. Each monomer has eight parallel β-sheets and ten α-helices. The carboxy terminal of each monomer has ‘S’ shape motif for intermolecular interaction. This motif is helpful in trimerization of arginase-I monomers with the help of hydrogen bonds, salt links and Vanderwaal’s interactions (Kanyo et al., 1996). The crystal structure of arginase-II is identical to crystal structure of arginase-I (Christianson et al., 2003, Kanyo et al., 1996). Additional salt bridge and hydrogen bond are reported in monomer of arginase-II in comparison to rat and human liver arginase-I (Cama et al., 2003).
Perozich et al., (1997) aligned the arginase sequences of 31 different organisms and with the help of crystal structure proposed by Kanyo et al., (1996) stated that arginase protein is conserved in at least 25 organisms (Perozich et al., 1997). They reported 32 residues as conserved residues and conservative substitutions. Glycine and Proline are more abundant in these conserved residues. Glycine and Proline are playing important in the folding of proteins. The catalytic site of this arginase is highly conserved so that their function is not much influenced during the course of evolution.
The activity of arginase in particular S-nitrosylation of Cys-303 stabilizes the arginase trimer resulting in a six fold decrease in its Km for arginine. The Cys 168 is conserved in mammalian arginase-I and arginase-II but Cys 303 is present in arginase-I not in arginase-II suggested that S-nitrosylation may not alter the activity of arginase-II (Santhum et al., 2007; 2008).
The native form of arginase-II contains complete mitochondrial targeting sequence whereas the arginase-I lost its mitochondrial targeting sequence and becomes cytosolic in mammals during the course of evolution (Cederbaum et al., 2004), while mitochondrial targeting sequence were predicted in both isoforms arginase-I and arginase-II in Cyprinus carpio (Joerink et al., 2006).
Arginase from ureotelic species has been reported to be trimeric with molecular weights of 100 -120 kDa, and sub-unit molecular weight of about 30 kDa (D’Costanzo et al., 2005). Molecular mass of native arginase from the liver of elasmobranchs, Squalus acanthias, and teleost, Clarias batrachus, were observed to be 100 and 87 kDa, respectively (Casey and Anderson, 1985; Singh and Singh, 1990). In contrast, arginase from uricotelic species has molecular weights between 220 -280 kDa. The number of subunits reported for arginase from different organisms ranges from one to eight such as a monomer in earthworm, a trimer in mammalian liver, hexamer in iris bulbs and bacteria, octamer in planarian and land snails (Jenkinson et al., 1996). In Mycoplasma arginine, the subunit of arginase is 60 kDa which is largest among all known subunit molecular weight of arginase
Optimum pH of arginase from elasmobranchs and teleosts ranges from 9.5 to 9.8, but varries from 9.5 to 10.5 in mammalian arginase (Jenkinson et al., 1996). An exception of optimum pH for Ox erythrocyte arginase has been reported to be 11.5 (Patil et al., 1990). The variation of activity with pH suggests the presence of an ionizable group at the catalytic site. The arginase from liver of beef, rabbit and buffalo were stable at 55°C (Dabir et al., 2005).
The Km for mammalian arginase exhibited wide variation from 1-20 mM for rat liver arginase, and 4-45 mM for other tissues. In teleost Km of arginase for L-arginine is 9.1 mM (Carvajal et al., 1987), and in Clarias batrachus 12.5 mM (Singh and Singh, 1990), Squalus acanthias, 1.2 mM (Casey and Anderson, 1982). Urea synthesis in this dogfish occurs mainly for osmoregulation, where low Km value for arginine favors formation of urea as an osmolyte. However, arginase from uricotelic species has higher Km values between 100 to 200 mM (Mora et al., 1965). The variation in mM concentration of L-arginine may also be due to variation in assay procedures and non-physiological conditions used in study (Pace et al., 1980; Ganganta and Bond, 1986). Substrate inhibition observed at higher concentrations of L-arginine was reported earlier for buffalo liver arginase (Dabir et al., 2005).
Several inhibitors were studied for arginase but no isoforms specific inhibitor is available till now. At lower concentration of N-Omega-Hydroxy-L-Arginine (NOHA), is reported to inhibit the arginase-I but at high concentration it inhibited arginase-II. Ornithine is also reported as the competitive inhibitor of arginase (Srivastava and Ratha, 2013).
The catalytic centre of arginase have manganese (Mn2+) that binds with the hydroxide molecule and help the hydrolytic cleavage (Dowling et al.,2008). Besides Mn2+, Co2+, and Ni2+ were also reported to enhance the enzymatic activity of arginase (Ash, 2004; Srivastava and Ratha, 2013).
The gene of ARG I is located on the 6q23 chromosome of Homo sapiens and have 11.5 kb long gene contains 8-exons. The promoter region of human liver arginase contains binding sites of several transcription factors such as TATA box, CTF/NF1, ‘CAAT’ box, GRE, CRE and EC sequences (Patterton and Shi, 1994; Haraguchi et al., 1987). The gene of ARG II is reported in 14q24 chromosome in human that produces 1.5 kb long transcript coded for 354 aa long protein (Vockley et al., 1996). The gene of ARG II contains 8-exons similar to ARG I. The 5’ UTR of murine ARG II have binding site for numerous transcription factors such as CRE-BP2, NF-KB, and AP1. In contrast to ARG I sequence, TATA-box like sequence is absent from 5’ UTR of ARG II.
In vertebrates, such as rat liver, the arginase is reported in cytosol for synthesis of urea and in mitochondria for glutamate and proline. A major difference between eukaryotic microbes and higher vertebrates are different environmental conditions. These adaptations for the survival lead the progressive loss of some of the more complex biosynthetic capabilities. Accordingly, there may be a close relationship between the loss of the capacity to synthesize arginine and the acquisition of the capacity to synthesize urea in the course of metazoan.
Figure 5 Arginine biosynthetic pathway in teleosts, amphibian and mammals
Arginase has been observed in microorganisms (Issaly and Issaly, 1974; Moreno-Vivan et al., 1992; Schrell et al.,1989; Szumanski and Boyle, 1990; Patchett et al.,, 1991; Chan and Cossins, 1973), plants (Kolloffel and Dijke, 1975; Kang and Cho, 1990), invertebrates (Reddy and Campbell,1968; Tramell and Campbell, 1972; Iino and Shimadate, 1986), and vertebrates (Grazi et al., 1975; Cheung and Raijman, 1981; Terayama et al., 1982; Skrzypek-Osiecka et al.,1983; Casey and Anderson, 1985; Dkhar et al., 1991; Venkatakrishnan and Reddy, 2010). Although it catalyzes hydrolysis of arginine to ornithine and urea, physiological functions of the products are diverse in different organisms (Jenkinson et al., 1996). In vertebrates, two major isoforms have been observed (Spector et al., 1985; Grody et al., 1989) however, only one type of arginase has been suggested in micro-organisms, plants and invertebrates (Chan and Cossins, 1973; Kolloffel and Dijke, 1975; Schrell et al., 1989; Szumanski and Boyle, 1990; Kang and Cho, 1990; Patchett et al., 1991; Moreno-Vivan et al., 1992). The genes of arginase are distinct and evolved independently (Patterson and Shi, 1994; Jenkinson et al., 1996; Perozich et al., 1998). Multiple forms of arginase have also been proposed (Tarrab et al., 1974; Grazi et al., 1975; Herzfeld and Raper, 1976; Porembska et al., 1980; Cheung and Raijman, 1981; Porembska and Zamecka, 1984; Cederbaum et al., 2004, Nissim et al., 2005) that needs reconsideration (Häussinger, 1990; Jenkinson et al., 1996; Wu and Morris, 1998; Cederbaum et al., 2004; Dowling et al., 2008) including mitochondrial membrane-bound arginase.
Physiological Function of Arginase
Arginase was also found critical for synthesis of glutamate, proline, and polyamine (Gotoh et al., 1996, Cederbaum et al., 2004). Among other nitrogen metabolizing enzymes, arginase was extensively studied because its isoforms proved important in deciphering structural and functional evolution of enzyme. There are reports in light of isoforms and role of arginase in the regulation of nitric oxide synthesis suggested that the conventional understanding of physiological role of the arginase in urea genesis may therefore need to be reconsidered (Mishra and Mishra, 2015).
Figure 6 Schematic representation of metabolic role of Arginase
(A) Arginase a potential regulator of the O-U cycle
Compared to other urea cycle enzymes, arginase has been widely distributed regulating important pathophysiological activities (reviewed by Jenkinson et al., 1996; Cederbaum et al., 2004). It is a potential role as a regulator of the synthesis of ornithine, urea, creatine, polyamines, glutamate, proline, NO and agmatine (Wu and Morris, 1998; Morris, 2007; Wu et al., 2009). Different isoforms are initially described on the basis of mode of nitrogen detoxification. In all other organisms’ urea is either a way of nitrogen detoxification or as source of carbon di-oxide and ammonia (Kolloffel and Dijke, 1975; Kang and Cho, 1990). After the enhanced sensitivity of the assay procedure, arginase-II was reported as the mitochondrial isoform involved in the metabolic pathways other than the nitrogen-detoxification. However, in the ureoosmotic fish arginase-II is involved in the synthesis of the urea for the osmotic balance to the marine water (Casey and Anderson, 1985).
(B) Role of arginase in cellular proliferation, differentiation and neurological complications
Polyamines are the important factor for the cellular proliferation and differentiation. Arginase appeared as a regulatory point of the cellular proliferation by providing the substrate ornithine. The polyamines are the product of the ornithine catalyzed by the Ornithine Decarboxylase (ODC). It is the key rate-limiting enzyme for the biosynthesis of polyamines (putrescine, spermidine, and spermine) which play a pivotal role in the control of DNA, RNA, and protein synthesis during cell growth, differentiation, and transformation of cells (Jenkinson et al.,, 1996). The roles of polyamines in cellular proliferation appeared an area of interest to study the regulation of pool of arginine and the role of arginase as a potential molecular marker of carcinogenesis (Feun et al., 2015). The elevated levels of arginase-I and polyamines are reported (Kropf et al., 2005), but the mechanism of regulation of pathological conditions by arginase needs attention.
Ornithine gets converted into glutamate and proline by Ornithine Amino Transferase (OAT). Proline is an essential component of structural proteins like collagens (Gordon and Hahn, 2010) and glutamate act as neurotransmitter and also help in synthesis of another neurotransmitter Gamma Amino Butyric Acid (GABA). It is the only way of incorporation of the ammonia into the metabolic pool that regulates ammonia metabolism. Glutamate is synthesized by amination of α-ketoglutarate by Glutamate Dehydrogenase (GDH), and further converted to glutamine by Glutamine Synthetase (GS). Increased glutamate and glutamine concentration is responsible several neurological implications such as accumulation of the glutamine lead to swelling of astrocytes, glutamine induced excitation, neuroinflamation etc. (Marcaida et al., 1992; Willard –Mack et al., 1996; Butterworth, 2000; Rao and Norenberg, 2001; Rao et al., 2005; Tanigami et al., 2005; Rao et al., 2005; Cagnon and Braissant, 2007; Ljubisavljevic et al., 2014). Lange et al., (2004) reviewed the role of arginase in the brain and neurodegeneration. They found age dependent expression of arginase-I in different regions of rat hippocampus. Lee et al., (2003) showed up regulation of arginase-I by cAMP and interferon γ. But there is need to study the transcriptional and translation regulation of arginase isoforms in the brain.
(C) Role of arginase in endothelial dysfunction, vascular and cardiovascular diseases
The arginase has been observed critical in the endothelial, vascular and cardiovascular diseases after the existence of Nitric oxide. Nitric oxide is synthesized from arginine by nitric oxide synthetase (NOS) and the new centre of attraction in arginine degradation pathway. NOS get reciprocally regulated by sharing L-arginine as a common substrate (Morris, 2009). NOS is found in three main forms –the two consecutive and Ca2+ dependent forms nNOS and eNOS found in brain/ neuronal cells and Ca2+ independent inducible form (iNOS) found in activated macrophages and neutrophils that play an important role in host defense mechanisms (Ni et al., 2001). Availability of arginine is important for NO biosynthesis. NO, thus, plays important role in regulating many physiological processes falling into two broad categories: cell signaling such as endothelium used nitric oxide to signal the surrounding smooth muscle to relax and thus, dilating the artery and increasing the blood flow and host defense mechanisms (Li et al., 2001). Role of arginase in regulating the rate of NO synthesis from arginine has been reported by controlling the arginine concentration in situ (Mori and Gotoh, 2004; Mori, 2007). NOS have higher Km than arginase suggested the tight regulation of these two proteins (Cederbaum et al., 2004).
Role of arginase has been reported in cardiovascular diseases (Chen et al., 2013), gets up-regulated in vascular diseases (Durante et al., 2007; Mori, 2007; Lucas et al., 2014), endothelial dysfunction (Toque et al., 2013). Arginase expression in these cells can be induced by IL-4 and IL-3 (Wei et al., 2001), TGF-β (Durante et al., 1997) and mechanical strain (Durante et al., 2000). Arginase is not modulate the NO synthesis, while function in synthesis of proline and polyamine for cell proliferation and collagen synthesis (Durante et al., 2007). The elevated arginase expression in vascular smooth muscle is observed as an important factor in development of intimal hyperplassia followed by vascular injury (Marinova, 2008). Morris et al., (2009) reviewed and concluded that a wide range of agents induces arginase expression when administered in cultured cells –LPS, LPS+TNF-a, TNF-a, H2O2, thrombin, high glucose, LDL, genistein, cocoa flavonoid, simvastatin. Whereas, hypertension, ischaemia-reperfusion, intimal hyperplasia, and ageing are some conditions which induced the endothelial arginase level. It has been found that the endothelial NO synthesis depends on the activity of mitochondrial arginase and L-arginine carriers in cell membrane (Topal et al., 2006; Yang et al., 2006). Yang et al., (2006) suggested the role of the arginine transporter (y+) have important role in the arginine homeostasis and endothelial cells have arginine transporter. They studied the effect of thrombin on arginase and NOS isoforms in Human Umbilical Vein Endothelial Cells (HUVEC). They observed the upregulation of arginase-II but not arginase-I in thrombin induced HUVEC at protein level. However, at the transcriptional level the neither arginase-I nor arginase-II was changed. Besides this no change were found in NOS isoforms at transcriptional as well as protein level. They suggest thrombus clotting factor II induces the arginase-II at activity as well as protein level. Ming and colleagues (2004) were also observed up regulation of arginase-II by induction of thrombin in endothelial cells. However, Berkowitz et al., (2003) reported the induction of arginase-I in endothelial dysfunction. Bachetti et al., (2004) suggested the presence of arginase-I and arginase-II both are present and functionally active in endothelial cells and they have important role in the cell cycle and their inhibition leads to the growth inhibition in human. It seems that the precise consequences of expression of arginase in endothelial cells will depend upon the animal species, the relative activities of enzymes such as NOS, and nature of the various stimuli to which the cells are exposed.
(D) Role of arginase in Cancer
The arginase directly influences the status of different diseases as a key regular of arginine homeostasis due to its high affinity and specificity for arginine (Lind et al., 2004). In recent years arginase is looked for the potential marker of carcinoma (Munder et al., 2009; Dzik 2014) because it is involved in the synthesis of the polyamines, a cell proliferation agent and also regulated the arginine homeostasis. The involvement of arginase is reported in prostate cancer (Kedia et al., 2012), gall bladder cancer (Shukla et al., 2009), breast cancer (Singh et al., 2000; Porembska et al., 2003), hepatocellular carcinoma (Chrzanowska et al., 2014) and Pancreatic cancer (Ino et al., 2013). The simple diagnosis method and the mark differentiation of the arginase isoforms make them to be explored as marker for diagnosis. However, differential expression of arginase isoforms is again intriguing in case of malignancies. The presence of the arginase-I and arginase-II isoforms in the serum of HCC and liver carcinoma patients was observed (Chrzanowska et al., 2014). However, they did not find arginase-II in normal serum. They suggested arginase-II as a potential marker of the cancer diagnosis. It has also been reported in the case of the colorectal cancer (Porembska et al., 2003) where presence of arginase-I and arginase-II was present in tissue but arginase-I was detected in the serum only. Several ways were proposed that targets arginase and arginine for the therapeutics for different malignancies. Use of inhibitors for the arginase is the first and the most common method proposed for the treatment of the cancer. The N-Omega-Hydroxy-L-Arginine (NOHA), Norvaline were reported as useful for cancer treatment (Singh et al., 2000; Wheatley et al., 2003) Second method used in the therapeutics of the cancer related to arginase is arginine deprivation method. Campbell and Wheatley reported the use of arginine deprivation method is useful for the enzymatic degradation of arginine in the different malignant cell culture. Depletion of arginine, an important substrate for the cellular growth and differentiation, inhibits the growth of malignant cells.
Recently, the use of recombinant arginase (HuArgI(Co)-PEG5000) has been suggested for the therapeutics of the cancer (Tanios et al., 2013). Specifically for acute myeloid Leukemia cells shown arginine auxotrophy. They can be a selectively targeted. The HCC rhArg-PEG induced arginine depletion regulates cell cycle arrest in the at the G2/M or S phase by transcriptional regulation of cyclins or cyclin dependent kinases (CDKs). They proposed the application of the recombinant arginase alone or in combination with the existing chemotherapeutics drugs could be an effective therapeutic strategy against HCC (Lam et al., 2008). The recombinant human arginase-I has also been suggested as a promising candidate for cancer therapy without any adverse immune response (Stone et al., 2012).
(E) Role of arginase in Diabetes
The arginase activity is diminished in endothelial cells of the diabetic rat however which isoforms is reduces is not identified. In contrast, the arginase activity and expression of arginase-I isoforms is elevated in aortas of streptozotocin diabetic rats (Romero et al., 2008) but not determine that it is present in endothelium and/or vascular smooth muscle. Kampfer et al., (2003) suggested the presence of arginase-II along with the arginase-I in the diabetes induced skin repair. However, they reported arginase-I as the predominant isoform in diabetes induced skin repair.
(F) Role of arginase in immuno-modulation
Arginase is not directly involved in the immune related responses but being the key regulator of the arginine homeostasis it is observed an interesting candidate for immune modulation studies of several diseases. Arginine supplementation was reported as major uses in the T-cell proliferation. Kitayama et al., (2014) reported to the high arginase-I expression in mesothelial cells. The increased arginase-I suppressed the T-cell proliferation by inhibition of the CD3ζ chain expression and L-arginine supplementation restores the suppression of T-cell proliferation. Similarly role of L-arginine and arginase was observed in the T-cell proliferation (Bhatt et al., 2014; Kang et al., 2014). The arginase and its role of immunity is an emerging area to study.
Mitochondrial membrane bound arginase:
Two major isoform of arginase is well known and characterized. Besides these two, other isoforms of arginase is also reported; differ in ionic charge and immunological properties. The most study of arginase isoform is done in mammals and more than two isoforms are proposed but only two were characterized till now summarized in the table given below.
Table Shows different arginase variants on the basis of Charge and immunological properties.
The mitochondrial membrane bound arginase is one of them that were proposed on the basis of their sub-cellular localization. The mitochondrial membrane bound arginase is extracted by 150 mM KCl and have 60% of the mitochondrial arginase activity, and proposed to have role in ureagenesis. The mitochondrial membrane bound arginase is associated to other organelle, like nucleus, endoplasmic reticulum but enriched in mitochondrial fraction most (Skrzypek-Osiecka et al., 1980). Cheung and Raijman, (1981) first demonstrated the association of mitochondrial membrane bound arginase to outer mitochondrial membrane and show the mitochondrial membrane bound arginase activity that is 60% of the total mitochondrial arginase activity. Later on, Nissim et al., (2005) studied role of mitochondrial membrane bound arginase in ureogenesis. They also suggested the 90% involvement of mitochondrial membrane bound arginase activity associated to the outer mitochondrial membrane.
The isoform are evolved due to physiological force towards the different metabolic pathway. The gene duplication event is responsible for the two isoform of arginase that is cytosolic arginase-I and mitochondrial arginase-II. The presence of mitochondrial arginase is known as the ancestor enzyme, from which the cytosolic form is evolved. Distribution of these two isoforms was different at organism, tissue and cellular level.
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