A Lipopolysaccharide- and β-1,3-Glucan-binding Protein from Hemocytes of the Freshwater Crayfish Pacifastacus leniusculus
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.1337
ISSN1083-351X
AutoresSo Young Lee, Ruigong Wang, Kenneth Söderhäll,
Tópico(s)Aquaculture disease management and microbiota
ResumoA lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to β-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and β-1,3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the β-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish. A lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to β-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and β-1,3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the β-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish. lipopolysaccharide phenoloxidase prophenoloxidase activating system β-1,3-glucan-binding protein lipopolysaccharide- and β-1,3-glucan-binding protein 3,4-dihydroxy phenylalanine polyacrylamide gel electrophoresis bovine serum albumin hemocyte lysate supernatant high performance liquid chromatography Vertebrates and invertebrates are capable of initiating several kinds of defense mechanisms after recognition of bacterial and fungal cell wall molecules, such as lipopolysaccharides (LPS),1peptidoglycans, and β-1,3-glucans (1.Rietschel E. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 3.Yang R.-B. Mark M.R. Gray A. Huang A. Xia M.Z. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-289Crossref PubMed Scopus (1100) Google Scholar). In the case of humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock (4.Parillo J.E. N. Engl. J. Med. 1993; 328: 1471-1477Crossref PubMed Scopus (1503) Google Scholar). Invertebrate animals lack antibodies and hence an adaptive immune response, and instead they have efficient innate immune systems to defend themselves against invading foreign materials (5.Hoffmann J.A. Kafatos F.C. Janeway Jr., C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2153) Google Scholar). The defense system of invertebrates is based on both cellular and humoral immune responses (6.Smith V.J. Söderhäll K. Lackie A.M. Immune Mechanisms in Invertebrate Vectors. Oxford University Press, Oxford, U. K.1986: 59-79Google Scholar). The former includes encapsulation (7.Kobayashi M. Johansson M.W. Söderhäll K. Cell Tissue Res. 1990; 260: 13-18Crossref Scopus (111) Google Scholar, 8.Cho M.Y. Lee H.S. Lee K.M. Homma K.-i. Natori S. Lee B.K. Eur. J. Biochem. 1999; 262: 737-744Crossref PubMed Scopus (39) Google Scholar, 9.Asgari S. Theopold U. Wellby C. Schmidt O. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3690-3695Crossref PubMed Scopus (94) Google Scholar), phagocytosis (10.Foukas L.C. Katsoulas H.L. Paraskevopoulou N. Metheniti A. Lambropoulou M. Marmaras V.J. J. Biol. Chem. 1998; 273: 14813-14818Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), and nodule formation (11.Koizumi N. Imamura M. Kadotani T. Yaoi K. Iwashana H. Sato R. FEBS Lett. 1999; 443: 139-143Crossref PubMed Scopus (165) Google Scholar). The clotting system of arthropods (12.Iwanaga S. Kawabata S.-i. Muta T. J. Biochem. (Tokyo). 1998; 123: 1-15Crossref PubMed Scopus (255) Google Scholar,13.Hall M. Wang R. Antwerpen R.V. Sottrup-Jensen L. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1965-1970Crossref PubMed Scopus (189) Google Scholar), the synthesis of a broad spectrum of potent antimicrobial proteins in many insects (14.Hoffmann J.A. Reichhart J.-M. Hetru C. Curr. Opin. Immunol. 1996; 8: 8-13Crossref PubMed Scopus (292) Google Scholar, 15.Bulet P. Hetru C. Dimarcq J.-L. Hoffmann D. Dev. Comp. Immunol. 1999; 23: 329-344Crossref PubMed Scopus (825) Google Scholar) and crustaceans (16.Destoumieux D. Bulet P. Loew D. Van Dorsselaer A. Rodriguez J. Bachère E. J. Biol. Chem. 1997; 272: 28398-28406Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 17.Schnapp D. Kemp G.D. Smith V.J. Eur. J. Biochem. 1996; 240: 532-539Crossref PubMed Scopus (173) Google Scholar, 18.Kawano K. Yoneya T. Miyata T. Yoshikawa K. Tokunaga F. Terada Y. Iwanaga S. J. Biol. Chem. 1990; 265: 15365-15367Abstract Full Text PDF PubMed Google Scholar), and the prophenoloxidase activating system (proPO system) (2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar) belong to the last immune response. Moreover, the humoral immune response is also triggered by LPS or β-1,3-glucans (2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar, 19.Fearon D.T. Locksley R.M. Science. 1996; 272: 50-53Crossref PubMed Scopus (1434) Google Scholar) as in vertebrates. Therefore, the proteins involved in the recognition of LPS, peptidoglycans, and β-1,3-glucans have been named pattern recognition proteins (20.Medzhitov R. Janeway Jr., C.A. Cell. 1997; 91: 295-298Abstract Full Text Full Text PDF PubMed Scopus (1973) Google Scholar), and they are involved in various ways in the biological defense mechanisms in both invertebrates and vertebrates. Recently, LPS- (21.Jomori T. Natori S. J. Biol. Chem. 1991; 266: 13318-13323Abstract Full Text PDF PubMed Google Scholar, 22.Lee W.-J. Lee J.-D. Kravchenko V.V. Ulevitch R.J. Brey P.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7888-7893Crossref PubMed Scopus (191) Google Scholar, 23.Koizumi N. Morozumi A. Imamura M. Tanaka E. Iwahana H. Sato R. Eur. J. Biochem. 1997; 248: 217-224Crossref PubMed Scopus (85) Google Scholar, 24.Muta T. Miyata T. Misumi Y. Tokunaga F. Nakamura T. Toh Y. Ikehara Y. Iwanaga S. J. Biol. Chem. 1991; 266: 6554-6561Abstract Full Text PDF PubMed Google Scholar) and/or β-1,3-glucan-binding proteins (25.Duvic B. Söderhäll K. J. Biol. Chem. 1990; 265: 9327-9332Abstract Full Text PDF PubMed Google Scholar, 26.Cerenius L. Liang Z. Duvic B. Keyser P. Hellman U. Palva E.T. Iwanaga S. Söderhäll K. J. Biol. Chem. 1994; 269: 29462-29467Abstract Full Text PDF PubMed Google Scholar, 27.Beschin A. Bilej M. Hanssens F. Raymarkers J. Van Dyck E. Revets H. Brys L. Gomez J. De Baetselier P. Timmermans M. J. Biol. Chem. 1998; 273: 24948-24954Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 28.Seki N. Muta T. Oda T. Iwaki D. Kuma K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; 269: 1370-1374Abstract Full Text PDF PubMed Google Scholar), peptidoglycan recognition proteins (29.Kang D. Lui G. Lundström A. Gelius E. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10078-10082Crossref PubMed Scopus (407) Google Scholar, 30.Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 31.Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 11854-11858Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), lectins (32.Inamori K. Saito T. Iwaki D. Nagira T. Iwanaga S. Arisaka F. Kawabata S. J. Biol. Chem. 1999; 274: 3272-3278Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33.Kawabata S. Iwanaga S. Dev. Comp. Immunol. 1999; 23: 391-400Crossref PubMed Scopus (87) Google Scholar, 34.Vasta G.R. Quesenberry M. Ahmed H. O'Leary N. Dev. Comp. Immunol. 1999; 23: 401-420Crossref PubMed Scopus (205) Google Scholar), and hemolin (35.Sun S.C. Lindström I. Boman H.G. Faye I. Schmidt O. Science. 1990; 250: 1729-1732Crossref PubMed Scopus (215) Google Scholar, 36.Su X.-D. Gastinel L.N. Vaughn D.E. Faye I. Poon P. Bjorkman P.J. Science. 1998; 281: 991-995Crossref PubMed Scopus (150) Google Scholar, 37.Mendoza H.L. Faye I. Dev. Comp. Immunol. 1999; 23: 359-374Crossref PubMed Scopus (44) Google Scholar) have been found in several different species of invertebrates, and their function in the immune response has been studied. For instance, in the horseshoe crab Tachypleus tridentatus, LPS or β-1,3-glucans both bind specifically to pattern recognition proteins, and as a result the coagulation cascade is activated (24.Muta T. Miyata T. Misumi Y. Tokunaga F. Nakamura T. Toh Y. Ikehara Y. Iwanaga S. J. Biol. Chem. 1991; 266: 6554-6561Abstract Full Text PDF PubMed Google Scholar, 28.Seki N. Muta T. Oda T. Iwaki D. Kuma K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; 269: 1370-1374Abstract Full Text PDF PubMed Google Scholar). In addition, the opsonic effect (26.Cerenius L. Liang Z. Duvic B. Keyser P. Hellman U. Palva E.T. Iwanaga S. Söderhäll K. J. Biol. Chem. 1994; 269: 29462-29467Abstract Full Text PDF PubMed Google Scholar) and degranulation of blood cells (38.Barracco M.A. Duvic B. Söderhäll K. Cell Tiss. Res. 1991; 266: 491-497Crossref Scopus (61) Google Scholar) by the β-1,3-glucan-binding protein (β-GBP) in the crayfish Pacifastacus leniusculus, the opsonic effect of the LPS-binding protein in the cockroachPeriplaneta americana (39.Jomori T. Natori S. FEBS Lett. 1992; 296: 283-286Crossref PubMed Scopus (97) Google Scholar), and the hemocyte nodule formation by the LPS-binding protein in the silkworm Bombyx mori (11.Koizumi N. Imamura M. Kadotani T. Yaoi K. Iwashana H. Sato R. FEBS Lett. 1999; 443: 139-143Crossref PubMed Scopus (165) Google Scholar) have already been reported as special biological properties of pattern recognition proteins. In particular, the proPO system is an important non-self-recognition system in invertebrates which can be activated by LPS or peptidoglycan from bacteria and β-1,3-glucans from fungi (2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). Non-self-molecules are recognized by endogenous pattern recognition proteins and their receptors, and then they cause activation of the proPO system (2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar). The active form of proPO, phenoloxidase (PO), is produced by a serine proteinase known as the proPO-activating enzyme. Subsequently, PO oxidizes DOPA to dopaquinone, which is converted to melanin through several non-enzymatic steps. The generated PO plays an important role as it can melanize pathogens (2.Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1102) Google Scholar), sclerotize the cuticle (40.Sugumaran M. FEBS Lett. 1991; 295: 233-239Crossref PubMed Scopus (35) Google Scholar), and heal wounds (41.Lai-Fook J. J. Insect Physiol. 1996; 12: 195-226Crossref Scopus (94) Google Scholar) in invertebrates. Since proPO was first cloned from the crayfish P. leniusculus (42.Aspán A. Huang T.-s. Cerenius L. Söderhäll K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (229) Google Scholar), a large number of invertebrate proPOs have been structurally determined, and recently, the primary structure of proPO-activating enzyme has been reported from three different insects (43.Lee S.Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S.I. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (109) Google Scholar, 44.Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (238) Google Scholar, 45.Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). However, so far only two groups have reported that a LPS- and β-GBP, the coelomic cytolytic factor-1, from the earthworm Eisenia foetida (27.Beschin A. Bilej M. Hanssens F. Raymarkers J. Van Dyck E. Revets H. Brys L. Gomez J. De Baetselier P. Timmermans M. J. Biol. Chem. 1998; 273: 24948-24954Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and the peptidoglycan recognition protein from B. mori (30.Yoshida H. Kinoshita K. Ashida M. J. Biol. Chem. 1996; 271: 13854-13860Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) appear to be involved in the activation of the proPO system. In this paper, we describe the purification of a LPS- and β-1,3-glucan-binding protein (LGBP) from crayfish blood cells, its cDNA cloning, and its role in the proPO system of the crayfishP. leniusculus. Freshwater crayfish, P. leniusculus, were purchased from Berga Kräftodling, Södermanland, Sweden, and kept in an aquarium with tap water at 10 °C. Only intermoult male crayfish were used in these experiments. Hemocyte lysate supernatant (HLS) was prepared by collecting hemolymph from 200 crayfish in anticoagulant buffer (0.14 m NaCl, 0.1 m glucose, 30 mm trisodium citrate, 26 mm citric acid, 10 mm EDTA, pH 4.6) (46.Leonard C. Söderhäll K. Ratcliffe N.A. Insect Biochem. 1985; 15: 803-810Crossref Scopus (173) Google Scholar). The hemocytes were spun down at 4 °C and 800 × g for 10 min, and then the hemocytes were homogenized with CAC buffer (10 mm sodium cacodylate, 0.1 m CaCl2, pH 6.5). After centrifugation at 4 °C and 25,000 × g for 30 min, the supernatant was applied to a Blue-Sepharose CL-6B column (1 × 12 cm), previously equilibrated with CAC buffer. The flow-through containing LGBP was collected, and it was loaded to a phenyl-Sepharose CL-4B (1 × 4 cm) equilibrated with CAC buffer and washed with the same buffer. Bound proteins were eluted with 65% ethylene glycol in CAC buffer. The eluted proteins were concentrated on a Centricon concentration filter (Amicon, Inc.) to a final volume of 0.5 ml. As a final purification step, Sephacryl S-200 gel filtration column (0.8 × 100 cm) equilibrated with CAC buffer was used. 10% SDS-PAGE was carried out by the method of Laemmli (47.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar). Samples were denatured by heating them for 4 min at 95 °C in 2% (w/v) SDS and 0.1% dithiothreitol, and then the gels were stained according to the method of Fairbank et al.(48.Fairbank G. Steck T.L. Wallach D.L.H. Biochemistry. 1971; 10: 2606-2617Crossref PubMed Scopus (6175) Google Scholar). A low molecular mass calibration kit for electrophoresis (Amersham Pharmacia Biotech) was used for size markers: rabbit muscle phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and bovine milk α-lactalbumin (14.4 kDa). The purified LGBP was separated by 10% SDS-PAGE under reducing conditions. After Coomassie Blue staining, a band corresponding to LGBP was excised and homogenized in phosphate-buffered saline. Antibody against LGBP was raised by three injections of 20 μg of purified LGBP each time with Freund's adjuvant (complete for the first injection, incomplete for the other two injections). To purify the antibody to be used for immunoblotting experiment, the purified LGBP was electrophoresed and transferred onto a nitrocellulose membrane. The region of LGBP on the filter was excised and treated with 5% skim milk in 20 mm Tris/HCl, pH 7.9, at 4 °C for 1 h and then incubated in 2-fold dilution anti-LGBP antiserum with rinse solution containing 10 mm Tris/HCl, pH 7.9, 150 mm NaCl, 1 mm EDTA, 0.1% Triton X-200, 1% NaN3, and 0.5% skim milk at 4 °C for 12 h with gentle shaking. The strip was washed carefully with rinse solution and subsequently cut into pieces. The antibody bound to LGBP was eluted with 0.2 m glycine HCl, pH 2.8, and then the eluted antibody solution was neutralized immediately with 1m KOH, and bovine serum albumin (BSA) was added to a final concentration of 0.1%. To purify the antibody for PO activity test, the anti-LGBP antiserum was loaded to protein A-Sepharose CL-4B equilibrated with 50 mm Tris/HCL, pH 7.0, and washed with the same buffer. Bound anti-LGBP antibody was eluted with 0.1m glycine HCl, pH 2.8. The eluted anti-LGBP antibody was concentrated on a Centricon concentration filter. For immunoblotting, the proteins were subjected to 10% SDS-PAGE under reducing conditions and electrotransferred to nitrocellulose membranes in transfer buffer (25 mm Tris/HCl, 190 mmglycine, 20% MeOH) for 2 h at 280 mA on ice. All of the following steps were performed at room temperature. The membrane was subsequently blocked in TTBS (0.1% Tween 20 in 20 mm Tris/HCl, 150 mm NaCl, pH 7.4) containing 3% BSA for 1 h and incubated with antibody in TTBS containing 0.1% BSA for 1 h 30 min. A 2,000-fold dilution of affinity-purified antibody was used in immunoblotting. Then the membrane was washed with TTBS once for 15 min and three times for 5 min. The anti-rabbit IgG peroxidase-conjugated IgG diluted 1:10,000 with TTBS containing 0.1% BSA was incubated for 1 h, washed with TTBS once for 15 min and four times for 5 min. For detection, the enhanced chemiluminescence (ECL) Western blotting reagent kit (Amersham Pharmacia Biotech) was used. To determine the internal amino acid sequence of peptide fragments of LGBP, the protein was subjected to 10% SDS-PAGE under reducing conditions, stained with 0.2% Coomassie Blue in 50% methanol, and destained with 30% methanol. The band corresponding to LGBP was excised and treated with lysyl endopeptidase according to Wilm et al. (49.Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Nature. 1996; 379: 466-469Crossref PubMed Scopus (1507) Google Scholar). The resulting digest was subjected to reverse phase high performance liquid chromatography (HPLC, Pharmacia Smart chromatography system) using a phase μRPC C2/C18 column (MIC-15-03-MRP, Amersham Pharmacia Biotech). The HPLC was performed with a linear gradient of 0–50% acetonitrile in 0.1% trifluoroacetic acid for 75 min at a flow rate of 30 μl/min, and the most prominent peaks were sequenced using an Applied Biosystem 476A sequencer. For determination of the NH2-terminal amino acid sequence, the protein was electrotransferred onto a polyvinylidene difluoride membrane. The membrane was stained with Coomassie Blue, destained, washed with distilled water, and dried. The LGBP band was cut into small pieces and subjected to an Applied Biosystem 476A automated protein sequencer for amino acid sequencing. Fluorescein isothiocyanate-labeled LPS (smooth types) from Salmonella abortus (Sigma) and smooth types of LPS fromEscherichia coli serotype 055:B5 (Sigma) were used for LPS binding activity of LGBP. Microtiter 96-well plates were coated with purified LGBP (200 μl/well of 10 μg/ml in CAC buffer, pH 6.5) overnight at 4 °C. Excess binding sites were blocked with 1% BSA in CAC buffer at 37 °C for 2 h. The same concentration (10 μg) of BSA was used as control. After washing three times with CAC buffer, different doses of fluorescein isothiocyanate-labeled LPS were added in 100 μl of CAC buffer containing 0.1% BSA, incubated for 3 h at 37 °C, and then the plates were washed three times with CAC buffer. 100 μl/well 50 mm Tris/HCl containing 50 mmNaCl, pH 8.5, was added for measuring the bound fluorescence using a fluorescence multiwell plate reader (Wallac 1420 multilabel counter) at emission/excitation 485 nm/530 nm. This experiment was repeated twice with similar results. In another method, curdlan, a linear polymer of glucose units linked with β-1,3-linkages (Wako), laminarin, which consists of β-1,3-glucan chain with occasional β-1,6-linked glucose units (Calbiochem), peptidoglycan of Staphylococcus aureus(Fluka), or LPS of E. coli serotype 055:B5 (Sigma) was used for testing binding activity to LGBP. 4 μg of purified LGBP was incubated with 100 μg of curdlan, laminarin, LPS, or peptidoglycan in CAC buffer for 1 h at 4 °C. The supernatant was taken, and the pellets of curdlan, laminarin, LPS, or peptidoglycan were washed three times with CAC buffer. The bound protein was eluted with 30 μl of SDS-PAGE sample loading buffer (60 mm Tris/HCl, pH 6.8, containing 2% SDS, 1% glycerol, 0.01% bromphenol blue, and 0.1% dithiothreitol) and treated by heating at 95 °C for 4 min. The supernatant was treated by trichloroacetic acid for protein precipitation. The precipitated proteins were dissolved with 30 μl of SDS-PAGE sample loading buffer. To investigate LGBP binding activity to LPS, β-1,3-glucans, or peptidoglycan, the eluted proteins and the supernatants were applied to 10% SDS-PAGE and subsequently developed for immunoblotting using anti-LGBP antibody as a probe. Six pairs of nested degenerate primers were synthesized according to amino acid sequences of four lysyl endopeptidase-derived peptide fragments of LGBP. Two cDNA fragments, both coding for LGBP, were amplified by polymerase chain reaction from the crayfish hemocyte cDNA library using each combination of 12 degenerate primers, one of which was labeled with [α-32P]dCTP by random priming using the Megaprime labeling kit (Amersham Pharmacia Biotech) and was used as a probe to screen more LGBP-specific clones. From an initial screening of approximately 120,000 recombinants of the crayfish hemocyte cDNA library λ-phage resulted in more than 300 positive clones. The largest one that was identified by restriction enzyme digestion was cultured and amplified, and the recombinant DNA was purified by using Wizard λ preparation DNA purification system (Promega). The insert was digested out by the restriction enzyme EcoRI (Amersham Pharmacia Biotech) and subcloned into EcoRI-digested pBluescript II (SK+) plasmid (Stratagene). It was subsequently sequenced in double strands by an Applied Biosystems PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer). The cDNA sequence was analyzed with the MacVector 4.1.4 software (Kodak). The nucleotide sequence, and the deduced amino acid sequence was compared with the BLAST program (National Center Biotechnology International, Bethesda, MD). Total RNA was extracted from crayfish hemocytes by using Trizol LS reagent (Life Technologies) according to the manufacturer's instructions. Approximately 20 μg of total RNA was fractionated on a 1% agarose gel in the presence of formaldehyde and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) by capillary blotting following standard procedures. The 0.24–9.5-kilobase RNA ladder (Life Technologies) was electrophoresed simultaneously and stained with ethidium bromide. The cDNA probes, spanning the coding region and 3′-untranslated region, respectively, were labeled with [α-32P]dCTP using the Megaprime labeling kit (Amersham Pharmacia Biotech). Northern blot hybridization was performed overnight at 65 °C in a solution composed of 5 × SSC, 5 × Denhardt's solution, 0.5% (w/v) SDS, and 100 μg/ml denatured salmon sperm DNA. The membrane was subsequently washed twice with 2 × SSC and 0.1% SDS at room temperature for 10 min, once with 1 × SSPE and 0.1% SDS at 65 °C for 15 min, and finally twice with 0.5 × SSC and 0.1% SDS at 65 °C for 10 min. The washed membrane was used for autoradiography. To confirm the involvement of LGBP in the crayfish proPO system, a polyclonal antibody against LGBP was used, and PO activity was assayed according to Aspán and Söderhäll. (50.Aspán A. Söderhäll K. Insect Biochem. 1991; 21: 363-373Crossref Scopus (134) Google Scholar). Briefly, 30 μg of HLS was incubated for 1 h at 4 °C with different concentrations of anti-LGBP antibody purified by protein A column chromatography and 10 μg of l-DOPA (3-(3,4-dihydroxylphenyl)-l-alanine). These mixtures were incubated at room temperature for 5 h in a final volume of 30 μl of 30 mm sodium cacodylate, pH 6.5, with or without 10 μg of laminarin, curdlan, LPS, or peptidoglycan. The oxidation ofl-DOPA was measured at 490 nm. For reconstitution of PO activity, 3 μg or 15 μg of purified LGBP, previously incubated with laminarin for 20 min at room temperature, was incubated with HLS, in which PO activity had been previously inhibited by addition of anti-LGBP antibody. The recovered PO activity was measured after addition of l-DOPA by spectrophotometry at 490 nm. The concentration of protein was measured with the Bradford method (51.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), using BSA as a standard protein. LGBP was purified from crayfish hemocytes. The hemocyte lysate of crayfish was first subjected to a Blue-Sepharose column chromatography, and the flow-through fraction, containing LGBP, was then purified by hydrophobic chromatography on phenyl-Sepharose followed by gel filtration on Sephacryl S-200 (Fig.1 A). The purity of LGBP in the different fractions was ascertained by SDS-PAGE (Fig. 1 B). Hemocyte lysate supernatants from 200 crayfish with a protein content of about 35 mg of protein gave 210 μg of purified LGBP. The purified LGBP ran as a single band of approximately 36 kDa and 40 kDa in 10% SDS-PAGE under reducing and nonreducing conditions, respectively (Fig.2, A and B).Figure 2SDS-PAGE of purified LGBP and immunoblotting analysis. 2 μg of LGBP was precipitated with trichloroacetic acid and then subjected to SDS-PAGE. Panel A, 10% SDS-PAGE of purified LGBP under reducing conditions; panel B, 10% SDS-PAGE of purified LGBP under nonreducing conditions; panel C, immunoblotting used for localization of LGBP. The samples were prepared from crayfish hemocyte lysate supernatant, and plasma and was then analyzed by immunoblotting using an affinity-purified antibody against LGBP. Lane 1, 20 μg of hemocyte lysate supernatant; lane 2, 30 μg of plasma; lane 3, 1 μg of purified LGBP. The arrows indicate the position of the size marker proteins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Anti-LGBP antibody was used to confirm the localization of LGBP in crayfish hemolymph using immunoblotting. Fig. 2 C shows that the anti-LGBP antibody could recognize LGBP in hemocytes but not in plasma. This result suggests that LGBP exists only in the hemocytes. Two different methods were used for testing the binding activity of purified LGBP to LPS, β-1,3-glucans, or peptidoglycan. First, to examine the LPS binding activity fluorescein isothiocyanate-labeled LPS was used, and 10 μg of purified LGBP was immobilized to microtiter plates. When the fluorescence intensity of LGBP was compared with that of control BSA fluorescence intensity, the binding activity of LGBP to LPS was gradually increased in a dose-dependent manner (Fig. 3). This result shows that LGBP exhibits LPS binding activity. In a second approach, the purified LGBP was incubated with curdlan, laminarin, LPS, or peptidoglycan to demonstrate the binding activity of LGBP. Fig.4 shows the result of an immunoblot of the corresponding LGBP binding activity to curdlan, laminarin, LPS, or peptidoglycan. In the pellets, LGBP is shown to bind strongly to LPS and curdlan, less to laminarin (lanes 2, 3, and4 in Fig. 4), and not at all to peptidoglycan (lane 5). LGBP could not be found in the supernatants that were incubated with LPS, laminarin, or curdlan, (lanes 6,7, and 8 in Fig. 4), whereas the supernatant solution of peptidoglycan showed a 36-kDa LGBP band (lane 9). These results clearly show that LGBP has β-1,3-glucan binding activity
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