Up-regulation of Neurohemerythrin Expression in the Central Nervous System of the Medicinal Leech, Hirudo medicinalis, following Septic Injury

We report here some results of a proteomic analysis of changes in protein expression in the leech Hirudo medicinalis in response to septic injury. Comparison of two-dimensional protein gels revealed several significant differences between normal and experimental tissues. One protein found to be up-regulated after septic shock was identified, through a combination of Edman degradation, mass spectrometry, and molecular cloning, as a novel member of the hemerythrin family, a group of non-heme-iron oxygen transport proteins found in four invertebrate phyla: sipunculids, priapulids, brachiopods, and annelids. We found by in situ hybridization and immunocytochemistry that the new leech protein, which we have called neurohemerythrin, is indeed expressed in the leech central nervous system. Both message and protein were detected in the pair of large glia within the ganglionic neuropile, in the six packet glia that surround neuronal somata in each central ganglion, and in the bilateral pair of glia that separate axonal fascicles in the interganglionic connective nerves. No expression was detected in central neurons or in central nervous system microglia. Expression was also observed in many other, non-neuronal tissues in the body wall. Real-time PCR experiments suggest that neurohemerythrin is up-regulated posttranscriptionaly. We consider potential roles of neurohemerythrin, associated with its ability to bind oxygen and iron, in the innate immune response of the leech nervous system to bacterial invasion. We report here some results of a proteomic analysis of changes in protein expression in the leech Hirudo medicinalis in response to septic injury. Comparison of two-dimensional protein gels revealed several significant differences between normal and experimental tissues. One protein found to be up-regulated after septic shock was identified, through a combination of Edman degradation, mass spectrometry, and molecular cloning, as a novel member of the hemerythrin family, a group of non-heme-iron oxygen transport proteins found in four invertebrate phyla: sipunculids, priapulids, brachiopods, and annelids. We found by in situ hybridization and immunocytochemistry that the new leech protein, which we have called neurohemerythrin, is indeed expressed in the leech central nervous system. Both message and protein were detected in the pair of large glia within the ganglionic neuropile, in the six packet glia that surround neuronal somata in each central ganglion, and in the bilateral pair of glia that separate axonal fascicles in the interganglionic connective nerves. No expression was detected in central neurons or in central nervous system microglia. Expression was also observed in many other, non-neuronal tissues in the body wall. Real-time PCR experiments suggest that neurohemerythrin is up-regulated posttranscriptionaly. We consider potential roles of neurohemerythrin, associated with its ability to bind oxygen and iron, in the innate immune response of the leech nervous system to bacterial invasion. The nervous system is involved in the control of the major physiological functions leading to homeostasis, including immunity. Kakizaki et al. (1Kakizaki Y. Watanobe H. Kohsaka A. Suda T. Endocr. J. 1999; 46: 487-496Crossref PubMed Scopus (75) Google Scholar) have shown that circulating levels of vertebrate proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6) increase upon systemic endotoxemia. 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Biochem. 2000; 267: 2354-2361Crossref PubMed Scopus (19) Google Scholar), without careful analyses of the mechanisms involved. Among annelids, one of the best known species is the medicinal leech, Hirudo medicinalis, which has been intensively studied since the XIX° century (25Retzius G. Biologische Untersuchengen, Neue Folge. 2. Samson and Wallin, Stockholm, Sweden1891: 1-28Google Scholar, 26Ehrenberg C.G. Beobachtungen einer auffallenden bisher unbekannten Structur des Seelenorgans bei Menschen und Thieren. Koenigliche Akademie der Wissenschaften, Berlin1836Google Scholar). Its central nervous system is by now very well described, with some 400 neurons per segmental ganglia, most of them identified and characterized morphologically and physiologically (27Macagno E.R. J. Comp. Neurol. 1980; 190: 283-302Crossref PubMed Scopus (137) Google Scholar). 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Using a global approach based on two-dimensional gel electrophoresis, we have investigated whether the central nervous system of the leech H. medicinalis responds to bacterial challenge with detectable changes in its profile of protein expression. We report here that, among several significant observable changes, the leech nervous system responds to bacterial challenge by increasing the translation of a member of the Hemerythrin family of oxygen-carrying proteins. Animals—Adult Hirudo medicinalis specimens were obtained from Ricarimpex (France) and kept in artificial pond water for acclimatization for at least 1 week before being used in experiments. Two-dimensional Gel Electrophoresis—H. medicinalis central nervous systems were dissected out in leech Ringer (115 mm NaCl, 1.8 mm CaCl2, 4 mm KCl, 10 mm Tris maleate, pH 7.4), bathed for ex vivo septic shock in 2 × 108 bacteria from a mixture of Gram-positive and negative bacteria (Escherichia coli/Micrococcus luteus) or in phosphate-buffered saline for 6 h before being frozen in liquid nitrogen. Batches of 10 control or experimental central nervous systems were stored prior to molecular analysis at –80 °C. The initial steps in protein extraction consisted of three freezing/defrosting cycles, ultrasonic extraction, and 10% trichloroacetic acid/acetone precipitation. Dried pellets were then incubated 2 h at room temperature in “lysis” buffer (9.5 m urea, 2% Triton X-100, 65 mm β-mercaptoethanol, 1.25% SDS) (32Damerval C. De Vienne D. Zivy M. Thiellement H. Electrophoresis. 1986; 7: 52-54Crossref Scopus (648) Google Scholar). The protein concentration of each sample was determined using the Peterson method (33Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7134) Google Scholar). Isoelectrofocusing for the two-dimensional gel electrophoresis was performed with the IEFCell system (Bio-Rad). Immobilized pH 3–10 linear gradient strips were rehydrated in a reswelling solution containing 9 m urea, 2% Triton X-100, 10 mm dithiothreitol (DTT), 1The abbreviations used are: DTT, dithiothreitol; ESI-MS-MS, electrospray ionization-tandem mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time of flight mass spectrometry; CP, crossing point; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 0.2% v/v Bio-lyte, pH 3–10:pH 5–8:pH 7–9, 1:1:2 (Bio-Rad) and 350 μg or 1 mg of proteins from the nervous system of H. medicinalis (for analytical or preparative electrophoresis, respectively). Rehydration was passive for 6 h, then active at 50 V for 14 h at 20 °C. Isoelectrofocusing voltage was rising gradually until 8000 V and running until 180,000 V·h at 20 °C. After focusing, the strips were incubated with 130 mm DTT in equilibration buffer (6 m urea, 375 mm Tris-HCl, 2% SDS, 20% glycerol, 0.02% Coomassie Blue G-250, pH 8.8) twice 15 min to reduce the proteins according to Wu et al. (34Wu X. Ritter B. Schlattjan J.H. Lessmann V. Heumann R. Dietzel I.D. J. Neurobiol. 2000; 44: 320-332Crossref PubMed Scopus (7) Google Scholar). Then the proteins were carbamidomethylated for a further 15 min with 135 mm iodoacetamide in equilibration buffer. The immobilized pH 3–10 linear gradient strips were transferred on a 12% polyacrylamide gel containing 0.8% of cross-linker piperazine diacrylamide. Electrophoresis was performed in Tris/Tricine buffer according to the conditions defined by Schägger and von Jagow (35Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar). Proteins were running 1 h at 30 V and then at 150 V until Coomassie Blue reached the bottom of the gel. After electrophoresis, gels were silver-stained according to a modified protocol of Morrissey (36Morrissey J.H. Anal. Biochem. 1981; 117: 307-310Crossref PubMed Scopus (2940) Google Scholar) for Coomassie Blue stain according to Neuhoff (37Neuhoff V. Arold N. Taube D. Ehrhardt W. Electrophoresis. 1988; 9: 255-262Crossref PubMed Scopus (2354) Google Scholar). Two-dimensional Image Analysis—Four gels per condition were analyzed using the Progenesis™ v1.5 software program (Nonlinear dynamics, Newcastle upon Tyne, UK). The analysis protocol included spot detection and filtering, whole image warping on a reference gel, background subtraction, average gel creation, spot matching, and volume normalization against the total volume of all protein spots present in the gel. Each analysis step was manually validated. In creating the gel average, one spot absence in the gel series was allowed. Statistical significance was measured by Student's paired t test. Edman Degradation—Two-dimensional spots were excised and eluted according to Shaw (38Shaw C. Thim L. Conlon J.M. J. Neurochem. 1987; 49: 1348-1354Crossref PubMed Scopus (26) Google Scholar) from Coomassie Blue-stained electrophoresis gels. NH2-terminal amino acid sequencing was performed on a pulse-liquid automatic peptide PerkinElmer Life Sciences/Applied Biosystems Procise cLC-492 microsequencer. In-gel Digestion for Mass Spectrometry Analysis—Polyacrylamide gel pieces of Coomassie Blue-stained proteins were washed in 25 mm ammonium bicarbonate, 50% acetonitrile. After drying, they were placed on ice for 30 min in 50 μl of protease solution (sequence grade-modified trypsin, Promega, at 0.02 mg/ml in 25 mm ammonium bicarbonate). Excess of protease solution was then removed and replaced by 50 μl of 25 mm ammonium bicarbonate. Digestion was performed overnight at 30 °C. Peptide extraction was performed twice 15 min with 50% acetonitrile, 1% trifluoroacetic acid for further MALDI-MS analysis or with 50% acetonitrile, 1% formic acid for further ESI-MS-MS analysis. Trypsin digests were then lyophilized in a SpeedVac concentrator (Savant) and resuspended in 5 μl of 0.1% trifluoroacetic acid or 0.1% formic acid. Matrix-assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)—MALDI-TOF MS analysis of trypsin digests was performed on a Voyager DE Pro (Applied Biosystems) in reflector mode at an accelerating voltage of 20 kV. One microliter of trypsin digests was spotted on 1 μl of dried α-cyano-4-hydroxycinnamic acid, 15 mg/ml in acetone and covered with 1 μl of α-cyano-4-hydroxycinnamic acid (10 mg/ml) in 70% acetonitrile, 0.1% trifluoroacetic acid. About 300 laser shots were accumulated to obtain the final spectrum. Mass measurements were then finalized after peak smoothing and internal calibration using the two autolysis trypsin fragments 2211.1 and 842.51. Protein data base searching was performed using MS-Fit (prospector.ucsf.edu/ucsfhtml4.0/msfit.htm) according to the monoisotopic molecular weight of [M+H]+ peptides ions. ESI-MS/MS Analysis—After desalting the samples on a C18 Zip-Tip (Millipore, Bedford, MA), the samples were loaded into nanoES capillaries (Protana, Odense, Denmark) using a 5-μl on-column syringe. The capillaries were inserted into an Applied Biosystems Q-STAR Pulsar (Q-TOF-MS) using an ion spray source. Doubly charged peptides were selected, fragmented by N2 collision, and analyzed by MS-MS. The MS-MS sequence was manually assigned to search against Swiss-Prot, NCBI, and GenPept using Fasts software (fasta.bioch.virginia.edu/fasta_www/cgi/search_frm.cgi?pgm=fs) (39Mackey A.J. Haystead T.A. Pearson W.R. Mol. Cell. Proteomics. 2002; 1: 139-147Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Cloning and Sequencing—Total RNA extraction was performed with the TRIzol® kit (Invitrogen) according to manufacturer's conditions. First-strand cDNAs were prepared from these total RNAs with oligo(dT)12–18 for primer. Five micrograms of RNA were first denatured 10 min at 70 °C in the presence of 2 μl of 100 μm primer in a total volume of 26.5 μl. First-strand synthesis was generated in a 40-μl volume by adding 2 μl of deoxynucleotide triphosphate (dNTP) at 10 mm,1 μl of RNase inhibitor, 1 μl of 0.1 m DTT, 8 μlof5× buffer (250 mm Tris-HCl, pH 8.3, 375 mm KCl, 15 mm MgCl2), and 1.5 μl of Superscript II™ (200 units/μl, Invitrogen). Incubation was performed for 55 min at 42 °C, then RNA was hydrolyzed at 55 °C for 15 min in the presence of 1 μl of RNase H. To clone the 5′-end of the cDNA, we added a poly(A) tail to the cDNA first strand. For this, 8 μl of cDNA were mixed with 2 μlof5× buffer, 2 μl of 2 mm ATP, and 1 μl of terminal deoxynucleotide transferase (Invitrogen) in a total volume of 20 μl. The mixture was incubated 15 min at 37 °C followed by 10 min at 65 °C. PCRs were performed in 50-μl mixtures containing 2 μl of firststrand cDNA as a template in PCR buffer 1× (Q-Biogene), 1 μl of primer, and 1 μl of oligo d(T)12–18, each dNTP at 200 μm, and 1 unit of TaqDNA polymerase (PerkinElmer Life Sciences). The PCR condition involves initial heating at 94 °C for 5 min followed thereafter by 30 cycles of denaturation at 94 °C for 40 s, primer annealing at 50 °C for 1 min, and primer extension at 72 °C for 1 min. Amplification cycles were followed by a final extension at 72 °C for 7 min. PCRs were performed in an Eppendorf Mastercycler gradient. The final PCR mixtures were analyzed on 1% (w/v) agarose gel. The PCR products were ligated into the pGEM-T easy vector according to the manufacturer's instructions (Promega). Dideoxy sequencing reactions of the recombinant plasmids were analyzed with the T7 sequencing kit from Amersham Biosciences. The nucleotide sequences were analyzed on an ABI Prism® 310 Genetic Analyzer (Applied Biosystems). Real-time PCR—Reverse transcription was performed as described above. Specific forward and reverse primers were designed using the LightCycler Probe Design software (Roche Applied Science), based on sequence data from the hemerythrin nucleic acid sequence. The primer sequences were: HEMF1 5′-TTCAGGCTTCTCTCGG-3′ and HEMR1b 5′-GTCAACTTCGACAAATCTGC-3′. Primers for the control gene were designed from the sequence of a protein of the large ribosomal subunit available in a H. medicinalis expressed sequence tag data base: RPL7-aF2, 5′-AATGATGAGGTCAGGCA-3′; RPL7aR2b, 5′-GGATCTCTTCAGCCCTTT-3′. PCRs were set up according to the LightCycler manual (Roche Applied Science). A mastermix of the following reaction components was prepared as follows (final concentrations): 8.6 μl of water, 2.4 μl of MgCl2 (4 mm), 1 μl of forward primer (0.4 μm), 1 μl of reverse primer (0.4 μm), and 2 μl of LightCycler Fast Start DNA Master SYBR Green I (Roche Diagnostics). LightCycler mastermix (15 μl) was filled in the LightCycler glass capillaries, and 5 μl of cDNA was added as PCR template. All primers were highly purified and salt-free (40Rajeevan M.S. Ranamukhaarachchi D.G. Vernon S.D. Unger E.R. Methods. 2001; 25: 443-451Crossref PubMed Scopus (267) Google Scholar). The following LightCycler run protocol was used: denaturation program (95 °C, 10 min), amplification and quantification programs repeated 40 times (95 °C for 15 s, annealing temperature for 5 s, 72 °C for 13s), melting curve program (60–95 °C with a heating rate of 0.1 °C/s and continuous fluorescence measurement), and a cooling step to 40 °C. Analysis of melting curves allowed optimization of annealing temperatures for each amplification product. Single highly specific amplification products were obtained using annealing temperatures ranging from 1 °C below to 1 °C above the Tm of primer pairs. For each reaction, the crossing point (CP) (defined as the cycle number at which the noise band intersects the fluorescent curves) was determined using the “Fit Point Method” of the LightCycler software 3.3 (Roche Diagnostics). PCRs were all set in triplicate, and the mean value of the three CPs was calculated. Intra-assay variation (test precision) of CPs was assessed by the coefficient of variation = (S.D. ± mean) × 100 (40Rajeevan M.S. Ranamukhaarachchi D.G. Vernon S.D. Unger E.R. Methods. 2001; 25: 443-451Crossref PubMed Scopus (267) Google Scholar, 41Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25597) Google Scholar). In addition, a no-template control (H2O control) was analyzed for each mastermix. To calculate amplification efficiencies (E) of each target cDNA, relative standard curves were generated using serial dilutions (1, 1:10, 1:50, 1:100, 1:500, and 1:1000) of a unique leech nervous system cDNA sample consisting of a pool of the three cDNAs to be analyzed (1:1:1). Standard curves were generated by the LightCycler software. They are based on the values of CPs and the log value of the standard cDNA dilution. Real-time PCR efficiencies (E) were calculated from the given slope of the standard curve according to the equation E = 10(–1/slope). For each sample, the level of expression of the target gene was compared with the expression of a large subunit ribosomal gene. As amplification efficiencies for each sample were 2, the expression ratio (R) is calculated according to the formula R = 2(CPribosomal–CPhemerythrin). In Situ Hybridization—A plasmid containing the 3′-end (641 bp) of hemerythrin was used as a template for the preparation of the probes. Digoxigenin-11-UTP and 35S-UTP-labeled antisense and sense riboprobes were generated from linearized cDNA plasmids by in vitro transcription using RNA labeling kits (Roche Applied Science) and 35S-UTP (Amersham Biosciences). Total animals or isolated nerve cords of H. medicinalis were fixed 2 h in a solution containing 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. After dehydration, tissues were embedded in Paraplast, and 8-μm sections were cut, mounted on poly-l-lysine-coated slides, and stored at 4 °C until use. Digoxigenin-labeled riboprobes (40–100 ng per slide) and 35S-labeled riboprobes (100 ng or 106 counts/min per slide) were hybridized to tissue sections as described previously (42Mitta G. Vandenbulcke F. Noel T. Romestand B. Beauvillain J.C. Salzet M. Roch P. J. Cell Sci. 2000; 113: 2759-2769PubMed Google Scholar, 43Munoz M. Vandenbulcke F. Saulnier D. Bachere E. Eur. J. Biochem. 2002; 269: 2678-2689Crossref PubMed Scopus (158) Google Scholar). Riboprobes were diluted in hybridization buffer containing 50% formamide, 10% dextran sulfate, 10× Denhardt's solution, 0.5 mg/ml tRNA from E. coli, 100 mm DTT, and 0.5 mg/ml salmon sperm DNA. Hybridization was carried out overnight at 55 °C in a humid chamber. Slides were then washed twice (2 × 30 min) with 2× SSC (standard saline citrate), treated with RNase A (20 μg/ml in 2× SSC) for 30 min at 37 °C, and consecutively rinsed twice for 30 min at 55 °C. The probes labeled with digoxigenin-UTP were revealed using alkaline phosphatase-conjugated antibodies as described previously (42Mitta G. Vandenbulcke F. Noel T. Romestand B. Beauvillain J.C. Salzet M. Roch P. J. Cell Sci. 2000; 113: 2759-2769PubMed Google Scholar). The 35S-UTP hybridization signal was visualized using autoradiography. Samples were coated by dipping in LM1 Amersham liquid emulsion, immediately dried, and exposed for a 10-day period. At the end of the exposure period, the autoradiograms were developed in D19b (Eastman Kodak Co.), fixed in 30% sodium thiosulfate (10 min at room temperature), stained with toluidine blue, and mounted with XAM mounting medium (Merck). Slides were observed under a Zeiss Axioskop microscope, and images were captured with a system equipped with the Leica IM 1000 software. Controls for in situ hybridization consisted of replacing antisense riboprobe with sense riboprobe. RNase control sections were obtained by adding a preincubation step with 10 μg/ml RNase A prior to hybridization. Immunocytochemistry—A potentially immunogenic region of hemerythrin (HIKGTDFKYKGKL) (His108–Leu120) was chemically synthesized, coupled to ovalbumin, and then used to immunize two New Zealand rabbits according to the protocol of Agrobio (La Ferté St Aubin, France) to recover antisera. Tissue sections obtained as decribed above were preincubated 1 h in TBS (0.1 m Tris, pH 7.4, 0.9% NaCl) containing 1% normal goat serum, 1% ovalbumin, and 0.05% Triton X-100. Then, sections were incubated overnight at 20 °C in TBS containing 1:400 rabbit anti-hemerythrin antiserum, 1% normal goat serum, 1% ovalbumin, 0.05% Triton X-100. After washes in TBS, sections were incubated for 2 h with a goat anti-rabbit fluorescein isothiocyanate-tagged antiserum (Jackson ImmunoResearch) diluted 1:100 in TBS. After washes, slides were mounted in glycerol containing 25% TBS and 0.1% p-phenylenediamine. Labeled cells were observed using a Leica laser scanning microscope (TCS NT) equipped with a Leica (DMIRBE) inverted microscope and an argon/krypton laser. Fluorescein isothiocyanate signal was detected using a 488 nm band pass excitation filter and a 575–640 nm pass barrier filter. Images were acquired sequentially as single transcellular optical sections and averaged over 16 scans/frame. Controls consisted of incubations of anti-hemerythrin immunserum preadsorbed with the His108–Leu120 synthesis peptide. Neurohemerythrin Is Up-regulated following Sepsis—Proteins from leech nervous systems, normal or subjected to sepsis, were separated by two-dimensional gel electrophoresis and stained with silver nitrate for image gel analysis using Progenesis™ software. The two-dimensional electrophoretic profile of leech nervous system proteins is illustrated in Fig. 1A. Among the changes observed as a result of sepsis, the increase in the signal of the spot labeled 11/3 was particularly noticeable and was chosen for further analysis. A quantitative assay of the size of the signal (see “Experimental Procedures”) revealed that the change in the annotated protein spot 11/3 was significant (p < 0.05; Student's paired t test). The normalized volume of this protein spot, which corresponds to a protein of about 14–15 kDa and a pI between 5 and 6 (Fig. 1A), increased by about 40% over controls following bacterial challenge (Fig. 1B). To identify the 11/3 protein, we first performed an in-gel digestion with trypsin, followed by MALDI-TOF mass spectrometry analysis. This yielded a peptide mass fingerprint for the 11/3 protein, but this was insufficient information for the immediate identification of the protein because of the current lack of deta