Immulectin-2, a Lipopolysaccharide-specific Lectin from an Insect, Manduca sexta, Is Induced in Response to Gram-negative Bacteria
2000; Elsevier BV; Volume: 275; Issue: 48 Linguagem: Inglês
10.1074/jbc.m003021200
ISSN1083-351X
AutoresXiao‐Qiang Yu, Michael R. Kanost,
Tópico(s)Insect symbiosis and bacterial influences
ResumoA lipopolysaccharide-specific lectin, immulectin-2, was isolated from plasma of the tobacco hornworm,Manduca sexta. Immulectin-2 has specificity for xylose, glucose, lipopolysaccharide, and mannan. A cDNA clone encoding immulectin-2 was isolated from an Escherichia coli-inducedM. sexta larval fat body cDNA library. The cDNA is 1253 base pairs long, with an open reading frame of 981 base pairs, encoding a 327-residue polypeptide. Immulectin-2 is a member of the C-type lectin superfamily. It consists of two carbohydrate recognition domains, which is similar to the organization of M. sextaimmulectin-1. Immulectin-2 was present at a constitutively low level in plasma of control larvae and increased 3–4-fold after injection of Gram-negative bacteria or lipopolysaccharide. Immulectin-2 mRNA was detected in fat body of control larvae, and its level increased dramatically after injection of E. coli. The concentration of immulectin-2 in plasma did not change significantly after injection of Gram-positive bacteria or yeast, even though its mRNA level was increased by these treatments. Compared with immulectin-1, immulectin-2 has a more restricted specificity for binding to Gram-negative bacteria. Immulectin-2 at low physiological concentrations agglutinatedE. coli in a calcium-dependent manner. It also bound to immobilized lipopolysaccharide from E. coli. Binding of immulectin-2 to lipopolysaccharide stimulated phenol oxidase activation in plasma. The properties of immulectin-2 are consistent with its function as a pattern recognition receptor for detection and defense against Gram-negative bacterial infection in M. sexta. A lipopolysaccharide-specific lectin, immulectin-2, was isolated from plasma of the tobacco hornworm,Manduca sexta. Immulectin-2 has specificity for xylose, glucose, lipopolysaccharide, and mannan. A cDNA clone encoding immulectin-2 was isolated from an Escherichia coli-inducedM. sexta larval fat body cDNA library. The cDNA is 1253 base pairs long, with an open reading frame of 981 base pairs, encoding a 327-residue polypeptide. Immulectin-2 is a member of the C-type lectin superfamily. It consists of two carbohydrate recognition domains, which is similar to the organization of M. sextaimmulectin-1. Immulectin-2 was present at a constitutively low level in plasma of control larvae and increased 3–4-fold after injection of Gram-negative bacteria or lipopolysaccharide. Immulectin-2 mRNA was detected in fat body of control larvae, and its level increased dramatically after injection of E. coli. The concentration of immulectin-2 in plasma did not change significantly after injection of Gram-positive bacteria or yeast, even though its mRNA level was increased by these treatments. Compared with immulectin-1, immulectin-2 has a more restricted specificity for binding to Gram-negative bacteria. Immulectin-2 at low physiological concentrations agglutinatedE. coli in a calcium-dependent manner. It also bound to immobilized lipopolysaccharide from E. coli. Binding of immulectin-2 to lipopolysaccharide stimulated phenol oxidase activation in plasma. The properties of immulectin-2 are consistent with its function as a pattern recognition receptor for detection and defense against Gram-negative bacterial infection in M. sexta. lipopolysaccharide carbohydrate recognition domain high performance liquid chromatography immulectin mannose-binding protein polyacrylamide gel electrophoresis polymerase chain reaction Tris buffer Tris-buffered saline base pairs bovine serum albumin Insects have a rapid and effective system for defense against microbial infections, which shares many characteristics with the innate immune system of vertebrates (1Vilmos P. Kurucz É. Immunol. Lett. 1998; 62: 59-66Crossref PubMed Scopus (195) Google Scholar, 2Gillespie J.P. Kanost M.R. Trenczek T. Annu. Rev. Entomol. 1997; 42: 611-643Crossref PubMed Scopus (1099) Google Scholar, 3Hoffmann J.A. Kafatos F.C. Janeway Jr., C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2144) Google Scholar). Proteins that specifically bind to microbial components play an important role in nonself-recognition and clearance of invading microbes. Such proteins are known as pattern recognition receptors, because they bind to certain molecular patterns present in the array of carbohydrate components on the surface of microorganisms (4Janeway Jr., C.A. Cold Spring Harbor Symp. Quant. Biol. 1989; 54: 1-13Crossref PubMed Google Scholar). These microbial patterns include lipopolysaccharide (LPS)1 and peptidoglycan from bacterial cell walls, and β-1,3-glucan from fungal cell walls. Because of their ability to bind to terminal sugars on glycoproteins and glycolipids, lectins are primary candidates for pattern recognition receptors in innate immunity. Animal C-type lectins (calcium-dependent lectins) have been reported to be important in pathogen recognition and cellular interactions (5Weis W.I. Taylor M.E. Drickamer K. Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (881) Google Scholar). Collectins, a subgroup of the C-type lectin superfamily, play a key role in the first line of defense against infection (6Epstein J. Eichbaum Q. Sheriff S. Ezekowitz R.A.B. Curr. Opin. Immunol. 1996; 8: 29-35Crossref PubMed Scopus (231) Google Scholar, 7Holmskov U. Malhotra R. Sim R.B. Jensenius J.C. Immunol. Today. 1994; 15: 67-74Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Collectins contain a carbohydrate recognition domain (CRD) connected to a collagen-like domain (8Thiel S. Reid K.B.M. FEBS Lett. 1989; 250: 78-84Crossref PubMed Scopus (151) Google Scholar). The most extensively studied collectin is the serum mannose-binding protein (MBP). MBP can activate the complement system through a recently discovered pathway, the lectin pathway (9Matsushita M. Microbiol. Immunol. 1996; 40: 887-893Crossref PubMed Scopus (87) Google Scholar). Activation of the complement system by MBP is associated with C1r/C1s-like proteases (10Matsushita M. Fujita T. J. Exp. Med. 1992; 176: 1497-1502Crossref PubMed Scopus (555) Google Scholar, 11Thiel S. Vorup-Jensen T. Stover C.M. Schwaeble W. Laursen S.B. Poulsen K. Willis A.C. Eggleton P. Hansen S. Holmskov U. Reid K.B.M. Jensenius J.C. Nature. 1997; 386: 506-510Crossref PubMed Scopus (746) Google Scholar). MBP also functions directly as an opsonin to increase the efficiency of phagocytosis of bacteria (12Kawasaki N. Kawasaki T. Yamashina I. J. Biochem. 1989; 106: 483-489Crossref PubMed Scopus (148) Google Scholar,13Kuhlman M. Joiner K. Ezekowitz A.B. J. Exp. Med. 1989; 169: 1733-1740Crossref PubMed Scopus (388) Google Scholar). Recently, C-type lectins have been isolated from a few insect species. These C-type lectins function in insect innate immune system by participating in hemocyte nodule formation (14Koizumi N. Morozumi A. Imamura M. Tanaka E. Iwahana H. Sato R. Eur. J. Biochem. 1997; 15: 217-224Crossref Scopus (83) Google Scholar, 15Koizumi N. Imamura M. Kadotani T. Yaoi K. Iwahana H. Sato R FEBS Lett. 1999; 443: 139-143Crossref PubMed Scopus (164) Google Scholar), activating prophenol oxidase in hemolymph (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar), and opsonization (17Jomori T. Natori S. FEBS Lett. 1992; 296: 283-286Crossref PubMed Scopus (97) Google Scholar). Among these insect lectins is a group of C-type lectins that contain two tandem CRDs. Lectins of this new type include immulectin-1 from the tobacco hornworm, Manduca sexta (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar), and LPS-binding lectins from the silkworm, Bombyx mori (15Koizumi N. Imamura M. Kadotani T. Yaoi K. Iwahana H. Sato R FEBS Lett. 1999; 443: 139-143Crossref PubMed Scopus (164) Google Scholar) and the fall webworm,Hyphantria cunea (18Shin S.W. Park S.-S. Park D.-S. Kim M.G. Kim S.C. Brey P.T. Park H.-Y. Insect Biochem. Mol. Biol. 1998; 28: 827-837Crossref PubMed Scopus (130) Google Scholar, 19Shin S.W. Park D.S. Kim S.C. Park H.Y. FEBS Lett. 2000; 467: 70-74Crossref PubMed Scopus (43) Google Scholar). We report here an LPS-specific C-type lectin, immulectin-2 from M. sexta, which contains two CRDs and binds to Gram-negative bacteria and to LPS and stimulates phenol oxidase activation in hemolymph. The synthesis of M. sexta immulectin-2 (IML-2) is induced in fat body after injection of Gram-negative bacteria or LPS. M. sexta eggs were initially obtained from Carolina Biological Supply, and larvae were reared using artificial diet as described by Dunn and Drake (20Dunn P.E. Drake D. J. Invertebr. Pathol. 1983; 41: 77-85Crossref Scopus (182) Google Scholar). Larvae in the second or third day of the fifth instar were injected with Micrococcus lysodeikticus (Sigma) (150 μg/larva), LPS from Escherichia coli 0111:B4 (Sigma) (20 μg/larva), formalin-killed E. coli strain XL1-blue, Pseudomonas aeruginosa ATCC27853, Serratia marcescens strain (obtained from James Urban, Division of Biology, Kansas State University) (all bacteria at 108 cells/larva), orSaccharomyces cerevisiae (107 cells/larva) suspended in 50 μl of water or with 50 μl of saline (0.85% NaCl) as a control. Hemolymph was collected using a 27-gauge needle at several time intervals post-injection. Hemocytes were removed by centrifugation at 5,000 × g for 5 min, and plasma samples were stored at −20 °C. Plasma (200 ml) collected 24 h post-injection of E. coli was dialyzed against 4 liters of 20 mm Tris-HCl, pH 7.4, 100 mm NaCl, 2 mm CaCl2 for 24 h at 4 °C. After removal of a precipitate by centrifugation for 10 min at 10,000 × g, dialyzed plasma was loaded onto an equilibrated mannan-agarose column (Sigma) (1.6 × 5.0 cm) at a flow rate of 0.5 ml/min. The column was washed with the starting Tris buffer containing 2 mm CaCl2 untilA 280 of the eluant was less then 0.01. The bound protein was then eluted with 5 mm EDTA in the starting Tris buffer lacking CaCl2. Protein fractions were analyzed by SDS-PAGE. Purified IML-2 (600 μg) was used as antigen for the production of polyclonal rabbit antiserum (Cocalico Biologicals, Inc.). Purified IML-2 (2.0 μg) was denatured in 20 mm phosphate buffer, pH 7.2, 1% SDS, 2% (v/v) 2-mercaptoethanol by heating at 100 °C for 3 min. The denatured protein was then incubated with 1 unit of N-glycosidase F or 1 milliunits of O-glycosidase (Roche Molecular Biochemicals) in 50 μl of 50 mm phosphate buffer, pH 7.2, 0.1% SDS, 0.5% (v/v) Nonidet P-40, and 0.5% (v/v) 2-mercaptoethanol for 24 h at 37 °C. Treated and untreated protein samples were then analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. The mass of IML-2 purified from plasma was determined by matrix-assisted laser desorption ionization mass spectrometry at Keck facility, Yale University. IML-2 was also subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane (0.2 μm; Bio-Rad). The protein bands were visualized by staining with Amido Black. IML-2 bands were cut out, and amino-terminal sequences were determined by automated Edman degradation using an Applied Biosystems model 473A Protein Sequencing System at the Biotechnology Core Facility of Kansas State University. 25 μg of purified IML-2 was analyzed by gel filtration HPLC (Bio-Sil SEC 250, 300 × 7.8 mm; Bio-Rad). The column was eluted with 50 mm sodium phosphate, pH 6.8, 150 mm NaCl at 1.0 ml/min. Protein peaks detected by A 280 were collected and dried (SpeedVac, Savant). Samples of proteins from the peaks were analyzed by immunoblotting, using rabbit anti-IML-2 antiserum. A molecular mass standard curve was generated by plotting log of the mass of a set of standards (thyroglobulin, 670 kDa; IgG, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1.35 kDa; Bio-Rad) against retention time. An M. sexta larval fat body cDNA library (24 h after injection of E. coli) in λ ZAPII (Stratagene) was screened using antiserum to IML-2 by the method of Ausubel et al. (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Green Publishing Associates and Wiley-Interscience, New York1991Google Scholar). Positive clones were purified to homogeneity and subcloned by in vivo excision of pBluescript phagemids. The nucleotide sequences of the cDNA clones were determined from double-stranded plasmid DNA templates by the dideoxynucleotide method using an automated DNA Sequencer (Iowa State University DNA sequencing facility). The cDNAs were sequenced using subcloned restriction fragments and oligonucleotide primers derived from previously determined sequences. Because the longest cDNA clone (pM13) was incomplete at the 5′ end, rapid amplification of cDNA ends was used to obtain the 5′ end sequence of IML-2. Rapid amplification of cDNA ends reactions were performed as described by Frohman (22Frohman M.A. Innis M.A. Gelfand D.G. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA1990: 28-38Google Scholar). Briefly, 5 μg of total RNA fromE. coli-induced larval fat body was reverse transcribed to cDNAs by Moloney murine leukemia virus reverse transcriptase using oligo(dT) as primer. The cDNAs were then tailed with dCTP using terminal transferase. For nested polymerase chain reactions (PCRs), the reaction was set up as follows: 5 μl of 10× PCR buffer A (Fisher), 3 μl of 10 mm dNTPs, 3 μl of 25 mm MgCl2, 25 pmol of each primer, 1 μl of cDNA pools or first round PCR product, 1 unit of Taq DNA polymerase (Fisher), and water to bring the total volume to 50 μl. For the first round PCR, the tailed cDNA pools were used as template, and a sequence-specific primer PMR5 (5′-GAT GGA TCC CAT TTG TGA GGT-3′) and Anchor primer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3′) (Life Technologies, Inc.) were used. For the second round PCR, the first round PCR product was used as template, and a sequence-specific primer PMR7 (5′-AAC GGA TCC CTC AAG ATG GCA-3′) and universal amplification primer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3′) (Life Technologies, Inc.) were used as primers. PCR reactions were performed as follows: denaturing for 30 s at 94 °C, annealing for 40 s at 50 °C (first round) or 58 °C (second round), and extension for 40 s at 72 °C for a total of 40 cycles. A PCR product of 280 bp was obtained from nested PCR reactions and purified by low melting point agarose gel electrophoresis. The purified PCR product was cloned into plasmid vector pGEMR-T (Promega). Plasmid DNA containing the insert was prepared, and the insert was sequenced as described above. Sequence analysis was performed using the GCG Sequence Analysis Software Package, version 7.3.1 (23Genetics Computer Group Program Manual for the GCG Package , Version 7. Madison, WI1991Google Scholar), and IBI Pustell programs. Plasma samples collected from the fifth instar larvae injected with bacteria, yeast, or saline as described above, were analyzed by SDS-PAGE by the method of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar), and IML-2 was identified by immunoblotting. 4-μl cell-free plasma from each larva at different time intervals of post-injection was separated on 12% SDS-PAGE, and proteins were transferred to nitrocellulose membrane. The membrane was blocked with 5% dry skim milk and then incubated with rabbit antiserum to IML-2 (1:2000 dilution). Antibody binding was visualized by a color reaction catalyzed by alkaline phosphatase conjugated to goat anti-rabbit IgG (Bio-Rad). The IML-2 band intensities were measured using Kodak Digital Science one-dimensional gel analysis software, and the amount of IML-2 in plasma was estimated using known concentrations of purified IML-2 as standards. For each group, plasma from four individual larvae was analyzed. Total RNA from fat body or hemocytes collected 24 h after injection of saline (0.85% NaCl), M. lysodeikticus, S. cerevisiae, or E. coli(strain XL1 blue) was prepared by the method described previously (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar). RNA samples (20 μg) were separated by agarose gel electrophoresis in the presence of formaldehyde (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2 nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), transferred to a positively charged nylon membrane (GeneScreen Plus; DuPont), and probed with IML-2 cDNA or M. sexta ribosomal protein S3 (rpS3) cDNA (26Jiang H. Wang Y. Kanost M.R. Insect Mol. Biol. 1996; 5: 31-38Crossref PubMed Scopus (55) Google Scholar) labeled by random primer extension with [α-32P]dCTP. Trypsinized and glutaraldehyde treated erythrocytes from human bloods group B or O were purchased from Sigma. All other erythrocytes were glutaraldehyde treated and were also from Sigma. These erythrocytes were trypsinized as described by Haqet al. (27Haq S. Kubo T. Kurata S. Kobayashi A. Natori S. J. Biol. Chem. 1996; 271: 20213-20218Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and suspended in Tris-buffered saline (TBS) (25 mm Tris-HCl, 137 mm NaCl and 3 mmKCl, pH 7.0) as a 10% suspension. For hemagglutination assay, erythrocytes were prepared as a 2% suspension in TBS. IML-2 was serially diluted 2-fold with 25 μl of TBS containing 5 mm CaCl2 in wells of a microtiter V-shape plate. Then 25 μl of 2% erythrocytes were added and mixed well. The plate was incubated for 1 h at 37 °C. Agglutinated erythrocytes formed a diffuse mat, whereas unagglutinated erythrocytes formed a clear red dot at the bottom of the well. To test carbohydrate specificity for IML-2, the hemagglutination assay was conducted by mixing IML-2 (1.0 μg/ml in TBS containing 5 mm CaCl2) with serial dilutions of various carbohydrates at room temperature for 30 min. Horse erythrocytes (2%) were then added, and the plate was incubated at 37 °C for 1 h before scoring for agglutination. Fluorescein isothiocyanate-labeled Staphylococcus aureus, E. coli, and S. cerevisiae (Molecular Probes) were suspended in TBS and used for the agglutination assay. IML-2 purified from plasma of E. coli-injected larvae was used in the assay performed as described previously (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar). To test whether the agglutination of E. coli requires calcium, fluorescein isothiocyanate-labeled E. coli was incubated with IML-2 (final concentration, 10 μg/ml) in TBS containing 1 mmEDTA, and the assay was performed as described by Yu et al.(16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar). Wells of a flat bottom 96-well assay plate (Costar, Fisher) were coated with LPS from E. coli 0111:B4 (Sigma) by a method modified from Tobias et al. (28Tobias P.S. Soldau K. Ulevitch R.J. J. Biol. Chem. 1989; 264: 10867-10871Abstract Full Text PDF PubMed Google Scholar) and Koizumi et al. (14Koizumi N. Morozumi A. Imamura M. Tanaka E. Iwahana H. Sato R. Eur. J. Biochem. 1997; 15: 217-224Crossref Scopus (83) Google Scholar). Briefly, LPS was suspended at 40 μg/ml in water and sonicated for 3 × 15 s, and 50 μl (2 μg) of LPS suspension was added to each well. The plate was then incubated at room temperature until the water evaporated completely. The plates were heated at 60 °C for 30 min and then blocked with 200 μl/well of 1 mg/ml BSA in Tris buffer (TB) (50 mm Tris-HCl, 50 mm NaCl, pH 8.0) for 2 h at 37 °C. The plates were then rinsed four times with 200 μl/well of TB. IML-2 diluted with TB containing 5 mmCaCl2 and 0.1 mg/ml BSA was added at 50 μl/well, and binding was allowed to occur for 3 h at room temperature. The plates were then rinsed four times with 200 μl/well of TB, and rabbit anti-IML-2 antiserum (diluted 1000-fold with TB containing 0.1 mg/ml BSA) was added at 100 μl/well. After incubation for 2 h at 37 °C, the wells were rinsed four times with 200 μl/well of TB. Alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 3000-fold with TB containing 0.1 mg/ml BSA was added at 100 μl/well and incubated for 2 h at 37 °C. The wells were washed again as described above, and 50 μl/well of 1 mg/ml p-nitro-phenyl phosphate (prepared in 10 mm diethanolamine, 0.5 mm MgCl2) was added and incubated at room temperature for 20 min. Absorbance at 405 nm of each well was determined using a microtiter plate reader (Bio-Tek Instrument, Inc.). LPS from E. coli 0111:B4 (Sigma), mannan (Sigma), or IML-2 purified from plasma of E. coli-injected larvae, all in TBS, were added separately or in combination to 40 μl of cell free hemolymph collected from naive fifth instar day 2 larvae. Total volume was adjusted to 50 μl with TBS, and the mixture was incubated at room temperature. At various times after mixing, 5-μl aliquots were removed and added to 0.7 ml of 2 mm dopamine in 50 mm sodium phosphate, pH 6.5, for measurement of phenol oxidase activity (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar). Absorbance at 470 nm was measured over 6 min. When plasma collected from M. sexta larvae 24 h after injection ofE. coli was passed through a mannan-agarose column, a protein that bound to mannan could be eluted with EDTA. This protein, designated IML-2, was more than 90% pure after this one-step affinity purification. Approximately 1.5 mg of purified IML-2 was recovered from 100 ml of plasma. IML-2 also bound to immobilized glucose (data not shown). Purified IML-2 appeared as two closely spaced bands at approximately 37 kDa (IML-2a) and 38.5 kDa (IML-2b) in analysis by SDS-PAGE (Fig. 1, lane 2). The masses of IML-2a and IML-2b determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry were 35,381 and 36,240 Da, respectively, indicating that SDS-PAGE analysis slightly overestimates the mass of the IML-2 isoforms. The amino-terminal sequences of the proteins recovered from these two bands were determined by Edman degradation. 24 residues were determined from IML-2a, whereas 10 residues were obtained from IML-2b. Both amino-terminal sequences were identical and perfectly matched the deduced amino acid sequence from an IML-2 cDNA clone described below (Fig. 2).Figure 2Nucleotide and deduced amino acid sequences of IML-2. The deduced amino acid sequence is shownbelow the cDNA sequence. Amino acid residues in the mature protein are assigned positive numbers, and those in the signal peptide are assigned negative numbers. A potential N-linked glycosylation site is marked with ♦. Cys residues that define C-type lectin short-form CRDs are marked with a ▴, whereas two extra cysteine residues in the long-form CRD2 are marked with ●. The amino-terminal sequence of the mature IML-2 was determined by Edman degradation. The sequence obtained from IML-2b (higher mass isoform) isdouble-underlined, and the sequence from IML-2a (lower mass isoform) begins at the same position but extends further (underlined). In the cDNA sequence, the polyadenylation sequence AATAAA is double-underlined.View Large Image Figure ViewerDownload (PPT) When purified IML-2 was analyzed by gel filtration HPLC, a major peak eluted at 10.87 min, which was just after the ovalbumin standard (10.24 min) (Fig. 3). A small peak eluting at 9.78 min and a peak eluting at the void volume of the column (6.50 min) were also observed. Fractions from all three peaks were identified as IML-2 by immunoblotting (data not shown). Using a standard curve generated by plotting log molecular mass of a set of standard proteinsversus retention times, the calculated masses of the three IML-2 peaks are: 39.5 kDa (10.87 min), 90 kDa (9.78 min), and >670 kDa (6.50 min). These results suggest that IML-2 is present mainly in monomeric form in solution, with lower amounts of dimers and oligomers. Fractions in a broad trough between the dimer and oligomer peaks also contained IML-2, indicating that IML-2 oligomers with different numbers of subunits may exist in solution. Purified IML-2 from plasma was used as an antigen for producing a rabbit polyclonal antiserum. We used this antiserum as a probe to screen an E. coli-induced M. sexta larval fat body cDNA library. From 1.2 × 105 λ phage screened, we obtained two positive clones, pM13 and pM18, which encoded IML-2. These two clones were identical in sequence. Clone pM13 had an insert of 1183 bp, which contained the complete 3′ end with a poly(A) tail, but it was not complete at the 5′ end. To obtain the full-length sequence, 5′ rapid amplification of cDNA ends was performed, and a fragment extending 70 bp farther at the 5′ end was cloned. The full-length sequence of IML-2 cDNA was 1253 bp long, with an open reading frame of 981 bp, encoding a 327-residue polypeptide (Fig. 2). The deduced amino acid sequence of IML-2 contains a 19-residue secretion signal peptide, confirmed by Edman degradation of the mature protein (Fig. 2). The calculated mass of the mature protein is 35,203 Da, which is less than the masses of IML-2a and IML-2b determined by mass spectrometry (35,381 and 36,240 Da). A potentialN-linked glycosylation site is present in the IML-2 sequence at Asn-253. Treatment of IML-2 with N-glycosidase F, which cleaves at N-linked glycosylation sites, resulted in two slightly more separated protein bands, with apparent molecular masses of 36 and 34 kDa (Fig. 1, lane 3). This result suggests that both IML-2 isoforms are N-glycosylated. Treatment of IML-2 with O-glycosidase did not change the mobility of the two protein bands (data not shown), indicating that IML-2 has no O-linked glycosylation. Analysis of the amino acid sequence deduced from the cDNA indicated that IML-2 is a member of the C-type lectin superfamily. It contains two C-type CRDs, an amino-terminal domain, CRD1 (residues 1–136), and a carboxyl-terminal domain, CRD2 (residues 137–288). This feature of IML-2 is similar to another M. sexta lectin, immulectin (now designated IML-1) (16Yu X.-Q. Gan H. Kanost M.R. Insect. Biochem. Mol. Biol. 1999; 29: 584-597Crossref Scopus (217) Google Scholar), and to lectins from other two insect species: LPS-binding proteins from the silkworm, B. mori (15Koizumi N. Imamura M. Kadotani T. Yaoi K. Iwahana H. Sato R FEBS Lett. 1999; 443: 139-143Crossref PubMed Scopus (164) Google Scholar), and the fall webworm, H. cunea (18Shin S.W. Park S.-S. Park D.-S. Kim M.G. Kim S.C. Brey P.T. Park H.-Y. Insect Biochem. Mol. Biol. 1998; 28: 827-837Crossref PubMed Scopus (130) Google Scholar). Fig.4 shows an alignment of these four insect C-type lectins with tandem CRD structure. IML-2 shows 55% identity toB. mori LPS-binding protein (BmLBP) and 47% to Hdd15 but only 27% to M. sexta IML-1. In comparison with vertebrate C-type lectins, CRD1 of IML-2 was most similar (26% identity) to rat macrophage asialoglycoprotein-binding protein (GenBankTM accession number P49301), whereas CRD2 was most similar (25% identity) to rat CD23 (GenBankTMaccession number S34198), an IgE receptor. IML-2 was present constitutively at a low level in plasma of naive larvae, with an average of 18.5 ± 8.5 μg/ml (measured from 36 larvae, with a range of 3.8 μg/ml to 36.5 μg/ml). After injection of E. coli, the concentration of both IML-2a and IML-2b in plasma consistently dropped within 2 h post-injection but then increased to the original level at 6 h and continued to increase up to 48 h post-injection (Fig.5). The level of IML-2a in hemolymph of naive larvae was significantly lower than that of IML-2b. The concentration of both IML-2a and IML-2b increased after injection ofE. coli, but the ratio of their concentrations changed, with a greater relative increase in IML-2a (Fig. 5 B). Northern analysis also showed that IML-2 mRNA was present at a low level in fat body of control larvae (injected with saline) and was induced to much higher level after injection of E. coli (Fig.6). IML-2 mRNA level was also increased after injection of Gram-positive bacteria (M. lysodeikticus) or yeast (S. cerevisiae). IML-2 mRNA was not detected in hemocytes of either control larvae or larvae injected with yeast or bacteria (Fig. 6). Injection of saline, M. lysodeikticus (Gram-positive), or S. cerevisiae (yeast) did not significantly change the concentration of IML-2 in plasma 24 h post-injection (Fig. 7). However, after injection of three different Gram-negative bacteria (E. coli, P. aeruginosa, and S. marcescens) or LPS from E. coli, IML-2 concentration increased 3–4-fold in plasma 24 h post-injection.Figure 6Northern hybridization of IML-2 mRNA. Samples of total RNA (20 μg) from hemocytes or fat body of larvae injected with saline (C), yeast (S. cerevisiae) (Y), M. lysodeikticus(M), or E. coli (E) were subjected to 1% agarose gel electrophoresis in the presence of 2.2 mformaldehyde. The RNA was transferred to a positively charged nylon membrane and probed with 32P-labeled IML-2 cDNA (A) or ribosomal protein S3 (rpS3) cDNA (B). The arrow points to the 1.3-kilobase IML-2 mRNA present in fat body of control larvae and induced in fat body after injection of microorganisms.View Large Image Figure ViewerDownload (PPT)Figure 7IML-2 concentration in plasma increased only after injection of gram-negative bacteria. Fifth instar day 2 larvae were injected with saline, Gram-positive bacteria (M. lysodeikticus), yeast (S. cerevisiae), LPS fromE. coli, or Gram-negative bacteria (E. coli,P. aeruginosa, and S. marcescens). Hemolymph was then collected at different times post-injection. Plasma (4 μl) was separated by SDS-PAGE (12%), and IML-
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