转录谱显示转铁蛋白在蜜蜂中的多重功能角色

Kucharski R, Maleszka R. 2003. Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera. 8pp. Journal of Insect Science, 3:27, Available online: insectscience.org/3.27 Journal of Insect Science insectscience.org Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera R. Kucharski and R. Maleszka Visual Sciences, Research School of Biological Sciences, The Australian National University, Canberra ACT 0200, Australia maleszka@rsbs.anu.edu.au Received 4 June 2003, Accepted 22 July 2003, Published 22 August 2003 Abstract Transferrins belong to a family of iron-binding proteins that have been implicated in innate immunity and in vitellogenesis in insects. Here we have sequenced and characterized a full-length cDNA encoding a putative iron-binding transferrin (AmTRF) in the honeybee. AmTRF shows high level of sequence identity with transferrins in both vertebrates and insects (26-46%) suggesting that the primary function of the predicted 712 amino acid protein is binding and transporting of iron. AmTRF is expressed ubiquitously, but particularly high levels of its mRNA are found in the central brain and in the compound eye. Using northern blotting and a microarray based approach we have examined the levels of AmTRF mRNA by expression profiling under a wide range of conditions including developmental stages, septic injury and juvenile hormone treatment. Increased expression of AmTRF is seen during early pupal stages, in the brain of mature foragers and in the abdomen of virgin queens, whereas treatment with juvenile hormone leads to a decrease of AmTRF levels in the abdomen. We show that a transcriptional response of transferrin to septic injury with E. coli is relatively moderate as compared to a dramatic up-regulation of an antibacterial polypeptide, Hymenoptaecin, under similar conditions. We conclude that major fluctuations of AmTRF mRNA in time and space are consistent with context-dependent functional significance and suggest broader multifunctional roles for transferrin in insects. Keywords: iron homeostasis, innate immunity, microarray, Hymenoptaecin, vitellogenesis, gene expression, context-dependent protein function Abbreviation: AmTRF Apis mellifera transferrin EST expressed sequence tag Cy-dUTP amino-propargyl-2’-deoxyuridine triphosphate coupled to cyanine fluorescent dye (Cy3 or Cy5). JH juvenile hormone Introduction Iron is one of the essential elements required by all organisms, but it is also a potent toxin because of its ability to produce free radicals in the presence of oxygen (Crichton et al., 2002, Nichol et al., 2002). In some tissues, such as brain or retina, where anti-oxidative defences are relatively low and oxygen consumption is very high, iron accumulation in specific regions is associated with a number of neurodegenerative diseases (Crichton et al., 2002). Hence, organisms must balance their nutritional requirements with the necessity to control this potential toxic property. In animals, iron-binding and transporting proteins provide an important way to minimise the reactivity of iron towards oxygen in addition to their facilitating role in iron metabolism. One class of iron-binding proteins that have attracted much attention in recent years belongs to a highly conserved family of transferrins. These proteins are well characterised in vertebrates, but in recent years there has been some progress in studies on their relatives in insects (Nichol et al., 2002). Despite a high level of sequence conservation of transferrins from different lineages there are significant differences in their biochemical properties as well as in their involvements in cellular functions. In vertebrates, transferrins are glycoproteins of approximately 80 kDa with two ferric-binding lobes, most likely resulting from a duplication of an ancient gene encoding a 40 kDa protein (Jamroz et al., 1993, Nichol et al., 2002). Most insect transferrins bind only one ferric ion because their C- terminal lobes have no iron-binding capacity. One notable exception is transferrin in Balberus discoidalis that has been found to contain two functional iron-binding sites (Jamroz et al., 1993). In addition to binding iron, mammalian transferrin has been shown to act as a growth factor and as a regulator of gene expression at the transcriptional level (Raivich et al., 1991, Espinosa-Jeffrey 2Kucharski R, Maleszka R. 2003. Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera. 8pp. Journal of Insect Science, 3:27, Available online: insectscience.org/3.27 et al., 2002). A correlation between myelination and transferrin synthesis and secretion in oligodendrocytes has also been demonstrated. In vertebrates, transferrin transports iron in blood, and many cells can access transferrin-bound iron by way of the transferrin receptor pathway. At present it is not known whether a similar pathway exists in insects. The lack of a gene encoding a protein similar to the vertebrate transferrin receptor in Drosophila was taken as evidence that insects may use a different transport mechanism or a different receptor (Nichol et al., 2002). Clearly, this issue needs to be re-examined as more insect genomes become available for comparative analyses. Transcriptional profiling is a powerful way to determine not only the functional state of the cell, but also to provide insights into the underlying biology and to identify hitherto unknown genes as well as new functions for previously described genes. As part of our ongoing study on molecular mechanisms underlying complex biological processes in the honeybee, Apis mellifera, we have found that a transcript encoding a putative transferrin was differentially expressed under a range of conditions including adult development, physical insults and following drug treatment (Kucharski and Maleszka, 2002). This finding prompted us to examine the spatio- temporal expression pattern of this transcript, designated AmTRF1, in more detail to gain a better understanding of its biological significance in the honeybee. On the basis of recent data Nichol et al. (2002) considered the following roles for transferrin in insects: as an iron binding protein, an antibiotic agent, a vitellogenic protein, and a protein repressible by juvenile hormone. Here, we show that the levels of transferrin mRNA in the honeybee are responsive to an even wider range of developmental and physiological conditions suggesting that the functional roles of transferrin are context- dependent and can only be viewed as part of a complex genetic network that is likely to include other related molecules and signalling factors. Materials and Methods Sample collection Foraging honeybee workers were captured near the hive entrance and snap-frozen in liquid nitrogen. To ensure that fully matured workers were harvested, only those that carried pollen or nectar were selected. We estimate their age to vary from 20-35 days. To obtain newly emerged honeybees a single brood frame was removed from the hive and incubated at 32° C (80% humidity). Individual insects were collected within 1-5 min after emergence and snap-frozen in liquid nitrogen. All dissections were done under permanent cooling (dry ice or liquid nitrogen). Pupae of different ages were collected on the basis of eye coloration. We estimate their approximate age to be 10-12 days (white eyes), 13-15 days (pink eyes) and 16-19 days (red eyes) respectively. Tissue dissection For all dissections the honeybees were snap-frozen in liquid nitrogen. Preparation of brain tissue was carried out under permanent liquid nitrogen cooling in the following manner. A drop of Tissue Tek O.C.T. Compound (Miles Scientific) was placed on a brass block protruding from a liquid nitrogen bath. While the drop was cooling, a bee head was removed from storage and placed immediately in the drop of Compound dorsal side uppermost. The head was held in position until the Compound had frozen around it. Following removal of the head capsule, the pigmented eyes were sliced away and stored separately. A transverse incision was made through the antenno-glomerular tract and the brain was prised gently out. To obtain individual compartments the brains were placed in a small plastic dish and separated under a dissection microscope into three regions: the mushroom bodies, the antennal lobes and the optic lobes. Dissections were performed on a brass block submerged in LN and brains were never allowed to thaw. Both thoraces and abdomens were separated from frozen bees using a pair of tweezers and a scalpel, whereas antennae and legs, which typically disintegrate from the bodies, where picked up from the bottom of the LN jar. Juvenile hormone and caffeine treatment Emerging adult insects were treated with juvenile hormone (JH) (typically within one hour after emergence) by applying 1 µl of JH (1.25% JH-III from Sigma (www.sigmaaldrich.com) in dimethyl formamide) on the thorax, transferred to a small cage containing a tube of honey and incubated at 32° C until desired age. To obtain caffeine-treated bees, a colony of 30 newly born individuals in a small cage was fed for 3 days with honey containing 10 mM caffeine. A similar colony, but fed only with pure honey was used as control. Light exposure Newly emerged bees were collected in a dark room illuminated with red light and transferred to a small cage containing a tube of honey (~50 individuals per cage). One cage was left in a dark incubator and the other one was placed between two light boxes and exposed to light for 24-48 hrs. Septic injury One microliter of either bacterial or yeast cells (10 x concentrated overnight cultures in sterile bee Ringer) were injected into the thorax of ice-chilled 3-4-day old bees using a 25 µl Hamilton syringe attached to a dispenser. We used E. coli strain XL1-Blue from Stratagen (www.stratagene.com) and S. cerevisiae strain Dip2 (Skelly and Maleszka, 1991). Aseptic injuries were done by either injecting 1 µl of sterile Ringer or by making a small incision in the thorax with a micro-scalpel. All bees were snap frozen in liquid nitrogen 6 hrs after treatment. Array preparation We employed spotted arrays containing either 2,500 or 9,000 cDNAs. The 9000-cDNA arrays were purchased from the University of Illinois at Urbana-Champaign. Their design is described elsewhere (Whitfield et al., 2002). The construction of the 2500-cDNA array as well as labelling and hybridisation were performed according to protocols established by Brown with minor modifications (http://cmgm.stanford.edu/pbrown). We used a standard, unannotated and non-normalised honeybee brain cDNA library made in lambda ZAP that was kindly provided by G. Robinson, Urbana-Champaign. Arrays were prepared by printing 4608 samples, in triplicates, on poly-lysine coated glass slides (Menzel) using a robotic device from Genetic Microsystems (model 3Kucharski R, Maleszka R. 2003. Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera. 8pp. Journal of Insect Science, 3:27, Available online: insectscience.org/3.27 418) (http://www.geneticmicro.com). These samples correspond to approximately 2500 unique cDNAs (Kucharski and Maleszka, 2002). RNA extraction, hybridisation and data analysis Total RNA was extracted from frozen tissues as described previously (Kucharski et al., 2000). Labelled probes were prepared by incorporating Cy3 and Cy5 dUTP (Amersham, www.apbiotech.com) during a reverse transcription of total RNA (SuperScript II, Life Technologies, www.synovislife.com). Hybridisation was conducted for 4-6 hrs at 62° C in a small volume (60-120 µl) of ExpressHyb buffer (Clontech, www.clontech.com) under a plastic coverslip. Following a washing step, slides were scanned with the Affymetrix 428 scanner and analysed using Affymetrix Pathways software v. 1.0 (www.affymetrix.com) and Microsoft Excel spreadsheets. Bioinformatics Database searches were performed at the National Center for Biotechnology Information (NCBI) using the BLAST server (www.ncbi.nlm.nih.gov). Additional searches were conducted at the Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign (titan.biotec.uiuc.edu/bee/ honeybee_project.htm) and at the TAGC University of Marseille (http://tagc.univ-mrs.fr). The presence of the signal peptide and the most likely cleavage site were predicted with SignalP VI.I (Nielsen et al., 1997). Northern blot analysis Total RNA was isolated using Trizol reagent from Gibco BRL (www.lifetech.com) followed by mRNA purification on Oligo (dt) 25 magnetic beads from Dynal (www.dynal.no). RNA samples were denatured by mixing with an equal volume of formamide, containing 0.05% bromophenol blue and 0.01% SybrGreen II, incubated at 90° C for 5-7 min and immediately chilled on ice. Electrophoresis was performed in small horizontal tanks (Hoeffer HE33) using 1.5% agarose gels submerged in TBE buffer (50 mM Tris-borate, 1 mM EDTA, pH 8. 2) at 20V/cm. Alternatively, the glyoxal based system (Ambion, www.ambion.com) was used for RNA separation. See the manufacturer’s instruction manual (Ambion, catalogue #1946) for details. Following electrophoretic resolution the gels were quantified with the Vistra FluoroImager (www.amershambiosciences.com) and then soaked in 1M ammonium acetate, 0.02 M NaOH and blotted onto Hybond N+ nylon membranes (Amersham, www.amershambiosciences.com) by capillary transfer. RNA was cross-linked to the membrane by UV irradiation and after a brief wash in 2x SSC the blots were pre- hybridised for 5-30 min. Hybridisation was carried out either at 68° C (ExpressHyb solution, Clontech, www.clontech.com), or at 42° C (UltraHyb buffer, Ambion) for 16 hrs using P32-labelled probes (RediPrime kit, Amersham). Blots were washed 3-4 times in 2x SSC, 0.1% SDS at 50° C and exposed to a phosphorstorage screen (Molecular Dynamics, www.moleculardynamics.com) without drying. Computer generated images (Molecular Dynamics, Phosphor-Imager 400S) of individual gels were analysed using ImageQuant (Amersham) software. Optical density measures for a given transcript were normalised against the corresponding optical density for loaded RNA. The expression levels were calculated and shown relatively to the highest hybridisation signal that was set as 100. Results AmTRF encodes a highly conserved transferrin In our previous study we have used a spotted cDNA microarray representing 2,500 transcripts in the honeybee brain to identify genes that are differentially expressed during adult development and as a result of caffeine treatment (Kucharski and Maleszka, 2002). Coupled with northern blot verification this approach revealed thirty-seven genes showing at least 2-fold change under our experimental conditions. One of these genes, represented by bEST92, was found to be up-regulated in the abdomen of mature worker bees. Subsequent sequencing and bioinformatics analyses revealed that bEST92 encodes a conserved transferrin. Here we present the molecular characterization and transcriptional profiles of the honeybee transferrin, designated AmTRF. AmTRF is transcribed as a single message of approximately 2400 base pairs that spans an open reading frame of 2136 nucleotides. This open reading frame encodes a 712 amino acid polypeptide with a predicted molecular weight of 78.6 kDa and a most likely cleavage site between positions 26 and 27 (IAA-QD) indicating a secreted protein (Fig. 1). AmTRF shares the highest sequence identity (45%) with two insect transferrins in the cockroach B. discoidalis and in the termite Mastotermes darwiniensis (Fig. 1). Like other transferrins in both insects and vertebrates the honeybee protein appears to be a product of intragenic duplication and contains relatively large number (25) of conserved cysteine residues. The level of amino Figure 1. Predicted amino acid sequence of the honeybee transferrin AmTRF. Residues conserved in the honeybee protein (Apis mellifera) and in two insect transferrins from the cockroach (Balberus discoidalis) and the termite (Mastotermes darwiniensis) are boxed and shaded in yellow. The leader peptide is typed in blue and the conserved amino acids implicated in iron binding are typed in red. GenBank accession No. Am: AY217097, Bd: AAA27820, Md: AAN03488. 4Kucharski R, Maleszka R. 2003. Transcriptional profiling reveals multifunctional roles for transferrin in the honeybee, Apis mellifera. 8pp. Journal of Insect Science, 3:27, Available online: insectscience.org/3.27 Figure 2. Microarray analysis of AmTRF expression under various conditions. Fold changes in the level of AmTRF message are shown for the following experiments: forager (F) head versus nurse (N) head, F brain v N brain, 8-day old brain v 7-day old brain, central brain (CB) v optic lobes (OL), F abdomen v N abdomen, mature queen (MQ) v virgin queen (VQ), early pupae head (ST1) v late pupae head (ST3), brains of light-exposed bees (L) v dark-kept (D) bees, brains of juvenile hormone treated bees (JH) v controls (CTRL), heads of caffeine and honey-fed bees (CAFF) v honey-fed bees (CTRL). Error bars represent standard deviations from 8 independent samples. Nurses refer to 1-day old bees, foragers to 20-30-days old bees. Figure 3. A. Northern blot (top panel) showing the expression of AmTRF in various tissues: antennae (An), compound eyes (E), optic lobes (Ol), mushroom bodies (Mb), antennal lobe (Al), thorax (T), legs (L) and abdomen (Ab). Equal amounts of poly(A)+RNA (~2 µg) were loaded per lane. B. Northern blot showing the expression of AmTRF in brains of newly emerged individuals (1 hr) and experienced foragers (21 days) and in the queen abdomen (V-virgin, M-mature, egg-laying). Lower panels in A and B show the relative expression levels calculated using ImageQuant software as described in materials and methods. acid positional identity between the N-terminal and C-terminal halves in AmTRF is 22%. The residues implicated in iron binding in insects (Jamroz et al., 1993, Thompson et al., 2003) are conserved only in the N-terminal half of AmTRF suggesting that its C-terminal lobe has no iron-binding capacity (Fig. 1). AmTRF is differentially expressed in time and space Transcriptional responses of AmTRF were investigated by means of both northern blots and microarray hybridizations using RNA populations representing major body parts, brain compartments, developmental stages, caffeine- or juvenile hormone- treated bees, and bees exposed to light. Spotted cDNA microarrays can be used to profile the expression of thousands of gene targets in a single experiment. However, they also can be used to evaluate changes in the expression pattern of a single gene by extracting data points from a series of microarray-based physiological and developmental studies. We used fluorescently labelled total RNAs to interrogate spotted arrays containing either 2,500 or 9,000 cDNAs. These arrays were developed in our lab (Kucharski and Maleszka, 2002) and by Whitfield et al. (2002) respectively. Figure 2 shows fold-changes in AmTRF levels measured across a series of experiments. The most profound difference (4.5 fold decrease) was found between the heads of late and early pupae (Fig. 2, pupal heads ST3 v ST1). This dramatic down-regulation of AmTRF expression in late pupae coincides with cellular reorganization of the insect nervous system during metamorphosis that involves both scrapping and recycling of the larval neurons to make way for new adult neurons (Levine, 1987). In our study, late and early pupal heads represent newly rewired adult brain and larval nervous system respectively. A two-fold increase was detected in the brains of older foragers as compared to brains of newly born individuals (in agreement with the northern blot analysis shown in Figure