A new nomenclature for the aldo-keto reductase superfamily

The aldo-keto reductases (AKRs) represent a growing oxidoreductase superfamily. Forty proteins have been identified and characterized as AKRs, and an additional fourteen genes may encode proteins related to the superfamily. Found in eukaryotes and prokaryotes, the AKRs metabolize a wide range of substrates, including aliphatic aldehydes, monosaccharides, steroids, prostaglandins, and xenobiotics. This broad substrate specificity has caused problems in naming these proteins. Enzymes capable of these reactions have been referred to as aldehyde reductase (ALR1), aldose reductase (ALR2), and carbonyl reductase (ALR3); however, ALR3 is not a member of the AKR superfamily. Also, some AKRs have multiple names based upon substrate specificity. For example, human 3α-hydroxysteroid dehydrogenase (3α-HSD) type I is also known as dihydrodiol dehydrogenase 4 and chlordecone reductase. To address these issues, we propose a new nomenclature system for the AKR superfamily based on amino acid sequence identities. Cluster analysis of the AKRs shows seven distinct families at the 40% amino acid identity level. The largest family (AKR1) contains the aldose reductases, aldehyde reductases, and HSDs. Other families include the prokaryotic AKRs, the plant chalcone reductases, the Shaker channels, and the ethoxyquin-inducible aflatoxin B₁ aldehyde reductase. At the level of 60% amino acid identity, subfamilies are discernible. For example, the AKR1 family includes five subfamilies: (A) aldehyde reductases (mammalian); (B) aldose reductases; (C) HSDs; (D) Δ⁴-3-ketosteroid-5β-reductases; and (E) aldehyde reductases (plant). This cluster analysis forms the basis for our nomenclature system. Recommendations for naming an aldo-keto reductase include the root symbol “AKR,” an Arabic number designating the family, a letter indicating the subfamily when multiple subfamilies exist, and an Arabic numeral representing the unique protein sequence. For example, human aldehyde reductase would be assigned as AKR1A1. Our nomenclature is both systematic and expandable, thereby allowing assignment of consistent designations for newly identified members of the superfamily.     

Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping

As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out in chronological order as they are reported to us. In addition, 20 human ALDH variant alleles that have been reported to date are listed herein and are recommended to be given numbers (or a number plus a capital letter) following an asterisk (e.g. 'ALDH3A2*2, ALDH2*4C'). It is anticipated that this eukaryotic ALDH gene nomenclature system will be extended to include bacterial genes within the next 2 years and that this nomenclature system will require updating on a regular basis; an ALDH Web site has been established for this purpose (http://++www.uchsc.edu/sp./sp./alcdbase/a ldhcov.html) and will serve as a medium for interaction amongst colleagues in this field.     

Association of the SULT1A1 R213H polymorphism with colorectal cancer

1. Sulphotransferases are a superfamily of enzymes involved in both detoxification and bioactivation of endogenous and exogenous compounds. The arylsulphotransferase SULT1A1 has been implicated in a decreased activity and thermostability when the wild-type arginine at position 213 of the coding sequence is substituted by a histidine. SULT1A1 is the isoform primarily associated with the conversion of dietary N-OH arylamines to DNA binding adducts and is therefore of interest to determine whether this polymorphism is linked to colorectal cancer. 2. Genotyping, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, was performed using DNA samples of healthy control subjects (n = 402) and patients with histologically proven colorectal cancer (n = 383). Both control and test populations possessed similar frequencies for the mutant allele (32.1 and 31%, respectively; P = 0.935). Results were not altered when age and gender were considered as potential confounders in a logistic regression analysis. 3. Examination of the sulphonating ability of the two allozymes with respect to the substrates p-nitrophenol and paracetamol showed that the affinity and rate of sulphonation was unaffected by substitution of arginine to histidine at position 213 of the amino acid sequence. 4. From this study, we conclude that the SULT1A1 R213H polymorphism is not linked with colorectal cancer in this elderly Australian population.     

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.     

Identification and characterization of two novel cytosolic sulfotransferases, SULT1 ST7 and SULT1 ST8, from zebrafish

Cytosolic sulfotransferases (SULTs) constitute a family of Phase II detoxification enzymes that are involved in the protection against potentially harmful xenobiotics as well as the regulation and homeostasis of endogenous compounds. Compared with humans and rodents, the zebrafish serves as an excellent model for studying the role of SULTs in the detoxification of environmental pollutants including environmental estrogens. By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative SULTs were identified. Sequence analysis indicated that these two putative zebrafish SULTs belong to the SULT1 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT1 ST7 and SULT1 ST8, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) cells. Purified GST-fusion protein form of SULT1 ST7 and SULT1 ST8 exhibited strong sulfating activities toward environmental estrogens, particularly hydroxylated polychlorinated biphenyls (PCBs), among various endogenous and xenobiotic compounds tested as substrates. pH-dependence experiments showed that SULT1 ST7 and SULT1 ST8 displayed pH optima at 6.5 and 8.0, respectively. Kinetic parameters of the two enzymes in catalyzing the sulfation of catechin and chlorogenic acid as well as 3-chloro-4-biphenylol were determined. Developmental expression experiments revealed distinct patterns of expression of SULT1 ST7 and SULT1 ST8 during embryonic development and throughout the larval stage onto maturity.     

Cloning and nucleotide sequences of crotamine genes

A cDNA library containing snake toxin genes was constructed in bacteriophage lambda by using mRNA isolated from the glands of the South American rattlesnake, Crotalus durissus terrificus. The first high-density screening of 400,000 plaques for crotamine-containing genes yielded over 800 positives when a labeled cDNA probe with sequence homology to crotamine was used. Four of these clones with insert sizes from 270 to 400 base pairs were chosen and their inserts subcloned into pGEM-3Z and sequenced. Nucleotide sequence analysis of the cloned cDNAs predicted the existence of multiple variants of the crotamine toxin. The different forms, identified from the DNA sequences, displayed discrepancies in amino acid sequence for crotamine when compared with previously published reports. Direct amino acid sequencing of commercially purified crotamine and CNBr fragments thereof confirmed the structures predicted by the nucleic acid sequences.     

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.     

内蒙古白绒山羊Izumo蛋白的基因编码区cDNA序列扩增及结构初步分析

目的  对内蒙古白绒山羊精子膜蛋白Izumo的基因编码区cDNA序列进行扩增并对其结构作初步分析。方法  以已知的内蒙古白绒山羊Izumo部分基因序列为模板,采用cDNA末端快速扩增技术扩增3′端和5′端未知序列,钓取基因编码区,并分析编码区氨基酸序列。结果  经序列扩增和拼接,得到1035 bp的Izumo编码区cDNA序列,后者编码由344个氨基酸组成的蛋白。氨基酸序列中包含免疫球蛋白样结构域。Izumo编码区的疏水性平均值为-0.181。找到4个位于Izumo蛋白胞外区的潜在磷酸化位点。结论  进一步确定Izumo属于免疫球蛋白超家族的一员,初步得出Izumo是一种亲水性蛋白,根据潜在磷酸化位点的位置,推断磷酸化可能不是Izumo蛋白发挥作用的机制。     

Regional divergence of phospholipase A2-like protein cDNAs between New Guinean and Australian Pseudechis australis

Snake Phospholipase A₂ (PLA₂) exhibits diverse pharmacological effects, such as hemolysis, myotoxicity, and neurotoxicity. In this study, we identified 10 novel PLA₂-like protein cDNAs, which we named Pr 1-10, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequence of Pr 1, which ortholog has not been reported in mulaga snake (Australian P. australis) yet, shows 78.8% identity with the ortholog in Australian tiger snake (Notechis scutatus scutatus). The amino acid sequences of Pr 2-10 are 92.4-99.3% identical with their orthologs and paralogs in Australian P. australis.     

Two metallothioneins in the fresh-water fish, crucian carp (Carassius cuvieri): cDNA cloning and assignment of their expression isoforms

Two metallothionein cDNAs (MT-A and MT-B) in the fresh-water fish crucian carp (Carassius cuvieri Temminck et Schlegel) were cloned. Sequence analysis of both cDNAs gave the structure of a coding region corresponding to 60 amino acid residues. The homology of their deduced amino acid sequences was completely conserved at the positions of the cysteine residue, but a significant difference existed in the size of their 3'-untranslated regions (130 base pairs for MT-A and 280 base pairs for MT-B). Direct amino acid sequencing of the MT-II isoform purified by HPLC was accomplished for up to 30 residues and its sequence was identical to that deduced from MT-B cDNA. This is the first case in vertebrates that N-terminal methionine in crucian carp MT-II was not blocked. By northern blot analysis, basal and cadmium chloride- or dexamethasone-induced MT-B (MT-II) mRNAs were detected time dependently after treatment. On the other hand, the expression of MT-A mRNA was extremely low. These results indicate that the MT isoform II in crucian carp is coded by the MT-B gene, and that the MT-B-dominant expression of mRNA in crucian carp may be due to the difference in the 3'-untranslated regions of MT mRNAs.     

Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants

OBJECTIVES: Completion of both the mouse and human genome sequences in the private and public sectors has prompted comparison between the two species at multiple levels. This review summarizes the cytochrome P450 (CYP) gene superfamily. For the first time, we have the ability to compare complete sets of CYP genes from two mammals. Use of the mouse as a model mammal, and as a surrogate for human biology, assumes reasonable similarity between the two. It is therefore of interest to catalog the genetic similarities and differences, and to clarify the limits of extrapolation from mouse to human. METHODS: Data-mining methods have been used to find all the mouse and human CYP sequences; this includes 102 putatively functional genes and 88 pseudogenes in the mouse, and 57 putatively functional genes and 58 pseudogenes in the human. Comparison is made between all these genes, especially the seven main CYP gene clusters. RESULTS AND CONCLUSIONS: The seven CYP clusters are greatly expanded in the mouse with 72 functional genes versus only 27 in the human, while many pseudogenes are present; presumably this phenomenon will be seen in many other gene superfamily clusters. Complete identification of all pseudogene sequences is likely to be clinically important, because some of these highly similar exons can interfere with PCR-based genotyping assays. A naming procedure for each of four categories of CYP pseudogenes is proposed, and we encourage various gene nomenclature committees to consider seriously the adoption and application of this pseudogene nomenclature system.     

Molecular cloning of phospholipases A2 from venom glands of Echis carpet vipers

Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A₂ (PLA₂) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA₂ protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA₂ enzymes. Comparison of the PLA₂ sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA₂-function distinctions between these Echis PLA₂ isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA₂-neutralizing antibodies with polyspecific potential.     

Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism

The human genome contains at least 17 genes that are members of the aldehyde dehydrogenase (ALDH) superfamily. These genes encode NAD(P)(+)-dependent enzymes that oxidize a wide range of aldehydes to their corresponding carboxylic acids. Aldehydes are highly reactive molecules that are intermediates or products involved in a broad spectrum of physiologic, biologic, and pharmacologic processes. Aldehydes are generated during retinoic acid biosynthesis and the metabolism of amino acids, lipids, carbohydrates, and drugs. Mutations in several ALDH genes are the molecular basis of inborn errors of metabolism and contribute to environmentally induced diseases.     

Structure of the precursor of Xenopus laevis endothelin-3 and phylogenetic analysis

Endothelin (ET)-related receptors homologous to mammalian receptors have been cloned from Xenopus laevis, indicating that ET-related ligands may be present in this species. Here we cloned cDNAs encoding preproendothelin-3 (PPET-3) from the X. laevis intestinal cDNA library. X. laevis ET-3 cDNA encodes 201 amino acids, including a 20-amino-acid putative signal sequence, as well as mature ET-3, big ET-3, and ET-3-like sequences. X. laevis ET-3 differs by one amino acid from mammalian ET-3, and is identical to frog ET-3 recently purified from Rana ridibunda. This sequence together with other published PPET sequences were used to analyze the phylogenetic relationship among all ET family genes. This is the first report of the cDNA encoding the precursor protein of ET-3 in a non-mammalian species.     

Antinociceptive and antiedematogenic properties and acute toxicity of Tabebuia avellanedae Lor. ex Griseb. inner bark aqueous extract

Background

The search for an optimal experimental model in pharmacology is recently focused on (mini)pigs as they seem not only to be an alternative source of cells and tissues for xenotherapy but also an alternative species for studies on drug metabolism in man due to similarities between (mini) pig and human drug metabolizing systems. The purpose of this work is to characterize minipig liver microsomal cytochromes P450 (CYPs) by comparing their N-terminal sequences with corresponding human orthologs.

Results

The microsomal CYPs exhibit similar activities to their human orthologous enzymes (CYP3A4, nifedipine oxidation; 2A6, coumarin 7-hydroxylation; 2D6, bufuralol 1'-hydroxylation; 2E1, p-nitrophenol hydroxylation; and 2C9, tolbutamide hydroxylation). Specific minipig CYP (2A, 2C and 3A) enzymes were partially purified and proteins identified by immunostaining (using antibodies against the respective human CYPs) were used for N-terminal amino acid sequencing. From comparisons, it can be concluded that the sequence of the first 20 amino acids at the N-terminus of minipig CYP2A is highly similar to human CYP2A6 (70% identity). The N-terminal sequence of CYP2C shared about 50% similarity with human 2C9. The results on the minipig liver microsomal CYP3A yielded identical data with those obtained for amino acid sequences of the pig CYP3A29 showing 60% identity with human CYP3A4.

Conclusions

Thus, our results further support the view that minipigs may serve as model animals in pharmacological/toxicological studies with substrates of human CYP enzymes, namely, of the CYP3A and CYP2A forms.     

Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily

Several novel UDP glycosyltransferase (UGT) genes, mainly UDP glucuronosyltransferases, have been identified in the human, mouse and rat genomes and in other mammalian species. This review provides an update of the UGT nomenclature to include these new genes and prevent the confusion that arises when the same gene is given different names. The new genes are named following previously established recommendations, taking into consideration evolutionary relatedness and the names already in general usage in the literature. The mammalian UGT gene superfamily currently has 117 members that can be divided into four families, UGT1, UGT2, UGT3 and UGT8. The 5-exon genes of the UGT1 family each contain a unique first exon, plus four exons that are shared between the genes; the exons 1 appear to have evolved by a process of duplication, leading to the synthesis of proteins with identical carboxyl-terminal and variable amino-terminal domains. Exon-sharing is also seen with the 6-exon UGT2A1 and UGT2A2 genes. However, UGT2A3 and those of the UGT2B (six exons), UGT3 (seven exons) and UGT8 gene families (five or six exons) do not share exons and most likely were derived by a process of duplication of all exons in the gene. Most UGT1 and UGT8 enzymes have been characterized in detail; however, the catalytic functions of the UGT3A enzymes and several UGT2 enzymes remain to be characterized.