Cloning of Fish Enzymes and Other Fish Protein Genes

ABSTRACT:?

Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.     

Cloning and characterization of cytoplasmic carbonic anhydrase from gills of four Antarctic fish: insights into the evolution of fish carbonic anhydrase and cold adaptation

Although carbonic anhydrase is a ubiquitous enzyme involved in a variety of physiological processes, the information on its evolution and cold adaptation among Antarctic fish is still limited: the only Antarctic fish carbonic anhydrase characterized up-to-date is from Chionodraco hamatus, a member of the Channichthyidae family. In this work, we characterized orthologous genes within two other fish families: Nototheniidae (Trematomus eulepidotus, Trematomus lepidorhinus, Trematomus bernacchii) and Bathydraconidae (Cygnodraco mawsoni). The cDNAs of epithelial gill carbonic anhydrases were cloned and sequenced. Both coding and deduced amino acid sequences were used in phylogenetic analyses. The group of enzymes preferentially expressed in fish erythrocytes (CAIIb) represented the most conserved variant. This result suggests that, although the two variants derived from the same ancestor, CAIIc genes have a more complex evolutionary history than CAIIb. The peculiar distribution of Antarctic CAs among fish CAIIcs suggests that the CAIIc gene appeared at different times through independent duplication events, even after the speciation that led to the differentiation of Antarctic fish families. Using the new CA sequences, we built homology models to trace the expected consequences of sequence variability at the protein structure level. From these analyses, we inferred that sequence variability in Antarctic fish CAs affect important physicochemical properties of these proteins and consequentially influence their reactivity. Furthermore, we searched and tested the validity of various potential molecular trademarks for cold adaptation: significant features that can be related to cold adaptation in fish CAs include reduction of positively charged solvent accessible surfaces and an increased flexibility of N-terminal and C-terminal regions.     

General review of fish protein hydrolysates

Fish protein hydrolysates are generally considered to be the liquefied products obtained from fish by the action of proteolytic enzymes under accelerated conditions of digestion. The proteolytic enzymes which are used for the digestion of fish proteins must be active either above the survival temperature of spoilage bacteria or outside the range of pH which would support their growth. By suitable selection of proteolytic enzymes and conditions for hydrolysis some control over the digestion can be achieved to give a range of products. This paper reviews the work done here and elsewhere on fish protein hydrolysates and discusses their potential for making more effective use of the fish resources.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Ribonucleases and angiogenins from fish

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities.     

Proteomic response to sublethal cadmium exposure in a sentinel fish species, Cottus gobio

The present study aimed at evaluating the toxicity of short-term cadmium (Cd) exposure in the European bullhead Cottus gobio, a candidate sentinel species. Several enzymatic activity assays (citrate synthase, cytochrome c oxidase, and lactate dehydrogenase) were carried out in liver and gills of fish exposed to 0.01, 0.05, 0.25, and 1 mg Cd/L for 4 days. Exposure to high Cd concentrations significantly altered the activity of these enzymes either in liver and/or in gills. Second, 2D-DIGE technique was used to identify proteins differentially expressed in tissues of fish exposed to either 0.01 or 1 mg Cd/L. Fifty-four hepatic protein spots and 37 branchial protein spots displayed significant changes in abundance in response to Cd exposure. A total of 26 and 12 different proteins were identified using nano LC-MS/MS in liver and gills, respectively. The identified differentially expressed proteins can be categorized into diverse functional classes, related to metabolic process, general stress response, protein fate, and cell structure for instance. This work provides new insights into the biochemical and molecular events in Cd-induced toxicity in fish and suggests that further studies on the identified proteins could provide crucial information to better understand the mechanisms of Cd toxicity in fish.     

Steroid catabolism in marine and freshwater fish

Steroids play important roles in regulating many physiological functions in marine and freshwater fish. Levels of active steroid in blood and tissues are determined by the balance between synthetic and catabolic processes. This review examines what is known about pathways of catabolism of steroids, primarily sex steroids, in marine and freshwater fish. Cytochrome P450 (P450) isoforms present in hepatic microsomes catalyze steroid hydroxylation to metabolites with lower or no activity at estrogen or androgen receptors. Important pathways of steroid catabolism to readily excreted metabolites are glucuronidation and sulfonation of hydroxyl groups. Estradiol, testosterone, DHEA and hydroxylated metabolites of these and other steroids readily form glucuronide and sulfate conjugates in those fish species where these pathways have been examined. Little is known, however, of the structure and function of the UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) enzymes involved in steroid conjugation in fish. Glucuronide and sulfate conjugates of steroids may be transported into and out of cells by organic anion transporter proteins and multi-drug resistance proteins, and there is growing evidence that these proteins play important roles in steroid conjugate transport and elimination. Induction or inhibition of any of these pathways by environmental chemicals can result in alteration of the natural balance of steroid hormones and could lead to disruption of the endocrine system. Recent studies in this area are presented, with particular focus on phase II (conjugative) pathways.     

Characterization and comparison of fatty acyl Delta6 desaturase cDNAs from freshwater and marine teleost fish species

Fish are the most important dietary source of the n-3 highly unsaturated fatty acids (HUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), that have particularly important roles in human nutrition reflecting their roles in critical physiological processes. The objective of the study described here was to clone, functionally characterize and compare expressed fatty acid desaturase genes involved in the production of EPA and DHA in freshwater and marine teleost fish species. Putative fatty acid desaturase cDNAs were isolated and cloned from common carp (Cyprinus carpio) and turbot (Psetta maximus). The enzymic activities of the products of these cDNAs, together with those of cDNAs previously cloned from rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), were determined by heterologous expression in the yeast Saccharomyces cerevisiae. The carp and turbot desaturase cDNAs included open reading frames (ORFs) of 1335 and 1338 base pairs, respectively, specifying proteins of 444 and 445 amino acids. The protein sequences possessed all the characteristic features of microsomal fatty acid desaturases, including three histidine boxes, two transmembrane regions, and N-terminal cytochrome b(5) domains containing the haem-binding motif, HPGG. Functional expression showed all four fish cDNAs encode basically unifunctional Delta6 fatty acid desaturase enzymes responsible for the first and rate-limiting step in the biosynthesis of HUFA from 18:3n-3 and 18:2n-6. All the fish desaturases were more active towards the n-3 substrate with 59.5%, 31.5%, 23.1% and 7.0% of 18:3n-3 being converted to 18:4n-3 in the case of turbot, trout, sea bream and carp, respectively. The enzymes also showed very low, probably physiologically insignificant, levels of Delta5 desaturase activity, but none of the products showed Delta4 desaturase activity. The cloning and characterization of desaturases from these fish is an important advance, as they are species in which there is a relative wealth of data on the nutritional regulation of fatty acid desaturation and HUFA synthesis, and between which substantive differences occur.     

Effects of organic pollutants on fish digestive enzymes: A review

A brief overview of data on the effects of organic pollutants of different chemical natures (organochlorine, organophosphorous and organotin compounds, naphthalene, formalin, nitrosoguanidine, glyphosate, and metylmercury) on the activity of fish enzymes hydrolyzing basic food components is given. It is shown that the xenobiotics listed above have different effects on fish digestive enzymes. The directions of toxicant-induced changes may differ depending on the fish species, type of hydrolyzed substratum, diapason of toxicant concentrations, and experimental conditions.     

Determination of the heterogeneous interactome between Edwardsiella tarda and fish gills

Edwardsiellosis caused by Edwardsiella tarda is a frequent occurrence throughout the world and has resulted in extensive losses in aquaculture. However, information regarding to protein-protein interaction between the pathogenic cells and host is not available although the portal of entry of the pathogen is determined. In this study, fish gill and bacterial pull-down approaches were used to isolate both bacterial outer membrane proteins that bind to gills and fish gill proteins that interact with bacterial cells, respectively. Eight interacting bacterial proteins and twelve interacting fish proteins were obtained. The genes of seven bacterial proteins were cloned and expressed for preparation of antibodies. The prepared antibodies were used to investigate protein-protein interactions between bacterial cells and fish gills. Five heterogeneous protein-protein interactions were determined. Moreover, the protective ability of three of the bacterial recombinant proteins, selected at random, was investigated in a mouse model where they showed significant protection. The gill proteins were highly homologous proteins with from humans and other animals where they are known to be involved in host immunity. These findings indicate that the heterogeneous interactome has significantly biological significance. Our results demonstrate a way to determine and understand the heterogeneous interaction between of E. tarda and gills.     

Comparison of in vitro systems of protein digestion using either mammal or fish proteolytic enzymes

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.     

Purification and properties of the hepatic glutathione S-transferases of the Atlantic salmon (Salmo salar)

Salmon from salt water have three classes of soluble hepatic glutathione S-transferases which can be separated from cytosol by affinity chromatography and chromatofocusing. The classes have different substrate specificities and kinetic properties. All the enzymes are dimeric proteins. There are immunologically distinct subunits of Mrs 22.4, 23.0 and 24.0 kDa. Fish killed in August have enzymes with different apparent isoelectric points and subunit compositions than fish killed in February. The glutathione S-transferase activity of fresh-water salmon is similar to that of February salt-water fish except that the former binds less avidly to S-hexylglutathione-Sepharose 6B.     

A new type of lectin discovered in a fish, flathead (Platycephalus indicus), suggests an alternative functional role for mammalian plasma kallikrein

A skin mucus lectin exhibiting a homodimeric structure and an S-S bond between subunits of ~40 kDa was purified from flathead Platycephalus indicus (Scorpaeniformes). This lectin, named FHL (FlatHead Lectin), exhibited mannose-specific activity in a Ca(2+)-dependent manner. Although FHL showed no homology to any previously reported lectins, it did exhibit ~20% identity to previously discovered plasma kallikreins and coagulation factor XIs of mammals and Xenopus laevis. These known proteins are serine proteases and play pivotal roles in the kinin-generating system or the blood coagulation pathway. However, alignment analysis revealed that while FHL lacked a serine protease domain, it was homologous to the heavy-chain domain of plasma kallikreins and coagulation factor XI therefore suggesting that FHL is not an enzyme but rather a novel animal lectin. On the basis of this finding, we investigated the lectin activity of human plasma kallikrein and revealed that it could indeed act as a lectin. Other genes homologous to FHL were also found in the genome databases of some fish species, but not in mammals. In contrast, plasma kallikreins and coagulation factor XI have yet to be identified in fish. The present findings suggest that these mammalian enzymes may have originally emerged as a lectin and may have evolved into molecules with protease activity after separation from common ancestors.     

Stress and interpopulation variation in glycolytic enzyme activity and expression in a teleost fish Fundulus heteroclitus

Northern populations of Fundulus heteroclitus have twofold greater activity of lactate dehydrogenase-B (LDH-B) than southern populations, but exposure to stress increases LDH-B in southern populations, abolishing this difference. To test whether differences in the activity of other hepatic glycolytic enzymes between populations are sensitive to stress, we injected fish with a pharmacological dose of cortisol in coconut oil (400 microg g(-1)) or exposed them to handling stress and measured the activities of all the glycolytic enzymes. At rest, liver phosphofructokinase (PFK) and aldolase (ALD) activities were greater in southern fish, whereas LDH-B activity was greater in northern fish. No other glycolytic enzymes differed in activity between populations in control fish. Cortisol injection and handling stress decreased PFK and ALD and increased LDH activities in the southern but not the northern population, such that the populations no longer differed in the activity of any enzyme following treatment. Unlike Ldh-B mRNA, Pfk and Ald mRNA levels did not parallel enzyme activity, suggesting complex kinetics or regulation at multiple levels. Plasma cortisol did not differ between populations at rest but was significantly different between populations in treated fish. These data suggest that differences in liver enzyme activity may be related to differences in stress hormone physiology between populations.     

鱼类对重金属胁迫的分子反应机理

水体中浓度超标的重金属对鱼类的正常生命活动产生了严重影响,甚至导致鱼类的死亡.鱼体受重金属离子胁迫时体内会产生一系列相关基因的表达变化,并合成相关的蛋白和酶,如热激蛋白、金属硫蛋白、转运铁蛋白、谷胱甘肽转移酶、过氧化氢酶、超氧化物歧化酶等以应对重金属对机体产生的危害.这是生物体对环境的一种应激保护性反应,是生物体对周围环境中过量重金属的一种防御性机制.通过鱼类在重金属胁迫下组织中相关基因的表达可以从分子水平上来阐述有毒物质对鱼体产生的影响.综述了重金属离子胁迫下鱼类机体的分子反应机理.     

环境DNA技术在鱼类生态学中的应用研究进展

全球渔业衰退是21世纪人类面临的重要挑战之一。为了有效地遏制鱼类资源的衰退,精确的鱼类生态调查是其首要任务。传统的鱼类监测以渔获物采集与形态学鉴定为主,往往耗时耗力且效果不佳,已无法满足现阶段大尺度上的精确调查。环境DNA （eDNA）技术作为一种近年来新兴的鱼类生态调查方法,其与传统方法相比具有灵敏度高、经济高效、采样受限小且对生态系统无干扰的优势,目前其已被广泛地应用于鱼类物种监测、多样性调查、生物量评估以及繁殖活动监测等方面的研究。然而,eDNA技术在鱼类生态学研究的具体应用中暴露出的一些问题将会影响其监测结果的精确性,诸如操作流程的不规范、基因数据库的不完善以及eDNA在环境中生态学过程的不明确等。鉴于上述原因,首先对eDNA技术的发展历程、分析流程以及eDNA技术在鱼类生态学研究领域中的研究进展进行了综述,而后着重分析了eDNA技术的发展当前所面临的困难与挑战,并提出了相应的解决方案,最后对eDNA技术未来在鱼类生态学研究领域中的发展趋势做出了展望。通过本研究,以期能够为eDNA技术在鱼类生态学领域中的准确应用提供理论基础。     

Monoclonal antibodies that immunoreact with a cation-stimulated plant membrane ATPase.

Hybridoma technology has been used successfully to generate monoclonal-antibody probes against protoplast membrane antigens. Hybridomas secreting monoclonal antibodies that either inhibit or stimulate a putative plasma-membrane marker enzyme, (K+ + Mg2+)-stimulated pH 6.5 ATPase, have been identified and cloned. The specificity of monoclonal-antibody probes on the activity of other phosphate-hydrolysing enzymes has also been examined. The production and identification of monospecific antibodies capable of immunoreacting with particular component proteins in a complex plant membrane mixture highlight the usefulness of hybridoma methodology for the enzymologist, especially since such monoclonal antibodies can be used in the purification of proteins by immunoaffinity techniques.     

Noncellular nonspecific defence mechanisms of fish

Fish tissues and body fluids contain naturally occurring proteins or glycoproteins of non-immunoglobulin (Ig) nature that react with a diverse array of environmental antigens and may confer an undefined degree of natural immunity to fish. They consist of microbial growth inhibitory compounds that include “acute phase” proteins such as transferrins, caeruloplasmin, and metallothionein. Their action is simply to chelate metal ions and deprive bacteria and other parasites of essential inorganic ion sources. Both serum and cellular interferons are found in fish, and this anti-virus protein has been demonstrated mainly in salmonids during viral disease studies. Enzyme-inhibitors (α2-macroglobulin and other α-globulins) thus far detected in fish appear to be antibacterial proteinases, and are involved in the inhibition of extracellular proteases secreted by fish pathogens. Fish also possess a variety of relatively specific lytic molecules that cause cell lysis, and some of these materials are hydrolase enzymes (lysozyme, chitinase, chitobiase) whose main actions are against bacteria and fungi. In addition, mucus contains trypsin-like proteinases which destroy gram-negative bacteria. Nonspecific lysins and agglutinins against erythrocytes and other cellular antigens are found in serum, eggs, and skin mucus. The lysins, including toxins, some of which are bacteriolytic in activity, are, in their mode of action, natural or spontaneous, antibody-independent and noncomplement-mediated. In contrast, specific hemolytic antibodies (Ig), which complex with antigens, bind complement, and cause complement-mediated immune lysis, are reported to exist. The agglutinins are generally reactive toward certain sugar residues on erythrocyte or bacterial cell walls, and in most cases act as lectins or lectin-like molecules. Natural lysins and agglutinins behave in a similar way as antigen-induced antibodies or Igs, but exhibit a high degree of cross-reactions, due to the occurrence of similar carbohydrate determinants on many types of microbial cell surface. As with mammals, both C-type (calcium-dependent) and S-type (thiol-dependent) lectins are present in fish. They more resemble invertebrate lectins than those of higher animals. Fish lectins appear to play antibacterial or antifungal roles and in some instances seem to be involved in egg-sperm fusion, polyspermy prevention, and embryo development. Natural, non-Ig precipitins (e.g. α-precipitin and C-reactive proteins) are found largely, but not exclusively, in fish serum and precipitate with simple monosaccharides or long chain polysaccharides of certain stereochemistry and glycosidic linkages. Their functions remain unknown, but C-reactive protein is induced following stress-induction and exposure to inflammatory agents. Many of the above mentioned “defence” substances are present in skin mucus and possess the capacity to react with potentially infective microorganisms including parasites. Mucus thus acts as an immediate defence barrier to invasion and/or colonisation of pathogens.     

Proteins from fish eggs that protect DNA from acid precipitation and inhibit DNA synthesis

We partially characterized proteins that inhibit DNA acid precipitation from various fish eggs (Sparus aurata, Dicentrarchus labrax, Mugil cephalus and Zeus faber). The active proteins were purified by acetone fractionation. The activity was found to be heat resistant. Of bivalent cations tested only Co(2+) and Cu(2+) exerted a profound promoting effect in the activity from all fish. The protein fraction from Sparus aurata inhibited DNA synthesis in PCR performed by different DNA polymerases. The possible role of DNA protective proteins in fish egg physiology is discussed.