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• W2022733092 abstract "Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response. Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response. Invertebrate animals do not have any adaptive immune system and have to rely on innate immune systems. Several such innate systems have been described such as the coagulation system (1Theopold U. Schmidt O. Söderhäll K. Dushay M. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), melanization synthesis (2Iwanaga S. Lee B.L. J. Biochem. Mol. Biol. 2005; 38: 128-150Crossref PubMed Google Scholar), and the production of antimicrobial peptides (3Ferrandon D. Imler J.L. Hetru C. Hoffmann J.A. Nat. Rev. Immunol. 2007; 11: 862-874Crossref Scopus (642) Google Scholar). The melanization reaction is an important component of the innate immune system of invertebrates and is essential for defense as well as for wound healing (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). In arthropods and most other invertebrates melanin synthesis is achieved by the prophenoloxidase (proPO)-activating 3The abbreviations used are: proPO, prophenoloxidase; MIP, melanization inhibition protein; PO, phenoloxidase; Tm, T. molitor; HLS, hemocyte lysate supernatant; l-DOPA, 3,4-dihydroxy-l-phenylalanine; PTU, phenylthiourea; TBS, Tris-buffered saline; MS, mass spectrometry; RACE, rapid amplification of cDNA ends; rMIP, recombinant MIP; dsRNA, double-stranded RNA; FReD, fibrinogen-related domain; TL, tachylectin; Pl, P. leniusculus; r-, recombinant; LPS, lipopolysaccharide; PGN, peptidoglycan. system, a proteolytic cascade similar to vertebrate complement (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). The proPO-activating system is initiated when microbial polysaccharides, such as lipopolysaccharides (LPS), β-1,3-glucans or peptidoglycans (PGN) are recognized by pattern recognition proteins, and the complexes formed induce activation of serine proteinase zymogens in the cascade (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). The final step in this process is the conversion of proPO into the active enzyme phenoloxidase (PO). To date ∼40 proPOs have been cloned and characterized, and several other constituent factors of the proPO system have recently been characterized (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). Active PO oxidizes o-diphenols into quinones that are toxic to microorganisms and melanin pigments are formed that also restrict the spreading of microorganisms within the host (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). Detailed studies of the molecular mechanism by which the activation is achieved has been performed in Tenebrio molitor (6Kan H. Kim C.H. Kwon H.M. Park J.W. Roh K.B. Lee H. Park B.J. Zhang R. Zhang J. Söderhäll K. Ha N.C. Lee B.L. J. Biol. Chem. 2008; 283: 25316-25323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), Holotrichia diomphalia (7Kim M.S. Baek M.J. Lee M.H. Park J.W. Lee S.Y. Söderhäll K. Lee B.L. J. Biol. Chem. 2002; 277: 39999-40004Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and Manduca sexta (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). In Tenebrio a complex of the peptidoglycan recognition protein Tm PGRP-SA and peptidoglycan fragments lead to the activation of proPO-activating factors, one of which (proPO-activating factor I) in its active form is mediating cleavage of proPO into an active enzyme (6Kan H. Kim C.H. Kwon H.M. Park J.W. Roh K.B. Lee H. Park B.J. Zhang R. Zhang J. Söderhäll K. Ha N.C. Lee B.L. J. Biol. Chem. 2008; 283: 25316-25323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 8Park J.W. Kim C.H. Kim J.H. Je B.R. Roh K.B. Kim S.J. Lee H.H. Ryu J.H. Lim L.H. Oh B.H. Lee W.J. Ha N.C. Lee B.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6602-66075Crossref PubMed Scopus (130) Google Scholar). A similar mechanism of proPO activation is implicated also by research done on M. sexta (9Gorman M.J. Wang Y. Jiang H. Kanost M.R. J. Biol. Chem. 2007; 16: 11742-11749Abstract Full Text Full Text PDF Scopus (91) Google Scholar). In crustaceans the proPO-activating system is studied in detail at the cellular and molecular level (5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). In crayfish the proPO-activating system is produced in hemocytes and stored in secretory granules similar to the clotting system of horseshoe crabs (1Theopold U. Schmidt O. Söderhäll K. Dushay M. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Upon challenge with microorganisms, the system is released into the hemolymph. Crayfish proPO is cleaved by the proPO-activating enzyme, a serine proteinase that is activated by another serine proteinase (4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). Although melanin formation is essential for host defense in crustaceans and insects, the process needs to be tightly regulated because of the danger to the animal of unwanted production of quinone intermediates and melanization in places where it is not appropriate (10Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman & Hall, New York1998: 135-172Google Scholar, 11Gillespie J. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1109) Google Scholar). Several proteinase inhibitors, such as serpins, have been described as responsible for preventing improper activation of the proPO system (for details see Ref. 4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). Pacifastin is a high molecular weight inhibitor consisting of one light chain containing the protease inhibitors and a heavy chain that contains three transferrin lobes (12Liang Z.C. Sottrup-Jensen L. Aspan A. Hall M. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6682-6687Crossref PubMed Scopus (124) Google Scholar). Pacifastin is highly efficient in inhibiting the crayfish proPO-activating enzyme (12Liang Z.C. Sottrup-Jensen L. Aspan A. Hall M. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6682-6687Crossref PubMed Scopus (124) Google Scholar), as is Drosophila serpin 27 in inhibiting the Drospohila proPO system and its proPO-activating enzyme (13Ligoxygakis P. Pelte N. Ji C. Leclerc V. Duvic B. Belvin M. Jiang H. Hoffmann J.A. Reichhart J.M. EMBO J. 2002; 21: 6330-6337Crossref PubMed Scopus (232) Google Scholar). In addition to inhibitors of the proPO system, activation of a number of endogenous factors acting as competitive inhibitors of PO activity has been described (5Cerenius L. Lee B.L. Söderhäll K. Trends Immunol. 2008; 29: 263-271Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). For example the so-called phenoloxidase inhibitors have been found in Musca domestica, and homologous basic lysine-rich peptides were also detected in mosquitoes and M. sexta (14Shi L. Li B. Paskewitz S.M. Insect Mol. Biol. 2006; 15: 313-320Crossref PubMed Scopus (16) Google Scholar, 15Lu Z.Q. Jiang B. Insect Biochem. Mol. Biol. 2007; 37: 478-485Crossref PubMed Scopus (30) Google Scholar). We have recently discovered a novel 43-kDa protein from the hemolymph of the beetle T. molitor (Tenebrio MIP) acting as a negative regulator of melanin synthesis (16Zhao M. Söderhäll I. Park J.W. Ma Y.G. Osaki T. Ha N.C. Wu C.F. Söderhäll K. Lee B.L. J. Biol. Chem. 2005; 280: 24744-24751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This protein, the target of which is presently unknown, is consumed during melanization. Interestingly, no similarity was found between Tenebrio MIP and any other known protein (16Zhao M. Söderhäll I. Park J.W. Ma Y.G. Osaki T. Ha N.C. Wu C.F. Söderhäll K. Lee B.L. J. Biol. Chem. 2005; 280: 24744-24751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Here, we now report the presence of a hemolymph protein with an apparently similar function as Tenebrio MIP in the crustacean Pacifastacus leniusculus. Interestingly, this protein has no sequence similarity to Tenebrio MIP. Instead, it is similar to vertebrate ficolin and horseshoe crab Tachylectin 5 (17Gokudan S. Muta T. Tsuda R. Koori K. Kawahara T. Seki N. Mizunoe Y. Wai S.N. Iwanaga S. Kawabata S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10086-10091Crossref PubMed Scopus (190) Google Scholar, 18Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 26: 623-633Crossref PubMed Scopus (147) Google Scholar). The crayfish MIP is very efficient in inhibiting activation of the proPO system and thus functions as an important regulatory molecule to prevent unwanted proPO activation. Animals, Collection of Plasma, and Microbial Organisms- Freshwater crayfish, P. leniusculus were purchased from Torsäng (Lake Vättern, Sweden) and kept in aquaria in aerated tap water at 10 °C. Only intermolt animals were used. Hemolymph was collected by bleeding from the abdominal hemocoel through a needle (0.8 mm) into sterile tubes on ice and centrifuged at 800 × g for 10 min at 4 °C to remove the hemocytes. The Gram-negative bacterium Hafnia alvei has earlier been isolated from P. leniusculus hemolymph 4I. Söderhäll, unpublished observation. and was cultured in LB broth. For fresh cultures the bacteria were grown with shaking at 260 rpm overnight at 37 °C and then diluted 1:100 and further cultured until the density reached OD600 of ≈0.5. Bacteria H. alvei were injected into the base of the fourth walking leg as earlier described (19Liu H.P. Jiravanichpaisal P. Cerenius L. Lee B.L. Söderhäll I. Söderhäll K. J. Biol. Chem. 2007; 282: 33593-33598Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Hemocyte Lysate Supernatant-Hemocyte lysate supernatant (HLS) was prepared by collecting hemolymph from 8 crayfishes in bleeding buffer (10 mm sodium cacodylate, 250 mm sucrose, pH 7.0). The hemocytes were spun down by centrifugation at 800 × g for 10 min at 4 °C and then homogenized in 10 mm sodium cacodylate, 5 mm CaCl2, pH 7.0. The homogenate was centrifuged at 25 000 × g for 20 min at 4 °C, and the supernatant was adjusted to a protein content of ∼1 mg/ml, kept on ice, and used as HLS within 1 h. Induction of proPO Activation and Assay of PO Activity-To confirm the involvement of crayfish MIP in the proPO system, 25 μl of HLS (1 mg/ml) was preincubated with 25 μl of MIP (wild type or MIP(-D4), a mutant protein lacking the tetraaspartic acid stretch, at 0.5–1 μg) or buffer for the control for 10 min at 20 °C. These mixtures were incubated with 25 μg of LPS-PGN (Sigma L3129 from Escherichia coli 0127:B8) and 25 μl of 3,4-dihydroxy-l-phenylalanine (l-DOPA, 3 mg/ml) for 5–20 min at 20 °C. For analysis of the effect of MIP on PO activity, HLS was preincubated with LPS-PGN for 25 min at 20 °C to fully activate proPO prior to the addition of MIP. Phenoloxidase activity was measured as the oxidation of l-DOPA at 490 nm and presented as the means ± standard deviation from four independent experiments. In some experiments the phenoloxidase inhibitor phenylthiourea (PTU) was preincubated with HLS for 5 min at 20 °C. Measurement of Proteinase Activity-To determine whether any activating proteinase was affected by MIP, LPS-PGN-activated amidase activity of HLS was assayed as the hydrolyzing activity toward the chromogenic peptide S-2222 (Suc-Ile-Gly (γPip) Gly-Arg-pNA; Chromogenix). Briefly, 25 μl of HLS was incubated with 25 μg of LPS-PGN and 100 μl of 100 mm Tris-HCl, pH 8.0, and 25 μl of 2 mm S-2222 at 30 °C for 30 min, and then the reaction was terminated by the addition of 50 μl of 50% acetic acid, and the absorbance at 405 nm was determined. The effect of MIP was determined by preincubation of MIP (0.5–1 μg) or buffer for the controls for 10 min at 20 °C prior to the addition of LPS. Detection of Crayfish MIP in Plasma and Determination of the Amino Acid Sequence-The proteins in plasma were precipitated with acetone and subjected to 12.5% SDS-PAGE or two-dimensional gel electrophoresis under reducing conditions. First-dimensional separation was performed according to pI, and second-dimensional separation was done according to molecular weight. The IPG strips (7 cm length, pI range between 3 and 11, nonlinear; GE Healthcare) were rehydrated with rehydration solution including 80 μg of protein extracted before, for 12 h or overnight at 20 °C. Using the IPGphor system (GE Healthcare), isoelectric focusing was performed at a total of 45.5 kVh at 20 °C. The cysteine sulfhydryls were reduced and carbamidomethylated, whereas the proteins were equilibrated in the two-dimensional loading buffer (glycerol, SDS, urea) supplemented with 1% dithiothreitol for 15 min at 20 °C, followed by 2.5% iodoacetamide in fresh equilibration buffer for an additional 15 min at 20 °C. After equilibration, the IPG strips were applied onto a 7-cm acrylamide gel (12.5%). SDS-PAGE was performed at 30 mA/gel for 50–60 min at 20 °C. All of the electrophoretic procedures were performed at room temperature. One two-dimensional gel was stained with Coomassie Brilliant Blue R-250, and the other gel was transferred to a polyvinylidene difluoride membrane for Western blot. An affinity-purified antibody against Tenebrio MIP (16Zhao M. Söderhäll I. Park J.W. Ma Y.G. Osaki T. Ha N.C. Wu C.F. Söderhäll K. Lee B.L. J. Biol. Chem. 2005; 280: 24744-24751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) was used for Western blot analysis. The proteins were separated by SDS-PAGE and transferred electrophoretically to a polyvinylidene difluoride membrane, blocked by immersion in 5% skimmed milk solution for 1 h. The membrane was then transferred to TBS (10 mm Tris-HCl, pH 7.5, containing 150 mm NaCl) containing the affinity-purified Tenebrio MIP antibody (50 ng/ml) and 1% bovine serum albumin and incubated at 20 °C for 1 h. After washing in T-TBS (TBS + 0.1% Tween 20) for 3 × 10 min, the ECL anti-rabbit IgG peroxidase-linked species-specific whole antibody from donkey (GE Healthcare) diluted 1:10000 with TBS + 1% bovine serum albumin was incubated for 1 h and washed with T-TBS for 3 × 10 min. For detection, the ECL Western blotting reagent kit (Amersham Biosciences) was used according to the manufacturer’s instructions. After comparing with the result of Western blot, selected spots from gels stained with Coomassie Brilliant Blue R-250 were excised and cleaved with trypsin by in-gel digestion. The peptides were analyzed by electrospray ionization mass spectrometry on a quadruple time-of-flight mass spectrometer (Waters Ltd.) using Masslynx software. SDS-PAGE and Western Blot-The proteins in plasma were precipitated with acetone and subjected to 12.5% SDS-PAGE under reducing conditions. To examine what happened to MIP during PO-induced melanization, we induced PO activity as described above. When melanin pigments were generated, the reaction mixture was centrifuged at 28,500 × g at 4 °C for 10 min. To confirm whether the disappeared bands on SDS-PAGE were specifically related to melanin synthesis, the inhibitor PTU was added to the same reaction mixture under the same conditions. After incubation, the proteins of the supernatant were precipitated with acetone and analyzed by Western blot after SDS-PAGE under reducing conditions as described above. cDNA Cloning and Nucleotide Sequencing of 43-kDa Crayfish MIP- Hepatopancreas total RNAs were extracted using GenElute™ mammalian total RNA miniprep kit (Sigma) and followed by RNase-free DNase I (Ambion, Austin, TX) treatment. cDNA was synthesized with ThermoScript (Invitrogen). Several sets of gene-degenerate primers based on the MS sequence results (VVMEDFDANK) were designed for 5′-rapid amplification of cDNA ends (RACE) and 3′-RACE. 5′-RACE was performed according to the manufacturer’s protocol (Invitrogen). Oligo(dT) was used to synthesize first strand cDNA. After poly(C) tail were assembled, PCR amplification was performed using the MIP-5R1 (5′-TTIGCRTCRAARTCYTCCAT-3′) and abridged anchor primer followed by nest PCR amplification with MIP-5R2 (5′-TCRAARTCYTCCATIACIAC-3′) and abridged universal amplification primer (AUAP) using the recommended conditions. For 3′ RACE, oligo(dT)-adapter was used to synthesize first strand cDNA. An initial amplification by PCR was carried out with primer MIP-3R1 (5′-GCGAGTACCAGGAAGGCTTT-3′) and oligo(dT) adapter. The nested PCR was performed with primer MIP-3R2 (5′-GCGTTCTCACCAGCTGAGAGT-3′). Amplified fragments were cloned into PCR 2.1-TOPO TA cloning vector (Invitrogen) and sequenced. Determination of MIP mRNA Localization-Total RNA extraction from different tissues (hepatopancreas, eyestalk, hemocytes, nerve tissue, heart, muscle, intestine, and hematopoietic tissue cells) was performed using TRIzol LS reagent (Invitrogen), followed by chloroform extraction and ethanol precipitation of the aqueous phase. Total RNA was treated by RNase-free DNase I (Ambion, Austin, TX) treatment. cDNA was synthesized with ThermoScript (Invitrogen) followed by PCR using primers specific for MIP (GenBank™ accession number EX571686). Crayfish 40 S ribosomal (R40s; GenBank™ accession number CF542417) primers were used in all PCR experiments as control. The primers used were as follows: MIP 217+, 5′-CCACTCACCTCAGCCGACAC-3′; 698-, 5′-TCTTCCATGACGACTCTCAGCT-3′; crayfish 40 S ribosomal protein gene 5+, 5′-CCAGGACCCCCAAACTTCTTAG-3′; and 364-, 5′-GAAAACTGCCACAGCC-3′. For detection of MIP in the RNA interference experiment MIP 1+, 5′-TACCAGGTACACTCTCATCTACC-3′; 701-, 5′-GTCCTCCATTACGACTCTCA-3′ were used. The PCR program was as follows: 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s for the MIP gene and 28 cycles for the R40s. The PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. Expression and Purification of Recombinant Crayfish MIP- The construction of the recombinant baculovirus vector and the expression of the recombinant MIP (rMIP) were performed according to manufacturer’s instructions (Invitrogen). The cDNA encoding the mature MIP was subcloned into pFastBac1 vector (Invitrogen) using BamHI and SalI enzyme sites. The recombinant virus for MIP expression was generated according to the manufacturer’s instruction manual (Bac-to-Bac Baculovirus expression system; Invitrogen). The recombinant virus was amplified using Spodoptera frugiperda 9 (Sf9; Invitrogen) cells in SF-900II serum-free medium (Invitrogen) at 27 °C. To produce the protein, Sf9 cells were grown in Sf-900II serum-free medium (Invitrogen) in a 75-cm2 tissue culture flask. The cells were infected with a cell density of 2 × 106 cells/ml at a multiplicity of infection of 10 and were incubated for 3–4 days. The supernatant was collected by centrifuging at 500 × g for 10 min. After discarding the pellets, the supernatant containing MIP protein was desalted by using a PD-10 desalting column (code 17-0851-01; Amersham Biosciences) and applied to a 1-ml HiTrap Q HP column (0.7 × 2.5 cm; Amersham Biosciences, Uppsala, Sweden) column, pre-equilibrated with 20 mm Tris-HCl buffer, pH 8.0, and then this column was washed with the same buffer until no material appeared in the effluent and then gradient eluted with 20 mm Tris-HCl, 1 m NaCl, pH 8.0 (100 ml, 0–50%) at a flow rate of 60 ml/h. Fractions of 1 ml were collected. The purified protein was subjected to 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 or transferred to polyvinylidene difluoride membrane to determine the purity by Western blot. Site-directed Mutagenesis-The cDNA encoding the mature crayfish MIP was subcloned into pFastBac1 vector (Invitrogen), and this plasmid was used for mutagenesis. Site-directed mutagenesis was done using the QuikChange site-directed mutagenesis kit (Stratagene). The mutants were obtained by deleting the four aspartic acids in the Asp-rich region in the cDNA clone: MIP(-D4) -858+:5′-GTTTTCTACCTACGACAAGAACAAAGATGGTAACTGCTC-3′; and MIP(-D4) -858-: 5′-GAGCAGTTACATCTTTGTTCTTGTCGTAGGTAGAAAAC-3′. The PCR was done as follows: 95 °C for 30 s, and 18 cycles of 95 °C for 30 s, 55 °C for 1 min, final extension at 68 °C, 6 min. The nicked vector DNA containing the desired mutations was then transformed into XL1-Blue supercompetent cells, and then mutant MIP(-D4) plasmids were purified and sequenced to verify the mutated sequence. The generation of the recombinant virus for crayfish MIP(-D4) expression and the purification of recombinant crayfish MIP(-D4) are similar to the methods used for crayfish MIP. Hemagglutinating Activity Assay-Hemagglutinating activity toward human erythrocytes type A, B or O of rMIP was determined in assay buffer containing 5 mm CaCl2 as described in Ref. 20Okino N. Kawabata S. Saito T. Hirata M. Takagi T. Iwanaga S. J. Biol. Chem. 1995; 270: 31008-31015Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar. RNA Interference Experiments-Oligonucleotide primers including T7 promoter sequences (italics) at the 5′ end were designed to amplify a 776-bp region of the P. leniusculus MIP gene: 217+, 5′-TAATACGACTCACTATAGGGCCACTCACCTCAGCCGACAC-3′; 993-, 5′-TAATACGACTCACTATAGGGCCAGGCCGCCATACTCGTTA-3′. Control 657-bp templates were generated by PCR using primers specific for the green fluorescent protein gene from the pd2EGFP-1 vector (Clontech, Palo Alto, CA) as follows: 63+, 5′-TAATACGACTCACTATAGGGCGACGTAAACGGCCACAAGT-3′; 719-, 5′-TAATACGACTCACTTAGGGTTCTTGTACAGCTCGTCCATGC-3′. To generate dsRNA, the PCR products purified by gel extraction (Qiagen) were used as templates for in vitro transcription using the MegaScript kit (Ambion, Austin, TX), and dsRNA was purified with the TRIzol LS reagent (Invitrogen) method. Small intermolt crayfish (20 ± 2 g, fresh weight) were used for in vivo RNA interference experiments. Briefly, 150 μg of MIP or green fluorescent protein control dsRNA dissolved in crayfish saline (0.2 m NaCl, 5.4 mm KCl, 1 mm CaCl2, 2.6 mm MgCl2, 2 mm NaHCO3, pH 6.8) (200 μl) was injected via the base of the fourth walking leg. The injection was repeated 24 h after the first dsRNA injection. The presence of MIP protein in the hemolymph of dsRNA-treated crayfish was analyzed by Western blot as described above. Homology Modeling-A homology model of Pacifastacus MIP protein was built using the structure of lectin called l-ficolin (pdbid 2J61) (18Garlatti V. Belloy N. Martin L. Lacroix M. Matsushita M. Endo Y. Fujita T. Fontecilla-Camps J.C. Arlaud G.J. Thielens N.M. Gaboriaud C. EMBO J. 2007; 26: 623-633Crossref PubMed Scopus (147) Google Scholar). An alignment of these two proteins was carried out using the PROBCONS web service (21Do C.B. Mahabhashyam M.S. Brudno M. Batzoglou S. Genome Res. 2005; 15: 330-340Crossref PubMed Scopus (879) Google Scholar) and manually edited. The MODELLER program version 9.2 (22Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10548) Google Scholar) was used to build three-dimensional models of crayfish MIP. The quality of the final model was checked using the ProCheck (23Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystalogr. 1993; 26: 283-291Crossref Google Scholar) and WhatCheck programs (24Rodriguez R. Chinea G. Lopez N. Pons T. Vriend G. Bioinformatics. 1998; 14: 523-528Crossref PubMed Scopus (311) Google Scholar). Tenebrio MIP Antibody Recognizes a Protein in Crayfish Plasma-We have earlier reported about the existence of MIP, a 43-kDa protein with no significant similarity to other reported proteins, acting as a negative regulator of melanization in the mealworm larvae. One characteristic of Tm MIP is the Asp-rich region including 11 adjacent Asp residues in its central part. To determine whether the freshwater crayfish P. leniusculus expresses a similar MIP protein, we used antibodies raised against Tm MIP in a Western blot experiment of crayfish hemocytes and plasma. As shown in Fig. 1A, a clear band was detected by the antibodies at a molecular mass of ∼43 kDa. To characterize further the protein that reacted with the MIP antibody, we performed a two-dimensional analysis of crayfish plasma, followed by Western blot. Two clear spots (and another two very weak) appeared at ∼43 kDa after immunoblotting of the two-dimensional gel (Fig. 1, B and C), and these spots were all subjected to amino acid analysis by matrix-assisted laser desorption ionization time-of-flight. The only peptide sequence that could be identified from these spots by tandem MS was VVMEDFDANK, showing no match with Tenebrio MIP or any other known protein. Thus we assumed that this crayfish protein might represent a new protein of unknown function. Cloning and Characterization of the cDNA for This Crayfish Protein-To determine the amino acid sequence of this crayfish protein, we designed degenerate primers to the peptide VVMED-FDANK and performed 5′-RACE and 3′-RACE using cDNA synthesized from hepatopancreas RNA. When we used hemocyte cDNA no transcript could be detected. However, we obtained a cDNA of 1764 base pairs from hepatopancreas, and the deduced amino acid sequence of the open reading frame of this cDNA is shown in Fig. 2A. This cDNA encodes a protein consisting of 326 amino acid residues with a signal" @default.
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• W2022733092 title "A Novel Protein Acts as a Negative Regulator of Prophenoloxidase Activation and Melanization in the Freshwater Crayfish Pacifastacus leniusculus" @default.
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