The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.     

Two newly identified membrane-associated and plastidic tomato HXKs: characteristics, predicted structure and intracellular localization

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.The nucleotide sequence data of LeHXK3 and LeHXK4 appear in the GenBank Nucleotide Sequence Database under accession numbers DQ056861 and DQ056862, respectively.M. Kandel-Kfir and H. Damari-Weissler contributed equally to this work.     

Duplication of the IGFBP-2 Gene in Teleost Fish: Protein Structure and Functionality Conservation and Gene Expression Divergence

Background

Insulin-like growth factor binding protein-2 (IGFBP-2) is a secreted protein that binds and regulates IGF actions in controlling growth, development, reproduction, and aging. Elevated expression of IGFBP-2 is often associated with progression of many types of cancers.

Methodology/Principal Findings

We report the identification and characterization of two IGFBP-2 genes in zebrafish and four other teleost fish. Comparative genomics and structural analyses suggest that they are co-orthologs of the human IGFBP-2 gene. Biochemical assays show that both zebrafish igfbp-2a and -2b encode secreted proteins that bind IGFs. These two genes exhibit distinct spatiotemporal expression patterns. During embryogenesis, IGFBP-2a mRNA is initially detected in the lens, then in the brain boundary vasculature, and subsequently becomes highly expressed in the liver. In the adult stage, liver has the highest levels of IGFBP-2a mRNA, followed by the brain. Low levels of IGFBP-2a mRNA were detected in muscle and in the gonad in male adults only. IGFBP-2b mRNA is detected initially in all tissues at low levels, but later becomes abundant in the liver. In adult males, IGFBP-2b mRNA is only detected in the liver. In adult females, it is also found in the gut, kidney, ovary, and muscle. To gain insights into how the IGFBP-2 genes may have evolved through partitioning of ancestral functions, functional and mechanistic studies were carried out. Expression of zebrafish IGFBP-2a and -2b caused significant decreases in the growth and developmental rates and their effects are comparable to that of human IGFBP-2. IGFBP-2 mutants with altered IGF binding-, RGD-, and heparin-binding sites were generated and their actions examined. While mutating the RGD and heparin binding sites had little effect, altering the IGF binding site abolished its biological activity.

Conclusions/Significance

These results suggest that IGFBP-2 is a conserved regulatory protein and it inhibits growth and development primarily by binding to and inhibiting IGF actions in vivo. The duplicated IGFBP-2 genes may provide additional flexibility in the regulation of IGF activities.     

Regulation of glucokinase activity in liver of hibernating ground squirrel Spermophilus undulatus

The kinetic properties of glucokinase (GLK) from the liver of active and hibernating ground squirrels Spermophilus undulatus have been studied. Entrance of ground squirrels into hibernation from their active state is accompanied by a sharp decrease in blood glucose (Glc) level (from 14 to 2.9 mM) and with a significant (7-fold) decrease of GLK activity in the liver cytoplasm. Preparations of native GLK practically devoid of other molecular forms of hexokinase were obtained from the liver of active and hibernating ground squirrels. The dependence of GLK activity upon Glc concentration for the enzyme from active ground squirrel liver showed a pronounced sigmoid character (Hill coefficient, h = 1.70 and S _0.5 = 6.23 mM; the experiments were conducted at 25°C in the presence of enzyme stabilizers, K⁺ and DTT). The same dependence of enzyme activity on Glc concentration was found for GLK from rat liver. However, on decreasing the temperature to 2°C (simulation of hibernation conditions), this dependency became almost hyperbolic (h = 1.16) and GLK affinity for substrate was reduced (S _0.5 = 23 mM). These parameters for hibernating ground squirrels (body temperature 5°C) at 25°C were found to be practically equal to the corresponding values obtained for GLK from the liver of active animals (h = 1.60, S _0.5 = 9.0 mM, respectively); at 2°C sigmoid character was less expressed and affinity for Glc was drastically decreased (h = 1.20, S _0.5 = 45 mM). The calculations of GLK activity in the liver of hibernating ground squirrels based on enzyme kinetic characteristics and seasonal changes in blood Glc concentrations have shown that GLK activity in the liver of hibernating ground squirrels is decreased about 5500-fold.     

A truncated Danio rerio PKZ isoform functionally interacts with eIF2α and inhibits protein synthesis

A protein kinase containing Z-DNA binding domains (PKZ), which resembles protein kinase R (PKR) in domain organization, was recently discovered to be a member of the eIF2α kinase family in fish. PKR has roles in antiviral immunity through inhibiting protein synthesis and activating NF-κB; therefore, it is thought that PKZ may have a similar role in fish antiviral immunity. In the present study, the roles of two Danio rerio PKZ isoforms (DrPKZ-A and DrPKZ-B) in eIF2α phosphorylation and protein synthesis regulation were explored. DrPKZ-A and DrPKZ-B possess N-terminal Z-DNA binding domains and a conserved eIF2α kinase domain; however, they have domains of differing lengths inserted between kinase subdomains IV and V. DrPKZ-A has an insert domain of 73 amino acids (aa), whereas DrPKZ-B has an insert sequence of only 10 aa, suggesting that DrPKZ-B could be a dysfunctional isoform or could interact with different substrates. Our results show that both DrPKZ-A and DrPKZ-B functionally interact with eIF2α and inhibit protein synthesis, although DrPKZ-B possesses attenuated kinase activity. Our results also show that deletion of the insert in either isoform results in the complete abrogation of kinase activity, suggesting that the insert is critical for PKZ kinase activity. Kinase activity appears to be independent of insert length but may depend on the presence of specific amino acids within the insert domain. Furthermore, the effects of the N-terminal regulatory domain on kinase activity were analyzed. Deletion of the N-terminus results in reduced kinase activity of these isoforms relative to the wild-type forms, indicating that the isolated kinase domain is sufficient for eIF2α phosphorylation and that DrPKZ-A and DrPKZ-B may be regulated in a similar manner. Overall, our results show that DrPKZ-B is a functional kinase in zebrafish and contribute to our understanding of the function of PKZ in fish.     

ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes,Recognizes 4-Hydroxyphenylacetic Acid,a Putative Reproductive Pheromone

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.     

Glucokinase contributes to glucose phosphorylation in d-lactic acid production by Sporolactobacillus inulinus Y2-8

Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial d-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve d-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and d-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3–1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus d-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5?kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and d-lactic acid production.     

Genetics of Yeast Glucokinase

Mutants of Saccharomyces cerevisiae lacking glucokinase (EC 2.7.1.2) have no discernible phenotypic difference from the wild-type strain; in a hexokinaseless background, however, they are unable to grow on any sugar except galactose. Reversion studies with glucokinase mutants indicate that the yeast S. cerevisiae has no other enzyme for phosphorylating glucose except the two hexokinases, P1 and P2, and glucokinase. Spontaneous revertants of hxk1 hxk2 glk1 strains collected on glucose regain any one of these three enzymes. The majority of glucokinase revertants synthesize species of enzyme activity that are kinetically or otherwise indistinguishable from the wild-type enzyme. In a few cases the reverted enzyme is very perceptibly altered in properties with a Km for glucose two orders of magnitude higher than that of the enzyme from the wild-type parent. These recessive, noncomplementing mutants, thus, define a single structural gene GLK1 of glucokinase. Yeast diploids lacking all of the three enzymes for glucose phosphorylation fail to sporulate. Heterozygosity of either of the hexokinase genes HXK1 or HXK2, but not GLK1, restores sporulation. The location of GLK1 on chromosome III was indicated by loss of this chromosome when hexokinaseless diploids heterozygous for glk1 were selected for resistance to 2-deoxyglucose; the homologue of chromosome III carrying GLK1, the mating-type allele and other nutritional markers on this chromosome was lost. Meiotic mapping of glucokinase executed with heterozygosity of one of the hexokinases indicated that the gene GLK1 defining the structure of glucokinase protein is located on the left arm of chromosome III 24 cM to the left of his4 in the order: leu2--his4--glk1. --Only two of 206 independent glucokinase mutants are nonsense ochre, both of which map at one end of the gene. In hxk1 only one of 130 isolates is a nonsense mutation, whereas in hxk2 none has been found among 220 independent mutants. These results raise the possibility that the protein products of these genes have some other essential function. --An earlier mapping result for hxk2 has been corrected. The new location is on the left arm of chromosome VII, 17 cM distal to ade5 in the order: lys5--ade5--hxk2.     

Distinct developmental expression of two elongase family members in zebrafish

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.     

Characterization of the transglutaminase gene family in zebrafish and in vivo analysis of transglutaminase-dependent bone mineralization

We have characterized the protein cross-linking enzyme transglutaminase (TGs) genes in zebrafish, Danio rerio, based on the analysis of their genomic organization and phylogenetics. Thirteen zebrafish TG genes (zTGs) have been identified, of which 11 show high homology to only 3 mammalian enzymes: TG1, TG2 and FXIIIa. No zebrafish homologues were identified for mammalian TGs 3-7. Real-time PCR analysis demonstrated distinct temporal expression profiles for zTGs in larvae and adult fish. Analysis by in situ hybridization revealed restricted expression of zTG2b and zFXIIIa in skeletal elements, resembling expression of their mammalian homologues in osteo-chondrogenic cells. Mammalian TG2 and FXIIIa have been implicated in promoting osteoblast differentiation and bone mineralization in vitro, however, mouse models lacking either gene have no skeletal phenotype likely due to a compensation effect. We show in this study that mineralization of the newly formed vertebrae is significantly reduced in fish grown for 5?days in the presence of TG inhibitor KCC-009 added at 3–5?days post fertilization. This treatment reduces average vertebrae mineralization by 30%, with complete inhibition in some fish, and no effect on the overall growth and vertebrae number. This is the first in vivo demonstration of the crucial requirement for the TG-catalyzed cross-linking activity in bone mineralization.     

Glucose repression may involve processes with different sugar kinase requirements.

Adding glucose to Saccharomyces cerevisiae cells growing among nonfermentable carbon sources leads to glucose repression. This process may be resolved into several steps. An early repression response requires any one of the three glucose kinases present in S. cerevisiae (HXK1, HXK2, or GLK1). A late response is only achieved when Hxk2p is present.     

Expanding the annotation of zebrafish microRNAs based on small RNA sequencing

MicroRNAs (miRs) are short non-coding RNAs that fine-tune the regulation of gene expression to coordinate a wide range of biological processes. Because of their role in the regulation of gene expression, miRs are essential players in development by acting on cell fate determination and progression towards cell differentiation and are increasingly relevant to human health and disease. Although the zebrafish Danio rerio is a major model for studies of development, genetics, physiology, evolution, and human biology, the annotation of zebrafish miR-producing genes remains limited. In the present work, we report deep sequencing data of zebrafish small RNAs from brain, heart, testis, and ovary. Results provide evidence for the expression of 56 un-annotated mir genes and 248 un-annotated mature strands, increasing the number of zebrafish mir genes over those already deposited in miRBase by 16% and the number of mature sequences by 63%. We also describe the existence of three pairs of mirror-mir genes and two mirtron genes, genetic features previously undescribed in non-mammalian vertebrates. This report provides information that substantially increases our knowledge of the zebrafish miRNome and will benefit the entire miR community.     

Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish

Reticulons (RTNs) are a family of evolutionary conserved proteinswith four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) presentin land vertebrates. While the exact functions of RTN1 to RTN3are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit theregeneration of severed axons in the mammalian central nervoussystem (CNS). This inhibitory function is exerted via two distinctregions, one within the Nogo-A–specific N-terminus andthe other in the conserved reticulon homology domain (RHD).In contrast to mammals, fish are capable of CNS axon regeneration.We performed detailed analyses of the fish rtn gene family todetermine whether this regeneration ability correlates withthe absence of the neurite growth inhibitory protein Nogo-A.A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish,and 30 in eight additional fish species. Phylogenetic and syntenicrelationships indicate that the identified fish rtn genes areorthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that severalparalogous fish genes (e.g., rtn4 and rtn6) resulted from genomeduplication events early in actinopterygian evolution. Accordingly,sequences homologous to the conserved RTN4/Nogo RHD are presentin two fish genes, rtn4 and rtn6. However, sequences comparableto the first 1,000 amino acids of mammalian Nogo-A includinga major neurite growth inhibitory region are absent in zebrafish.This result is in accordance with functional data showing thataxon growth inhibitory molecules are less prominent in fisholigodendrocytes and CNS myelin compared to mammals.     

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.