Affinity Proteomic Approach for Identification of an IgA-like Protein in

Shantou University, Shantou 515063, People's Republic of China. Received November 13, 2005. An unknown protein reacted with anti-human IgA, namely, ...
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Affinity Proteomic Approach for Identification of an IgA-like Protein in Litopenaeus vannamei and Study on Its Agglutination Characterization Yueling Zhang,†,§ Sanying Wang,† Anlong Xu,‡ Jun Chen,§ Bokun Lin,§ and Xuanxian Peng*,†,‡ Center for Proteomics and Department of Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China, State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshan University, Guangzhou 510275, People’s Republic of China, and Department of Biology, School of Sciences, Shantou University, Shantou 515063, People’s Republic of China Received November 13, 2005

An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat antihuman IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens. Keywords: IgA-like protein • affinity proteomics • hemocyanin • agglutinative activity • bacteria • erythrocyte

Introduction Shrimp farming is a significant source of world income.1 However, great loss of this farming has resulted from microbial infections under intensive aquaculture in recent years.2 Reports indicated that the understanding of shrimp immunity was very helpful for the control of microbial diseases and further development of sustainable shrimps.3-5 Recently, a large variety of circulating molecules related to immunological defense have been characterized in invertebrates,6-8 many of which belong to IgSF molecules that may be involved in adaptive immune response in mammals. These IgSF molecules are active against microbial infections in invertebrates, such as being implicated in cell-cell interactions, stimulating phagocytosis and encapsulation, serving as a viral receptor or as an opsonin, and even participating in primary adaptive immunity.9-14 IgSF molecules are of significance as the components of host defense system because invertebrates cannot produce specific antibodies and may rely mainly on innate defense against microbial infec* Corresponding author: Dr. Xuanxian Peng, School of Life Sciences, Xiamen University, Xiamen 361005, People’s Republic of China. Tel: +86592-218-7987. Fax: +86-592-2181015. E-mail: [email protected]. † Xiamen University. ‡ Zhongshan University. § Shantou University. 10.1021/pr0503984 CCC: $33.50

 2006 American Chemical Society

tions.15 Therefore, it is significant to determine an IgSF molecule and characterize its immune function in invertebrates. In agreement with other reports, our previous report indicated the existence of a protein which reacted with anti-human IgA, namely, IgA-like protein, in shrimp by using the methods of single radial immunodiffusion and Dot-ELISA,16 suggesting that the protein must share homologous sequences with some epitopes of human IgA, an IgSF molecule in human. However, information regarding its identification is not available. This is in part because the identification of an unknown protein is difficult and rather time-consuming based on traditional approaches. Recently, proteomic methodologies have become a powerful approach to characterize an unknown protein.17-19 The results we report here were obtained with the use of affinity proteomic methodologies for the identification of the IgA-like protein from Litopenaeus vannamei haemolymph. Our results indicated that the IgA-like protein was hemocyanin and its Iglike conserved domain could be bound to anti-human IgA. Furthermore, we interestingly found that L. vannamei hemocyanin could aggregate with eight species of pathogenic bacteria and four types of animal erythrocytes, suggesting its immune feature functioning as a defense molecule against pathogens. Journal of Proteome Research 2006, 5, 815-821

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research articles Materials and Methods Animal and Preparation of Shrimp Sera. Penaeid shrimps (L. vannamei), weighing 15∼20 g, irrespective of sex, were purchased from a local market and kept in aerated seawater. Haemolymph was drawn directly from the pericardial sinus using a sterile tube, and then allowed to clot overnight at 4 °C. The sera were collected after centrifuging at 3000g for 20 min and stored at -20 °C until analysis. Protein Purification by Affinity Chromatography. Protein purification by affinity chromatography was performed as previously described.18 Goat anti-human IgA (5 mg), which was purified by the methods of ammonium sulfate precipitation and ion-exchange chromatography, was covalently coupled to CNBr-activated sepharose 4B (0.5 mL) using carbonate buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 8.3), and the matrix was packed into a 1.0 mL syringe. L. vannamei sera (200 µL) were loaded onto the affinity column. Nonabsorbed proteins were washed with PBS (0.05 M, pH 7.0) until absorbance at 280 nm reached baseline. Bound proteins were eluted with 3 mL of glycine-HCl buffer (0.1 M, pH 2.2), and the eluates were neutralized with 300 µL of Tris-HCl buffer (1 M, pH 8.0). Then the eluted proteins were concentrated by PEG 20 000, separated by 1-D and 2-D electrophoresis (1-DE and 2-DE), and transferred to a poly(vinylidene difluoride) (PVDF) membrane for 1-D and 2-D immunoblotting. 1-DE and 2-DE and Their Immunoblottings. 1-DE of the bound fraction from affinity chromatography was carried out using a 3% stacking gel (pH 6.8) and a 10% separating gel (pH 8.9) in Tris-glycine buffer (pH 8.3). 2-DE of the bound fraction from affinity chromatography was performed as previously described .18 Briefly, proteins were homogenized in lysis solution (9.8 M urea (PA), 2% (w/v) NP-40, 2% carrier ampholyte, pH 3-9.5, 100 mM DTT) at room temperature for at least 30 min, and then separated by nonequilibrium pH gradient electrophoresis in gels containing 4% acrylamide using pH 3-9.5 ampholyte at 400 V for 18 h. After equilibration for 15 min in equilibration buffer (2% SDS, 100 mM DTT, 10% glycerol, 0.06 M Tris-HCl, pH 6.8), the tube gel was sealed to the top of the stacking gel (0.75 mm thick), which was placed on top of a 10% acrylamide separating gel. The gels were run at 4 °C and 60 V for 45 min then 120 V for 2∼3 h per gel until the bromophenol blue (BPB) dye front had migrated to within 1 cm from the bottom of the gel. Then Coomassie brilliant blue R-250 staining was performed. Following 1-DE and 2-DE, the gels were separately transferred to a PVDF membrane for 6 h at 200 mA in transfer buffer (25 mM Tris, 0.1 M glycine, and 20% methanol), and the membranes were blocked for 60 min with 5% skim milk in TBS (20 mM Tris, 150 mM NaCl, pH 7.4) at 37 °C. After rinsing three times with TBS for 5 min each, the PVDF membrane was incubated with goat anti-human IgA (Institute of Shanghai for Bio-products, Shanghai, China), at a dilution of 1:200 in TTBS (0.05% Tween-20, 20 mM Tris, 150 mM NaCl, pH 7.4) containing 5% skim milk for 2.5 h at 28 °C on a gently rocking shaker. The membrane was rinsed three times with TTBS for 10 min and incubated with rabbit antigoat IgG-HRP antibody (Sino-American Bio. Corp., Luoyang, China) at a dilution of 1:1000 in TTBS containing 5% skim milk for 1 h at 37 °C. Then the membrane was washed as above and developed with substrate (3′3-diminobenzidine, DAB) until optimum color developed. In-Gel Protein Digestion. The protein that reacted with goat anti-human IgA was excised from the 2-D gel and digested with trypsin as previously described.20 Briefly, the spot was washed 816

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several times with 50% acetonitrile. Gel pieces were dried in a vacuum centrifuge. The cysteine reduction and alkylation steps were done in 10 mM DTT for 30 min and 55 mM iodoacetamide for 45 min in the dark at room temperature. The gel pieces were then dried again and rehydrated in a minimal volume of 50 mM NH4HCO3, pH 8.0, containing 5 mM CaCl2 and 12.5 ng/µL sequencing grade modified trypsin (Promega) for 45 min in ice. The excess liquid was removed, and the gel pieces were immersed overnight in the same buffer (without enzyme) at 37 °C. Then the gel pieces were extracted in 20 µL of 20 mM NH4HCO3, pH 8.0, followed by three extractions in 20 µL of 5% formic acid in 50% acetonitrile. The resulting pooled eluates were dried prior to analysis by mass spectrometry. MALDI-TOF Mass Spectrometry. Following in-gel protein digestion, 0.5 µL of the peptide mixture was mixed with the matrix R-cyano-4-hydroxycinnamic acid (1:1) and spotted onto a stainless steel MALDI plate. An Applied Biosystems Voyager DE-STR mass spectrometer running in delayed-extraction reflectron mode was used to acquire MALDI-TOF data, in which the following parameters were used: accelerating voltage, 20 000; mass range, 800-3500; laser power, 2100. External calibration was done by using a mixture of angiotensin I (1296.19) and ACTH (fragment 18-39) (2465.68) under the same instrument setting. Selected peptide masses were submitted to Mascot (http://www. matrixscience.com/) for NCBInr and MSDB databases search. Search parameters used were the following: taxonomy, metazoa (animals); allowable missed cleavages, 2; variable modification, carbamidomethyl (C); Peptide tol.(, 150 ppm; monoisotopic, being chosen; Mass values, MH+. ESI-MS/MS. Partial sequence of peptides was determined by nanoflow ESI tandem MS using a quadrupole TOF mass spectrometer (Q-tof-2, Micromass). Proteins were digested with the same method as mentioned above except that peptides were resolved in 5% formic acid and then purified and condensed by Zip-tip instrument. Conditions used were cone voltage, 45 V; collision gas, argon; collision energy, 15-35 V. The ESI voltage used was 3.5 kV, and the ion source was maintained at 80 °C. Peptide fragmentation data searching was performed using the Mascot MS/MS ion search algorithm at http://www.matrixscience.com/. Preparation of Antiserum against IgA-like Proteins. IgAlike proteins (the mixture of proteins with 77, 75, and 64 kDa) were purified by affinity chromatography (a-hemocyanin). Estimation of the proteins was performed according to the method of Folin-phenol. The IgA-like proteins were suspended in 0.01 M PBS, pH 7.4, to a total volume of 2 mL and emulsified with an equal volume of Freund’s complete adjuvant. Multiple subcutaneous injections of the proteins totaling approximately 1 mg were administered in the back of four New Zealand white rabbits. Three booster injections using Freund’s incomplete adjuvant were given subcutaneously at 20-25 day intervals. The rabbits were bled from the ear vein at 10 days after the last boosting. Rabbit anti-shrimp a-hemocyanin from the bleedings was separated and stored at -20 °C until analysis. Dot-ELISA Analysis. A Dot-ELISA method was applied to compare the immune specificity between human sera, human IgA, L. vannamei sera, a-hemocyanin, and the protein reacted with anti-human IgA and isolated by electrophoresis (ehemocyanin). Nitrocellulose (NC) membranes were cut into desired size, soaked in TBS for 5 min, and then allowed to dry on a filter paper. All samples were prepared in TBS, and 1 µL of aliquots of each sample with 1 µg was spotted onto the NC

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Hemocyanin Is an IgA-like Protein

membrane using a regulatory pipet. After drying, the NC membrane was processed for indirect Dot-ELISA using goat anti-human IgA antiserum (1:200) or rabbit anti-shrimp ahemocyanin (1:400) as the primary antibody and rabbit antigoat IgG-HRP (1:1000) or goat anti-rabbit IgG-HRP (1:1000) as the secondary antibody, respectively. Cloning of Ig-like Conserved Domain of Hemocyanin (ICDH) Gene Fragment. 1. RNA Preparation and Reverse Transcription. Polyadenylated (poly A+) RNA was isolated from the hepatopancreas of live L. vannamei using an RNeasy mRNA kit (QIAGEN, Guangzhou, China) according to the supplier’s recommendations. First strand cDNA synthesis was performed with SMART RACE cDNA Amplification kit (CLONETECH, Guangzhou, China) following the manufacturer’s protocol. 2. Primers and PCR Amplification. Primers were designed based on the Ig-like domain nucleotide sequence of L. vannamei hemocyanin cDNA (accession X82502) searched from the NCBI database.21-23 SacI and PstI restriction sites were added to the ends of 5′- and 3′-primers, respectively, to enable easy insertion of the Ig-like domain into pQE-32. Two oligonucleotide primers were as following: primer I, 5′-CGGAGCTCTACCCCCATACACCAAAGC-3′; primer II, 5′-GGCTGCAGGAAGAGTTGTAAGCTTGAATC-3′. Two microliters of cDNA was used as template to amplify ICDH by PCR. Each reaction consisted of 0.4 µM of primers, 0.2 mM dNTP, 3.0 mM MgCl2, and 1 unit of Pfu DNA polymerase in 25 µL of reaction buffer as recommended by the manufacturer. The amplification cycles of 60 s at 94 °C, 60 s at 55 °C, and 60 s at 72 °C (36 cycles) were carried out in a UNO-Thermoblock (Vers.3.30 Biotron 1998). 3. Cloning and Sequencing. The PCR products were fractionated on a 0.75% agarose gel in TBE (0.045 M Tris-H3BO4, 0.001 M EDTA). The gel fragments were cut out and cleaned using the Geneclean II Kit (Q. BIOgene, CA). Then the genecleaned DNA and plasmid DNA of pQE-32 (QIAGEN, 3.4 kb) were separately digested with both SacI and PstI at 37 °C for 2 h. After purification, the digested target gene fragments were ligated into the pQE-32 vector according to the suppliers’ recommendations. The ligated products were transformed into XL1-Blue MRF cells. Single colonies were picked and cultured overnight at 37 °C. The plasmid DNA was extracted and digested with both SacI and PstI, and then applied on a 0.75% agarose gel to screen for positive clones. The sequences of the positive clones were confirmed by DNA sequencing with the dideoxy chain termination method using CEQTM 8000 Genetic Analysis System (Beckman). The nucleotide sequences were compared with the Pairwise BLAST program at http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html. Expression of the Recombinant Protein from ICDH and Immunoblotting Analysis. Expression of the recombinant ICDH protein was induced in XL1-Blue MRF Escherichia coli cells in log phase with 1 mM IPTG and grown for an additional 4 h. After the recombinant ICDH protein was purified, SDSPAGE and immunoblotting analysis were performed using the primary antibody, goat anti-human IgA (1:200) or rabbit antishrimp a-hemocyanin (1:400), and the secondary antibody, rabbit anti-goat IgG-HRP (1:1000) or goat anti-rabbit IgG-HRP (1:1000), respectively. Agglutination and Agglutination Inhibition Assays by Saccharides. Eight species of shrimp pathogenic bacteria including Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Aeromonas hydrophila, Aeromonas cavine, Aeromona sobria, and Pseudomonas fluorescens were selected for this analysis. These bacteria were separately

cultured in broth medium overnight at 28 °C. The cells were harvested, washed, and diluted to 108 cfu/mL in TBS-Ca2+ (0.05 M Tris, 0.75% NaCl, 0.05M CaCl2). Agglutination of the eight bacteria by a-hemocyanin was assayed at 37 °C for 30 min. The a-hemocyanin was diluted 2-fold in TBS-Ca2+, and 20 µL of each of these bacteria was added. Agglutination was observed in a light microscope and scored as positive (+) or negative (-) compared to a control placing the corresponding bacteria only in the TBS-Ca2+ buffer. Agglutinative titer was defined as the highest dilution of the test samples when the agglutination was appeared. The agglutination of bacteria by a-hemocyanin was further confirmed by an inhibition test using 10 saccharides, namely, glucose, mannose, galactose, lactose, sucrose, maltose, mannitol, inositol, N-acetylglucosamine, and N-acetylneuraminic acid. For this purpose, a-hemocyanin was diluted into 1:64 with TBS-Ca2+ and mixed separately with an equal volume of each of the 10 saccharides (200 mM) that were serially diluted with TBS-Ca2+ on a clean glass slide for 10 min at room temperature. Then V. parahaemolyticus suspension (108 cfu/mL) was added to each sample and incubated for 30 min at 37 °C. The minimal concentration of the testing saccharides was recorded when the agglutination activity was completely inhibited. Hemagglutination and Hemagglutination Inhibition Assays. Erythrocytes from healthy human donors, mice, herrings, and chickens were washed three times with TBS by centrifugation at 500g for 5 min and then diluted to 0.5% erythrocyte suspension in TBS-Ca2+. The suspension was directly applied for hemagglutination assay or diluted into 1:16 with TBS-Ca2+ for hemagglutination inhibition assay. The two inhibition assays were performed in the same condition as described above in the agglutination assays.

Results A Protein of 75 kDa Reacted with Anti-Human IgA. An IgAlike protein from L. vannamei haemolymph was separately isolated with the use of affinity chromatography, 1-DE, and 2-DE as shown in Figure 1. Three bands and proteins with 77, 75, and 64 kDa in 1-DE and 2-DE, respectively, were obtained, and their isoelectric points were estimated as 6.0. Furthermore, their immune reactivity was detected using 1-DE and 2-D immunoblotting with goat anti-human IgA. Only the band and protein with 75 kDa in 1-DE and 2-DE, respectively, were documented to be positive, and the other two were not bound with anti-human IgA, although they were strongly stained by Coomassie Brilliant Blue R-250 stains (Figure 1). The 75 kDa Protein Was Documented To Be Hemocyanin by Mass Spectrometry. The 75 kDa protein was cut out from a 2-DE gel and subjected to MALDI-TOF/MS analysis following digestion with trypsin. The following mass values were chosen for identification of the protein: 1065.6389, 1091.6754, 1110.7204, 1207.7585, 1235.7499, 1277.8964, 1302.8901, 1365.8226, 1448.9386, 1487.9692, 1503.9169, 1513.9909, 1529.8664, 1571.0177, 1591.9703, 1744.1866, 1784.1991, 1792.0072, 1801.1897, 1829.2105, 1839.2326, 1891.2402, 1912.3049, 2192.4047, and 2410.5309. Databases (NCBInr and MSDB) search results are shown in Table 1, indicating that the 75 kDa protein was L. vannimei hemocyanin (gi|854403, p < 0.05) or P. vannimei hemocyanin precursor (S55387, p < 0.05). To confirm the initial identification by peptide mass fingerprinting (PMF), tandem mass spectrometry was performed to analyze the peptides from the digested protein. When the ESI tandem MS data (Table 2) were subjected to database searching, the protein was identified to be P. Journal of Proteome Research • Vol. 5, No. 4, 2006 817

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Figure 1. 1-DE and 2-DE and their immunoblotting analysis of an IgA-like protein. (A) 1-DE and 1-D immunoblotting analysis of an IgA-like protein: lane 1, molecular mass markers; lane 2, 1-DE of an IgA-like protein; lane 3, 1-D immunoblotting analysis of an IgA-like protein using goat anti-human IgA antiserum (1:200) and rabbit anti-goat IgG-HRP (1:1000) as the primary and secondary antibodies, respectively. (B) 2-DE analysis of an IgA-like protein. (C) 2-D immunoblotting analysis of an IgA-like protein using goat anti-human IgA antiserum (1:200) and rabbit anti-goat IgG-HRP (1:1000) as the primary and secondary antibodies, respectively. Table 1. PMF Search Results of IgA-like Protein with 75 kDa Using Mascot Search with NCBInr and MSDB Databases rank

database

accession name

protein description

species

mass (Da)

score

expect

queries matched

percentage of match

sequence coverage

1 2

NCBInr MSDB

gi|854403 S55387

hemocyanin hemocyanin precursor

Litopenaeus vannamei Penaeus vannamei

74934 74934

82 82

0.0046 0.0032

13 13

52% 52%

21% 21%

Table 2. Matched Amino Acid Sequences of IgA-like Protein with 75 kDa Identified as P. vannamei Hemocyanin Precursor (gi|1085839) by ESI-MS/MS Analysis m/z

Mr (expt)

Mr (calc)

delta

miss

score

rank

start-end

sequence

474.24 489.28 501.27 533.25 546.27 554.78 483.57 492.24

946.47 976.55 1000.52 1064.48 1090.53 1107.54 1447.70 1473.70

946.48 976.56 1000.52 1064.48 1090.54 1107.56 1447.72 1473.71

-0.01 -0.01 -0.00 -0.00 -0.01 -0.02 -0.02 -0.00

0 0 0 0 0 0 0 0

17 12 45 59 58 46 13 39

1 5 1 1 1 1 3 1

410-417 27-34 70-78 291-298 566-575 640-648 278-290 312-325

DNLPPYTK DVLYLLNK LVQDLNDGK FEDVDDVAR FESATGLPNR VFEDLPNFK YGGQFPARPDNVK DAIAHGYIVDSEGK

vannimei hemocyanin precursor (gi|1085839, p < 0.05). At first, the three identified results about the 75 kDa protein seemed inconsistent. However, sequence alignment indicated that L. vannimei hemocyanin (gi|854403) was 100% identical to both of the P. vannimei hemocyanin precursors (S55387, gi|1085839). Therefore, this led us to conclude that the 75 kDa protein which reacted with anti-human IgA was hemocyanin. In addition, the bands and spots with 77 and 64 kDa were also identified as L. vannimei hemocyanin by MS. Human IgA and Hemocyanin Could Be Cross-Reacted with Anti-Human IgA and Anti-Shrimp A-Hemocyanin. For the determination of a similar epitope between L. vannamei hemocyanin and human IgA, human sera, human IgA, L. vannamei sera, a-hemocyanin, e-hemocyanin, and BSA were separately reacted with rabbit anti-shrimp a-hemocyanin or

Figure 2. A map of Dot-ELISA analysis, reacted with rabbit antishrimp a-hemocyanin (1:400, row: a) or goat anti-human IgA (1: 200, row: b) as the primary antibody, and then with goat antirabbit IgG-HRP (1:1000, row: a) or rabbit anti-goat IgG-HRP (1: 1000, row: b) as the secondary antibody, respectively. Lane 1, BSA; lane 2, human sera; lane 3, human IgA; lane 4, L. vannamei sera; lane 5, a-hemocyanin; lane 6, e-hemocyanin. The protein concentration was 1 mg/mL for each sample. 818

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anti-human IgA. The result indicated that these proteins, except for BSA, could separately be bound to the two antibodies, showing that the activity of L. vannamei sera and a-hemocyanin was significantly higher than that of e-hemocyanin, human IgA, and human sera against anti-shrimp a-hemocyanin, and the ability of human sera and human IgA was significantly stronger than that of L. vannamei sera, a-hemocyanin, and

Figure 3. A 0.75% agarose gel electrophoresis of restriction digest analysis of recombinant plasmid pQE-32-ICDH. Lane 1, DNA molecular mass markers; lane 2, restriction digest analysis of recombinant plasmid pQE-32-ICDH with both SacI and PstI.

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Hemocyanin Is an IgA-like Protein

Table 3. Agglutinative Activity of 350 µg/mL A-Hemocyanin with a Bacterium or Erythrocyte

Figure 4. SDS-PAGE and immunoblotting analysis of recombinant ICDH. Lane 1, molecular mass markers; lane 2, SDS-PAGE of the pellet from XL1-Blue MRF without being induced by IPTG; lane 3, SDS-PAGE of the pellet from XL1-Blue MRF induced by IPTG; lane 4, SDS-PAGE of purified recombinant ICDH; lane 5, the purified recombinant ICDH incubated with goat anti-human IgA (1:200) and then rabbit anti-goat IgG-HRP (1:1000); lane 6, the purified recombinant ICDH incubated with rabbit anti-shrimp a-hemocyanin (1:400) and then goat anti-rabbit IgG-HRP (1:1000).

e-hemocyanin against anti-human IgA (Figure 2). That the reaction by e-hemocyanin with anti-shrimp a-hemocyanin was dramatically weaker than that by L. vannamei sera and ahemocyanin did suggest the difference of antigenicity between the 77, 75, and 64 kDa hemocyanins because the antiserum was prepared from the mixture of the three hemocyanins rather than e-hemocyanin, 75 kDa protein. Recombinant Protein of the Ig-like Conserved Domain of L. vannamei Hemocyanin Could Be Reacted with AntiHuman IgA. Searches for conserved domain showed that L. vannamei hemocyanin (gi|854403) contained an Ig-like domain with 252 residues in its C-terminus.23 To test whether the region that reacted with anti-human IgA was located in the ICDH, the ICDH was cloned and then expressed in E. coli XL1-Blue MRF. A thick band approximately 777 bp in length in the recombinant plasmid pQE-32-ICDH was observed (Figure 3), whose sequence showed 100% identity with that of the reported L. vannamei ICDH,22 followed by obtaining a marked high-level expressed protein of about 35 kDa. Furthermore, immunoblotting test indicated that the purified recombinant ICDH could

bacterium or erythrocyte

agglutinative titera

agglutinative activity (µg/mL)b

specific activityc

Vibrio parahaemolyticus Vibrio alginolyticus Vibrio anguillarum Vibrio fluvialis Aeromonas hydrophila Aeromonas cavine Aeromona sobria Pseudomonas fluorescens fish erythrocyte chicken erythrocyte mouse erythrocyte human erythrocyte

1024 512 1024 512 512 512 512 256 32 32 32 64

0.34 0.68 0.34 0.68 0.68 0.68 0.68 1.37 10.94 10.94 10.94 5.47

2.93 1.47 2.93 1.47 1.47 1.47 1.47 0.73 0.09 0.09 0.09 0.18

a The highest dilution of the testing samples in the presence of V. parahaemolyticus. b Protein concentration/agglutinative titer. c Agglutinative titer/protein concentration.

react with rabbit anti-shrimp a-hemocyanin or goat antihuman IgA (Figure 4). These findings imply that the Ig-like conserved domain of L. vannamei hemocyanin can function as an antigen reacted with anti-human IgA. L. vannamei Hemocyanin Could Bind Both Bacteria and Erythrocytes Directly. Interestingly, agglutination could be observed when a-hemocyanin was separately bound to V. parahaemolyticus, V. alginolyticus, V. anguillarum V. fluvialis, A. hydrophila, A. cavine, A. sobria, and P. fluorescens (Figure 5 and Table 3). Their agglutinative activities were related to the bacterial species, ranging from 0.34-1.37 µg/mL. Meanwhile, the agglutination of erythrocytes by a-hemocyanin was evaluated. The erythrocytes of the four animals tested were all agglutinated, showing that their hemagglutinative activity varied from 5.47 to 10.94 µg/mL (Figure 5 and Table 3). Furthermore, an agglutination inhibition test was applied to evaluate the saccharide specificity in the agglutination of bacteria and erythrocytes by a-hemocyanin. Namely, glucose, mannose, galactose, lactose, sucrose, maltose, mannitol, inositol, N-acetylglucosamine, and N-acetylneuraminic acid were used for this analysis. Of the 10 saccharides, N-acetylneuraminic acid (25 mM) and N-acetylneuraminic acid (50 mM),

Figure 5. Agglutinative activity of a-hemocyanin. (A) Negative control using V. parahaemolyticus (3280×); (B) a-hemocyanin titer 1:64 with V. parahaemolyticus (3280×); (C) negative control using A. hydrophila (3280×); (D) a-hemocyanin titer 1:64 with A. hydrophila (3280×); (E) negative control using chicken erythrocytes (400×); (F) a-hemocyanin titer 1:16 with chicken erythrocytes (400×); (G) negative control using human erythrocytes (300×); (H) a-hemocyanin titer 1:16 with human erythrocytes (300×). Journal of Proteome Research • Vol. 5, No. 4, 2006 819

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Figure 6. Inhibition of agglutinative activity of a-hemocyanin by N-acetylneuraminic acid. (A) Negative control using V. parahaemolyticus (3280×); (B) with V. parahaemolyticus by 25 mM N-acetylneuraminic acid (3280×); (C) negative control using human erythrocytes (1200×); (D) with human erythrocytes by 25 mM N-acetylneuraminic acid (1200×). Table 4. Inhibition of Agglutinative Activity of A-Hemocyanin with V. parahaemolyticus and Human Erythrocyte by Saccharides minimum inhibitory concentration (mM) saccharide

V. parahaemolyticus

human erythrocyte

glucose mannose galactose lactose sucrose maltose mannitol inositol N-acetylglucosamine N-acetylneuraminic acid

Na Na Na Na Na Na Na Na Na 25

200 Na Na 200 Na Na 100 Na Na 50

a

N, not inhibited.

mannitol (100 mM), glucose (200 mM), and lactose (200 mM) could inhibit the agglutination with V. parahaemolyticus and human erythrocytes, respectively (Figure 6 and Table 4).

Discussion In the current study, we isolated three proteins of 77, 75, and 64 kDa from L. vannamei sera by affinity proteomics and immunoproteomics, in which the 75 kDa protein was determined to react with goat anti-human IgA and identified as hemocyanin by MS. Furthermore, Dot-ELISA results showed that the hemocyanin and human IgA could separately react with anti-shrimp a-hemocyanin and anti-human IgA, indicating that a part of L. vannamei hemocyanin shared a similar epitope with human IgA. Our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of a Forssman antigen, the crossing antigen between species. Interestingly, only the band and protein with 75 kDa were separately reacted with anti-human IgA, although the other two were also hemocyanin, suggesting that a portion of hemocyanin strongly reacted with anti-human IgA. Indeed, the conclusion is further supported by the results of Dot-ELISA in the current study. The Dot-ELISA reaction was significantly weaker in e-hemocyanin than L. vannamei sera and a-hemocyanin because anti-shrimp a-humocyanin was prepared by the mixture of the three bands rather than the purified band with 75 kDa. We do not know which portion reacted with human IgA and what is the biological significance of this, but this finding has certainly contributed to the approach of hemocyanin diversity. Nowadays, more and more evidence indicates that diversity is a characteristic feature in invertebrates.11,14,24 Our results from recombinant ICDH as an antigen indicated 820

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that the epitope(s) of L. vannamei hemocyanin which reacted with anti-human IgA was located in its C-terminus. Indeed, hemocyanin, an oxygen carrier in arthropod and mollusk, was determined as an IgSF molecule with an Ig domain in its C-terminus.23,25 Particularly, hemocyanin could be functionally converted into phenoloxidase-like enzyme as an important type of defense molecule,26-31 and showed antifungal, antibacterial, and antivirus activities.32-35 Consequently, it has been suggested that hemocyanin may possess some important immune functions in crustacea. We have very interesting findings from the observation of agglutination and agglutination inhibition tests in the current study. The agglutination of eight species of pathogens and four types of animal erythrocytes by hemocyanin was determined. Furthermore, the agglutination inhibition test was performed using V. parahaemolyticus and human erythrocytes, showing the agglutination between the bacterium and the erythrocyte could be stopped by N-acetylneuraminic acid (25 mM) and N-acetylneuraminic acid (50 mM), mannitol (100 mM), glucose (200 mM), and lactose (200 mM), respectively. The results are somewhat in agreement with the report that N-acetylated sugars were the most effective inhibitors of hemagglutination caused by a lectin from the haemolymph of L. setiferus.36 Reports indicated that invertebrates managed to survive with an innate immune response. A variety of bacterial and fungal cell wall components including lipopolysaccharide, peptidoglycan, and β-1,3-glucan were biologically active and elicited various innate immune reactions, resulting in microorganism agglutination, antibacterial peptides induction, prophenoloxidase activation, and so on.37-39 In the present study, our results indicated that L. vannamei hemocyanin could be bound to bacteria and animal erythrocytes directly. Thus, we could deduce that L. vannamei hemocyanin should have specific recognition motifs binding to the components of pathogens and erythrocytes. Apparently, the novel discovery that L. vannamei hemocyanin can bind directly with pathogens and animal erythrocytes suggests that L. vannamei hemocyanin has a new potential function. Future work is required for an understanding of the mechanism and biological significance. In conclusion, a type of IgA-like protein with 75 kDa from L. vannamei haemolymph was identified as hemocyanin. To the best of our knowledge, this is the first report that the portion of Ig-like domain of L. vannamei hemocyanin can react with anti-human IgA. Moreover, we found for the first time that hemocyanin showed agglutination with bacteria and erythrocytes directly, which could be stopped by N-acetylneuraminic acid and N-acetylneuraminic acid, mannitol, glucose, and lactose, respectively. Further studies are required to determine the binding motif of Ig-like domain with Ig and the significance

research articles

Hemocyanin Is an IgA-like Protein

of the interaction with pathogens and erythrocytes in host protection against microbial infections.

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Acknowledgment. We thank the Lee Foundation of Singapore and Prof. C. L. Hew, Department of Biological Sciences, National University of Singapore, Singapore, for support of ESI-MS/MS analysis. This work was sponsored by a grant from “863” project of China (No. 2005AA626013), Scientific Foundation of Xiamen, and Guangdong Natural Science Fund (Doctor’s Start-up Fund, No. 04300750).

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