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• W2057183941 abstract "Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph. Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph. Drosophila is a useful model system for the study of innate immunity (3Hultmark D. Curr. Opin. Immunol. 2003; 15: 12-19Crossref PubMed Scopus (475) Google Scholar, 4Hoffmann J.A. Nature. 2003; 426: 33-38Crossref PubMed Scopus (1136) Google Scholar). The humoral activity and induction of antimicrobial peptides in adults have attracted much attention (5Naitza S. Ligoxygakis P. Mol. Immunol. 2004; 40: 887-896Crossref PubMed Scopus (53) Google Scholar). Increasingly though, cellular reactions are being studied thanks to the availability of cell lines with similarity to hemocytes and the establishment of high throughput techniques and large scale mutagenesis projects (6Lavine M.D. Strand M.R. Insect Biochem. Mol. Biol. 2002; 32: 1295-1309Crossref PubMed Scopus (1153) Google Scholar, 7Rämet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Nature. 2002; 416: 644-648Crossref PubMed Scopus (591) Google Scholar, 8Pearson A.M. Baksa K. Ramet M. Protas M. McKee M. Brown D. Ezekowitz R.A. Microbes Infect. 2003; 5: 815-824Crossref PubMed Scopus (104) Google Scholar). Whole genome induction studies have confirmed the presence of several immune induction pathways, which differentially contribute to responses against different classes of microorganisms, different phases of an immune reaction, and cellular versus humoral immunity (9De Gregorio E. Spellman P.T. Rubin G.M. Lemaitre B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12590-12595Crossref PubMed Scopus (589) Google Scholar, 10Irving P. Troxler L. Heuer T.S. Belvin M. Kopczynski C. Reichhart J.M. Hoffmann J.A. Hetru C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15119-15124Crossref PubMed Scopus (331) Google Scholar, 11De Gregorio E. Spellman P.T. Tzou P. Rubin G.M. Lemaitre B. EMBO J. 2002; 21: 2568-2579Crossref PubMed Scopus (644) Google Scholar). Genes involved in antimicrobial responses can be grouped according to their induction pattern using cluster analysis (11De Gregorio E. Spellman P.T. Tzou P. Rubin G.M. Lemaitre B. EMBO J. 2002; 21: 2568-2579Crossref PubMed Scopus (644) Google Scholar, 12Boutros M. Agaisse H. Perrimon N. Dev. Cell. 2002; 3: 711-722Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). More recently, the systemic immune response has been studied at a posttranscriptional level using proteomics (13Vierstraete E. Verleyen P. Baggerman G. D'Hertog W. Van Den Bergh G. Arckens L. De Loof A. Schoofs L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 470-475Crossref PubMed Scopus (122) Google Scholar, 14Levy F. Bulet P. Ehret-Sabatier L. Mol. Cell. Proteomics. 2004; 3: 156-166Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 15Vierstraete E. Verleyen P. Sas F. Van den Bergh G. De Loof A. Arckens L. Schoofs L. Biochem. Biophys. Res. Commun. 2004; 317: 1052-1060Crossref PubMed Scopus (51) Google Scholar, 16Loseva O. Engström Y. Mol. Cell. Proteomics. 2004; 3: 796-808Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), summarized in Ref. 17Engström Y. Loseva O. Theopold U. Trends Biotechnol. 2004; 22: 600-605Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar. These studies were facilitated by the establishment of two-dimensional protein maps of the larval hemolymph (18Vierstraete E. Cerstiaens A. Baggerman G. Van den Bergh G. De Loof A. Schoofs L. Biochem. Biophys. Res. Commun. 2003; 304: 831-838Crossref PubMed Scopus (88) Google Scholar, 19Guedes S.d.M. Vitorino R. Tomer K. Domingues M.R. Correia A.J. Amado F. Domingues P. Biochem. Biophys. Res. Commun. 2003; 312: 545-554Crossref PubMed Scopus (47) Google Scholar). Hemolymph samples at different times after infection or the addition of immune elicitors were compared with unchallenged controls. Known immune proteins as well as candidate immune proteins were identified, including proteins with similarity to vertebrate complement components (thiol ester motif-containing proteins) (13Vierstraete E. Verleyen P. Baggerman G. D'Hertog W. Van Den Bergh G. Arckens L. De Loof A. Schoofs L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 470-475Crossref PubMed Scopus (122) Google Scholar, 14Levy F. Bulet P. Ehret-Sabatier L. Mol. Cell. Proteomics. 2004; 3: 156-166Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), a pattern recognition protein (β-glucan binding protein) (14Levy F. Bulet P. Ehret-Sabatier L. Mol. Cell. Proteomics. 2004; 3: 156-166Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), several serine proteases, serine protease inhibitors (14Levy F. Bulet P. Ehret-Sabatier L. Mol. Cell. Proteomics. 2004; 3: 156-166Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), and metabolic proteins (13Vierstraete E. Verleyen P. Baggerman G. D'Hertog W. Van Den Bergh G. Arckens L. De Loof A. Schoofs L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 470-475Crossref PubMed Scopus (122) Google Scholar, 14Levy F. Bulet P. Ehret-Sabatier L. Mol. Cell. Proteomics. 2004; 3: 156-166Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 15Vierstraete E. Verleyen P. Sas F. Van den Bergh G. De Loof A. Arckens L. Schoofs L. Biochem. Biophys. Res. Commun. 2004; 317: 1052-1060Crossref PubMed Scopus (51) Google Scholar), summarized in Ref. 17Engström Y. Loseva O. Theopold U. Trends Biotechnol. 2004; 22: 600-605Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar. Here we describe a proteomic approach for the identification of proteins involved in the Drosophila larval hemolymph clotting reaction, which is essential for both sealing wounds to avoid loss of hemolymph (hemostasis) and preventing dissemination of microbes into the hemocoel. Proteomics provides a particularly suitable means to study clotting that involves the fast interaction of cellular and humoral proteins, resulting in the precipitation of an insoluble matrix independent of and preceding changes in gene expression. This precipitation leads to a soft clot, which is hardened through the activity of cross-linking enzymes (summarized in Ref. 1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Candidates for cross-linking enzymes include phenoloxidase and transglutaminase. The formation of the soft clot is independent of hemolymph phenoloxidase (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), but the enzyme participates at later stages, leading to visible melanization of the clot (summarized in Ref. 1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Transglutaminases mediate covalent cross-linking between primary amines (e.g. a lysine residue) and glutamine residues (20Lorand L. Graham R.M. Nat. Rev. Mol. Cell. Biol. 2003; 4: 140-156Crossref PubMed Scopus (1213) Google Scholar). Clotting factor XIIIa, which is a transglutaminase, cross-links the vertebrate blood clot. Transglutaminases are also involved in clotting in non-insect arthropods (1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 21Li D. Scherfer C. Korayem A.M. Zhao Z. Schmidt O. Theopold U. Insect Biochem. Mol. Biol. 2002; 32: 919-928Crossref PubMed Scopus (94) Google Scholar). Our experimental approach in this study was to compare larval hemolymph before and after clotting by proteomics. In contrast to previous (immune) proteomic studies, we did not challenge larvae with microbial elicitors, and we removed hemocytes to focus on changes in the hemolymph after clotting. In addition to this comparison, we also identified transglutaminase substrates as a means to highlight clotting factors. Our results confirm and extend previous studies in the lepidopteran Galleria mellonella (21Li D. Scherfer C. Korayem A.M. Zhao Z. Schmidt O. Theopold U. Insect Biochem. Mol. Biol. 2002; 32: 919-928Crossref PubMed Scopus (94) Google Scholar) and Drosophila melanogaster (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) that involved direct isolation of the clot and subsequent identification of its components. They provide further molecular insight into clotting and the events in hemolymph after wounding. These events are the first line of defense against intruding microbes and set the stage for subsequent immune reactions. Flies—Drosophila w1118 cultures were kept at 18 °C in a 12-h light and 12-h dark cycle on standard cornmeal sucrose medium. Third instar larvae were used in this study. Pull-out Assay—To obtain a non-clotted plasma fraction (Fig. 3A), 10 animals were bled directly into anticoagulant Ringer's solution (Drosophila Ringer's solution containing 0.02 m EDTA and no CaCl2). In a first attempt to obtain clotted serum, animals were opened, left for 30 s, and then placed on an electron microscope grid (90-μm pore width), which acted as a filter to remove the clot. The flow-through fraction (serum) was collected in a capillary placed underneath the grid. Both samples were subsequently centrifuged for 10 min, first at 300 × g to remove hemocytes and then at 11,000 × g and prepared for PAGE analysis. Comparison between the samples revealed that the differences between them were not sufficiently reproducible to allow a proteomics approach. Therefore, clotting was performed as part of a pullout reaction (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) followed by centrifugation as described above. Briefly, the pull-out assay relies on the incorporation of paramagnetic beads into the clot and the subsequent removal of the clot with a magnet. Proteins in the remaining supernatant (serum) were precipitated with ice-cold chloroform:methanol and washed two times with ice-cold methanol. The pellet was dried in a vacuum centrifuge and dissolved in two-dimensional sample buffer (2-D Sample Preparation for Insoluble Proteins, catalog number 89866, Pierce). After 1 h the sample was desalted with the two-dimensional sample kit according to kit instructions. Prior to isoelectric focusing (IEF), 1The abbreviations used are: IEF, isoelectric focusing; BP, 5-biotinamide-pentylamine; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight. a rehydration solution (Amersham Biosciences) was added to the sample to a final volume of 125 μl (for 7-cm strips) and 250 μl (for 13-cm strips), as well as a trace of bromphenol blue. Two-dimensional Gel Electrophoresis—The solubilized protein sample was applied onto immobilized pH gradient strips (7- or 13-cm non-linear pH 3–10; Amersham Biosciences). IEF was performed with the IPGphor isoelectric focusing system (Amersham Biosciences) at 20 °C and 50 μA per immobilized pH gradient strip as follows. 7-cm strips underwent 12 h of rehydration and IEF for 1 h at 500 V, 1 h at 1000 V, and ∼3 h at 8000 V to reach a total of 12,035 volt hours. 13-cm immobilized pH gradient strips were rehydrated for 12 h followed by IEF for 1 h at 500 V, 1 h at 1000 V, and a final step at 8000 V to reach 25,600 volt hours. Prior to SDS-PAGE, strips were equilibrated for 15 min in equilibration buffer (0.05 m Tris-HCl, pH 8.8, 6 m urea, 30% glycerol, 2% SDS, and bromphenol blue) including 1% dithiothreitol followed by 15 min of incubation in equilibration buffer containing 2.5% iodoacetamide. SDS-PAGE was carried out at 20 °C for 75 min at 30 mA (7-cm strips) or 4 h at 30 mA (13-cm strips) on a 1-mm 10% minigel (Bio-Rad) or a 1.5-mm 12% 14 × 16-cm gel (Amersham Biosciences), respectively. Proteins were detected either with silver staining (a silver staining kit from Amersham Biosciences) or Coomassie staining (Brilliant Blue G-colloidal concentrate from Sigma) according to the manufacturers' protocols. The silver staining protocol was modified to be compatible with subsequent in-gel digestion for mass spectrometry analysis (22Yan J.X. Wait R. Berkelman T. Harry R.A. Westbrook J.A. Wheeler C.H. Dunn M.J. Electrophoresis. 2000; 21: 3666-3672Crossref PubMed Scopus (648) Google Scholar). Image Analysis—Gels were scanned with Fujifilm Luminescent PhosphorImage Analyzer LAS 1000 plus and analyzed with the PDQuest software. Protein spots were matched automatically across three gels from Drosophila plasma and four gels from serum. Gel images were normalized so that the total density in gel image on analyzed gels was made equal and thereafter subjected to statistical analysis with a Student's t test to determine whether the observed differences were statistically significant. Each of the spots was also checked by eye to exclude uncertain spots and to examine whether abundant proteins were constitutively reduced in the serum samples (for example, some isoforms of phenoloxidase differed in all pairwise comparisons between plasma and serum; see “Results”). To identify proteins that are developmentally regulated during the stages used in this analysis, plasma samples from early and late wandering larvae distinguished by gut clearance (23Andres A.J. Thummel C.S. Methods Cell Biol. 1994; 44: 565-573Crossref PubMed Scopus (156) Google Scholar) were compared on two-dimensional gels. Proteins that differed between the different stages were excluded from further analysis. MALDI-TOF Mass Spectrometry and Protein Identification—Spots were cut from the gel with sterile stainless steel scalpels and digested in-gel with sequencing grade-modified trypsin (Promega V511A) as described in (24Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7822) Google Scholar), except that the reduction and alkylation steps were omitted because cysteines were carbamidomethylated on the equilibration step of two-dimensional gel electrophoresis. Coomassie-stained samples were destained in 0.025 m NH4HCO3, dehydrated in CH3CN, and centrifuged in a vacuum centrifuge to complete dryness. Peptides were extracted in 50% CH3CN and 5% CF3COOH, dried in a vacuum centrifuge, and resuspended in 50% CH3CN and 0.1% CF3COOH. Silver-stained samples were prepared according to Gharahdaghi et al. (25Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (845) Google Scholar) with some modifications. In brief, samples were destained in a 1:1 solution of 0.03 m K3Fe(CN)6 and 0.1 m Na2S2O3 and then washed once in water and once in 50% CH3CN and 0.025 m NH4HCO3 followed by dehydration with 100% CH3CN. Finally, samples were dried in a SpeedVac. Proteins were in-gel digested as described above. Peptides were extracted in 50% CH3CN and 5% CF3COOH and dried completely in a SpeedVac. If necessary, the recovered peptides were purified and concentrated on C18Ziptips (Millipore) according to the manufacturer's instructions. Prior to mass spectrometry, peptides were resuspended in 70% CH3CN and 0.1% CF3COOH. Mass spectra were recorded in positive reflection mode by using an Applied Biosystems MALDI-TOF Voyager-DE STR mass spectrometer equipped with delayed ion-extraction technology. α-Cyano-4-hydroxycinnamic acid was used as the matrix. External calibration was performed using the Sequazyme peptide mass standard kit (Angiotensin I and ACTH, clips 1–17, 18–39, and 7–38; PerSeptive Biosystems), and autodigestion peaks of bovine trypsin were used for internal calibration. Peptide mass profiles were analyzed using Mascot (Matrix Science; www.matrixscience.com/) and Profound (prowl.rockefeller.edu/). Searches were performed using the NCBI data base. Search parameters for monoisotopic peptide masses included allowance for one missed enzymatic cleavage and accepted the carbamidomethylation of cysteine residues and the oxidation of methionine as modifications. The results of protein identification by Mascot were confirmed based on molecular mass/pI values, and further confirmation of protein identifications was obtained using Profound software. CG numbers of the genes and predicted protein function were obtained from FlyBase (flybase.bio.indiana.edu/). Transglutaminase Labeling of Hemolymph Proteins—Labeling of hemolymph proteins with 5-(biotinamido)pentylamine was performed essentially as described elsewhere (26Ikura K. Kita K. Fujita I. Hashimoto H. Kawabata N. Arch. Biochem. Biophys. 1998; 356: 280-286Crossref PubMed Scopus (33) Google Scholar). Briefly, larvae were bled into a buffer containing 0.05 m Tris-HCl, pH 7.5, 0.01 m dithiothreitol, and 0.005 m 5-(biotinamido)pentylamine (Pierce). After 10 min of incubation at 37°C, the reaction was stopped by adding EDTA to a final concentration of 0.05 m and centrifuged at 760 × g. The supernatant was dialyzed against 0.1 m Na2HPO4, pH 7.2, containing 0.15 m NaCl, centrifuged at 15000 × g, and analyzed in Western blots using peroxidase-conjugated streptavidin at a concentration of 0.2 μg/ml (Sigma). Preparation of Drosophila Serum—A dense fibrous clot forms immediately when Drosophila larvae are bled. The clot can be visualized by phase-contrast microscopy or fluorescence microscopy using lectins that bind to the glycoprotein-rich clot (Fig. 1, A and B) (1Theopold U. Schmidt O. Söderhäll K. Dushay M.S. Trends Immunol. 2004; 25: 289-294Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Although the reaction appears quite extensive, comparison of hemolymph before (plasma) and after clotting (serum) showed only a partial depletion of clot proteins, making a systematic proteomics comparison difficult (see “Experimental Procedures”). To allow more extensive clot formation and to quantitatively deplete clotting factors, we decided to use a method we had established previously for isolation of clot components (2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This method involves the incorporation of paramagnetic beads into the clot and the subsequent magnetic removal of the clot. Clotting factors can be extracted from the beads after extensive washing (Fig. 1, lane 1). When the supernatant of the reaction was applied sequentially to new batches of beads, almost no bead aggregation was observed in the second incubation, and no aggregation at all was seen in subsequent incubations (Fig. 1C, lanes 2–4). Thus, most clotting factors are depleted during the first pull-out reaction. In accordance with this observation, few proteins bind to the beads in the second and subsequent pull-out reactions (Fig. 1C). The pull-out method can thus be used to quantitatively deplete clotting factors from plasma, providing a serum fraction that is not capable of further clotting. Comparison between Plasma and Serum—To identify proteins that differ between plasma and serum, two-dimensional gel electrophoresis was used. We decided to first establish a two-dimensional reference map of plasma proteins, because available maps are all derived from complete hemolymph, which includes hemocytes. As shown in Fig. 2 and Table I, our map includes previously identified Drosophila hemolymph proteins as well as many proteins that had not been identified previously. A limited set of differences was observed comparing plasma and serum (see Fig. 3). Some differences could not be reproduced between replicas of the same treatment and were discarded. To avoid artifacts, we decided to only consider protein spots that differed at least 5-fold between serum and plasma (exceptions are indicated; see below and Table II). The results are shown in Fig. 3 and summarized in Table II. Several proteins were almost completely depleted from hemolymph after clotting. These included two isoforms of the CG15825 product, an immune-induced protein with two sets of internal repeats but no strong sequence homology to other known or predicted proteins (Fig. 3, spots a and b, and Fig. 4A). This protein had been isolated previously with the pull-out method (Fig. 2 and Ref. 2Scherfer C. Karlsson C. Loseva O. Bidla G. Goto A. Havemann J. Dushay M.S. Theopold U. Curr. Biol. 2004; 14: 625-629Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), supporting our proteomic approach. Four proteins depleted in serum were identified as different isoforms of the CG8502 product, a composite protein with a mucin-domain and a domain with similarities to cuticular proteins (Fig. 3, spots d–g, and Fig. 4B). Two splice variants of CG8502 have been identified (see FlyBase), accounting for some of the isoforms we observed. Additional variation may be due to differences in posttranslational modification, for example in the degree of glycosylation of the mucin domain. When the sequence of the CG8502 product was analyzed for mucin-type glycosylation (27Hansen J.E. Lund O. Tolstrup N. Gooley A.A. Williams K.L. Brunak S. Glycoconj. J. 1998; 15: 115-130Crossref PubMed Scopus (455) Google Scholar), 19 significant consensus sites for O-glycosylation were identified, all of which are clustered in the mucin domain shown in Fig. 4B. In addition to predicted proteins from both Anopheles gambiae and Drosophila, CG8502 shows sequence similarity to several known cuticular proteins from other insect orders (28Andersen S.O. Insect Biochem. Mol. Biol. 1998; 28: 421-434Crossref PubMed Scopus (90) Google Scholar). The alignment with the best score is shown in Fig. 4B. Although some cuticular proteins extend beyond the aligned region shown in Fig. 4B, none of them contains a mucin domain similar to the CG8502 product, making this a unique protein.Table IProteins identified in Drosophila plasmaSample no.Gene identityProtein nameTheoretical MrTheoretical pISequence coverageMolecular functionBiological processDa%01CG6206-PA123,2925.5418α-Mannosidase activity, hydrolysisCarbohydrate metabolism02CG14526-PA79,5785.6924Endothelin-coverting enzyme activityProteolysis, peptidolysis, signal transduction03CG14476-PB106,0646.0524α-Glucosidase activityPolysaccharide metabolism04CG6953-PAFat-spondin87,1716.3830Structural molecule activityCytoskeleton organization and biogenesis, ectoderm development05CG4725-PA86,3455.8426Endothelin-converting enzyme activityProteolysis, peptidolysis, signal transduction06CG5779-PADox-A179,4416.1446Monophenol monooxygenase activityMelanization, defense response07CG5779-PADox-A179,4416.1446Monophenol monooxygenase activityMelanization, defense response08CG5779-PADox-A179,4416.1446Monophenol monooxygenase activityMelanization, defense response09CG6186-PATransferrin 172,9646.6947Ferric ion binding, iron ion transporter activity, carrier activityIron ion homeostasisCG8193-PA1979,5206.4939Monophenol monooxygenase activityDefense response10CG8193-PA19Dox-A179,5206.4939Monophenol monooxygenase activityDefense responseCG5779-PA79,4416.1446Melanization, defense response11CG8193-PA1979,5206.4939Monophenol monooxygenase activityDefense response12CG5779-PADox-A179,4416.1446Monophenol monooxygenase activityMelanization, defense responseCG8193-PA1979,5206.4939Monophenol monooxygenase activityDefense response13CG8913-PAPeroxidase79,0866.9937Peroxidase activityDefense response, oxygen and reactive oxygen species metabolism14CG15825-PA1856,6715.972515CG15825-PA1856,6715.972516CG15825-PB1858,1126.452717CG5210-PA47K glycoprotein precursor50,7067.9939Chitinase-like, growth factor activity18CG1780-PAIdgf448,8027.6541Idgf activityCell-cell signaling, signal transduction19CG4559-PAIdgf349,4177.1234Idgf activity, NOT chitinase activity, hydrolase activityCell-cell signaling, signal transductionCG1780-PAIdgf448,8027.653520CG5779-PADox-A179,4416.1446Monophenol monooxygenase activityMelanization, defense response ferric ion binding, iron ion transporter activity, carrier activityCG1106-PAGelsolin, secreted form precursor98,5005.1119Actin binding, structural constituent of cytoskeletonCytoskeleton organization and biogenesis21CG11313-PA41,8818.2837Monophenol monooxygenase activator activity, trypsin activityProteolysis and peptidolysis22CG6058-PHAldolase39,8507.6067Fructose-biphosphate aldolase activityGlycolysis23CG9331-PA35,7486.2751Oxidoreductase activity24CG6058-PB19Aldolase39,2516.9759Fructose-biphosphate aldolase activityGlycolysis25CG8502-PC37,1486.3842Structural constituent of larval cuticle26CG8502-PC37,1486.3842Structural constituent of larval cuticle27CG8502-PA33,8116.3642Structural constituent of larval cuticle28CG8502-PA33,8116.3642Structural constituent of larval cuticle29CG5177-PA31,2916.6339Trehalose phosphatase activityDisaccharide metabolism30CG1803-PC18Regucalcin33,6806.0240Calcium ion bindingAnterior/posterior specification, calcium-mediated signaling31CG10467-PA1839,8886.1073Aldose-1 epimerase activityMonosaccharide metabolism32CG32031-PD18,19Arginine kinase40,1266.0461Arginine kinase activityPhosphorylation33CG5896-PA37,9917.5140Serine-type endopeptidase activity, trypsin activityProteolysis, peptidolysis34CG1780-PAIdgf448,8027.6538Idgf activityCell-cell signaling, signal transductionCG1106-PHGelsolin, secreted form precursor98,5005.1122Actin binding, structural constituent of cytoskeletonCytoskeleton organization and biogenesis35CG4475-PA18Chain A, crystal structure of Idgf247,0966.0948Idgf activity, NOT chitinase activity, hydrolase activity, hydrolyzing N-glycosyl compoundsCell-cell signaling, signal transduction36CG17654-PA18Enolase54,5898.6940Phosphopyruvate hydratase activityGlycolysis37CG5154-PAIdgf550,3616.3237Chitinase activity, hydrolase activity, hydrolyzing N-glycosyl compounds, growth factor activityCell-cell signaling, polysaccharide metabolism, signal transduction38CG8063-PA51,2257.0527Intramolecular isomerase activity" @default.
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