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• W2007375089 abstract "Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation. Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. In this study, goldfish granulin was one of the most commonly identified transcripts in the differential cross-screening of macrophage cDNA libraries and was preferentially expressed in proliferating macrophages. Unlike mammalian granulins, which possess 7.5 repeats of a characteristic signature of 12 cysteine residues, the goldfish granulin encoded a putative peptide possessing only 1.5 cysteine repeats. Northern blot and real-time PCR analyses indicated that goldfish granulin was expressed only in the hematopoietic tissues of the goldfish, specifically the kidney and spleen, and in activated peripheral blood mononuclear cells. We expressed granulin using a prokaryotic expression system and produced an affinity-purified rabbit anti-goldfish granulin IgG. Recombinant goldfish granulin induced a dose-dependent proliferative response of goldfish macrophages that was inversely related to the myeloid differentiation stage of the cells studied. The highest proliferative response was observed in macrophage progenitor cells and monocytes. This proliferative response of macrophages was abrogated by the addition of anti-granulin IgG. These results indicate that goldfish granulin is a growth factor that positively modulates cell proliferation at distinct junctures of macrophage differentiation. First identified as small (6 kDa) peptides, granulins are produced by the proteolysis of a larger precursor molecule by leukocyte derived elastase activity (1Bateman A. Belcourt D. Bennett H. Lazure C. Solomon S. Biochem. Biophys. Res. Commun. 1990; 173: 1161-1168Crossref PubMed Scopus (175) Google Scholar, 2Shoyab M. McDonald V.L. Byles C. Todaro G.J. Plowman G.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7912-7916Crossref PubMed Scopus (154) Google Scholar, 3Bateman A. Bennett H.P. J. Endocrinol. 1998; 158: 145-151Crossref PubMed Scopus (196) Google Scholar, 4He Z. Bateman A. J. Mol. Med. 2003; 81: 600-612Crossref PubMed Scopus (415) Google Scholar, 5Ong C.H. Bateman A. Histol. Histopathol. 2003; 18: 1275-1288PubMed Google Scholar, 6Sparro G. Galdenzi G. Eleuteri A.M. Angeletti M. Schroeder W. Fioretti E. Protein Expression Purif. 1997; 10: 169-174Crossref PubMed Scopus (29) Google Scholar, 7Zhu J. Nathan C. Jin W. Sim D. Ashcroft G.S. Wahl S.M. Lacomis L. Erdjument-Bromage H. Tempst P. Wright C.D. Ding A. Cell. 2002; 111: 867-878Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). The larger precursor is known by several names, including granulin/epithelin precursor (8Zanocco-Marani T. Bateman A. Romano G. Valentinis B. He Z.H. Baserga R. Cancer Res. 1999; 59: 5331-5340PubMed Google Scholar), proepithelin (9Plowman G.D. Green J.M. Neubauer M.G. Buckley S.D. McDonald V.L. Todaro G.J. Shoyab M. J. Biol. Chem. 1992; 267: 13073-13078Abstract Full Text PDF PubMed Google Scholar), acrogranin (10Baba T. Hoff H.B. II I Nemoto H. Lee H. Orth J. Arai Y. Gerton G.L. Mol. Reprod. Dev. 1993; 34: 233-243Crossref PubMed Scopus (122) Google Scholar), PC cell-derived growth factor (PCDGF) (11Zhou J. Gao G. Crabb J.W. Serrero G. J. Biol. Chem. 1993; 268: 10863-10869Abstract Full Text PDF PubMed Google Scholar), and progranulin. Granulins have a unique 12-cysteine motif that is arranged in four β-hairpins, stacked one upon another in a helical formation and connected via a central rod of disulfide bonds (12Bhandari V. Bateman A. Biochem. Biophys. Res. Commun. 1992; 188: 57-63Crossref PubMed Scopus (62) Google Scholar, 13Hrabal R.C.Z. James S. Bennett H.P. Ni F. Nat. Struct. Biol. 1996; : 747-752Crossref PubMed Scopus (138) Google Scholar, 14Tolkatchev D. Ng A. Vranken W. Ni F. Biochemistry. 2000; 39: 2878-2886Crossref PubMed Scopus (28) Google Scholar). Structurally, granulins are distinct from most growth factors, with the exception of the epidermal growth factor/transforming growth factor-α family (13Hrabal R.C.Z. James S. Bennett H.P. Ni F. Nat. Struct. Biol. 1996; : 747-752Crossref PubMed Scopus (138) Google Scholar). In addition to the mammalian granulins of human (1Bateman A. Belcourt D. Bennett H. Lazure C. Solomon S. Biochem. Biophys. Res. Commun. 1990; 173: 1161-1168Crossref PubMed Scopus (175) Google Scholar, 6Sparro G. Galdenzi G. Eleuteri A.M. Angeletti M. Schroeder W. Fioretti E. Protein Expression Purif. 1997; 10: 169-174Crossref PubMed Scopus (29) Google Scholar), rat (2Shoyab M. McDonald V.L. Byles C. Todaro G.J. Plowman G.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7912-7916Crossref PubMed Scopus (154) Google Scholar, 12Bhandari V. Bateman A. Biochem. Biophys. Res. Commun. 1992; 188: 57-63Crossref PubMed Scopus (62) Google Scholar), mouse (10Baba T. Hoff H.B. II I Nemoto H. Lee H. Orth J. Arai Y. Gerton G.L. Mol. Reprod. Dev. 1993; 34: 233-243Crossref PubMed Scopus (122) Google Scholar, 15Baba T. Nemoto H. Watanabe K. Arai Y. Gerton G.L. FEBS Lett. 1993; 322: 89-94Crossref PubMed Scopus (33) Google Scholar), and horse (16Couto M.A. Harwig S.S. Cullor J.S. Hughes J.P. Lehrer R.I. Infect. Immun. 1992; 60: 5042-5047Crossref PubMed Google Scholar), granulin-like proteins have been identified in a number of non-mammalian organisms including the nematode Caenorhabditis elegans (17Berks M. Genome. Res. 1995; 5: 99-104Crossref PubMed Google Scholar), the locust (18Nakakura N. Hietter H. Van Dorsselaer A. Luu B. Eur. J. Biochem. 1992; 204: 147-153Crossref PubMed Scopus (89) Google Scholar), the mussel (19Nara K. Matsue H. Naraoka T. Biochim. Biophys. Acta. 2004; 1675: 147-154Crossref PubMed Scopus (14) Google Scholar), the marine worm Hediste diversicolor (20Deloffre P. Cocquerelle C. Andries J.C. Bull. Soc. Zool. Fr. 1999; : 337-346Google Scholar), and teleosts (bony fish) (21Belcourt D.R. Lazure C. Bennett H.P. J. Biol. Chem. 1993; 268: 9230-9237Abstract Full Text PDF PubMed Google Scholar, 22Belcourt D.R. Okawara Y. Fryer J.N. Bennett H.P. J. Leukocyte Biol. 1995; 57: 94-100Crossref PubMed Scopus (20) Google Scholar, 23Uesaka T. Yano K. Yamasaki M. Ando M. Gen. Comp. Endocrinol. 1995; 99: 298-306Crossref PubMed Scopus (43) Google Scholar). Granulin-like motifs have also been identified in multiple thiol protease gene sequences from plants (24Avrova A.O. Stewart H.E. De Jong W.D. Heilbronn J. Lyon G.D. Birch P.R. Mol. Plant-Microbe Interact. 1999; 12: 1114-1119Crossref PubMed Scopus (75) Google Scholar, 25Tolkatchev D. Xu P. Ni F. J. Pept. Res. 2001; 57: 227-233PubMed Google Scholar). The mammalian progranulin genes are ubiquitously expressed in various tissues (9Plowman G.D. Green J.M. Neubauer M.G. Buckley S.D. McDonald V.L. Todaro G.J. Shoyab M. J. Biol. Chem. 1992; 267: 13073-13078Abstract Full Text PDF PubMed Google Scholar, 10Baba T. Hoff H.B. II I Nemoto H. Lee H. Orth J. Arai Y. Gerton G.L. Mol. Reprod. Dev. 1993; 34: 233-243Crossref PubMed Scopus (122) Google Scholar, 26Bhandari V. Giaid A. Bateman A. Endocrinology. 1993; 133: 2682-2689Crossref PubMed Scopus (69) Google Scholar, 27Bhandari V. Palfree R.G. Bateman A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1715-1719Crossref PubMed Scopus (210) Google Scholar, 28Daniel R. Daniels E. He Z. Bateman A. Dev. Dyn. 2003; 227: 593-599Crossref PubMed Scopus (131) Google Scholar, 29Daniel R. He Z. Carmichael K.P. Halper J. Bateman A. J. Histochem. Cytochem. 2000; 48: 999-1009Crossref PubMed Scopus (274) Google Scholar) and have been detected in epithelial and hematopoietic cell lines (26Bhandari V. Giaid A. Bateman A. Endocrinology. 1993; 133: 2682-2689Crossref PubMed Scopus (69) Google Scholar, 27Bhandari V. Palfree R.G. Bateman A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1715-1719Crossref PubMed Scopus (210) Google Scholar, 28Daniel R. Daniels E. He Z. Bateman A. Dev. Dyn. 2003; 227: 593-599Crossref PubMed Scopus (131) Google Scholar, 29Daniel R. He Z. Carmichael K.P. Halper J. Bateman A. J. Histochem. Cytochem. 2000; 48: 999-1009Crossref PubMed Scopus (274) Google Scholar) and neoplastic cells (30Davidson B. Alejandro E. Florenes V.A. Goderstad J.M. Risberg B. Kristensen G.B. Trope C.G. Kohn E.C. Cancer. 2004; 100: 2139-2147Crossref PubMed Scopus (88) Google Scholar, 31Donald C.D. Laddu A. Chandham P. Lim S.D. Cohen C. Amin M. Gerton G.L. Marshall F.F. Petros J.A. Anticancer Res. 2001; 21: 3739-3742PubMed Google Scholar, 32He Z. Bateman A. Cancer Res. 1999; 59: 3222-3229PubMed Google Scholar, 33Jones M.B. Michener C.M. Blanchette J.O. Kuznetsov V.A. Raffeld M. Serrero G. Emmert-Buck M.R. Petricoin E.F. Krizman D.B. Liotta L.A. Kohn E.C. Clin. Cancer Res. 2003; 9: 44-51PubMed Google Scholar, 34Jones M.B. Spooner M. Kohn E.C. Gynecol. Oncol. 2003; 88: S136-S139Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 35Liau L.M. Lallone R.L. Seitz R.S. Buznikov A. Gregg J.P. Kornblum H.I. Nelson S.F. Bronstein J.M. Cancer Res. 2000; 60: 1353-1360PubMed Google Scholar, 36Wang W. Hayashi J. Kim W.E. Serrero G. Clin Cancer Res. 2003; 9: 2221-2228PubMed Google Scholar). Progranulin was shown to be highly expressed in epithelial cells that exhibit rapid turnover, such as the columnar epithelium of the gastrointestinal tract (29Daniel R. He Z. Carmichael K.P. Halper J. Bateman A. J. Histochem. Cytochem. 2000; 48: 999-1009Crossref PubMed Scopus (274) Google Scholar) and the cells of the immune and nervous systems (21Belcourt D.R. Lazure C. Bennett H.P. J. Biol. Chem. 1993; 268: 9230-9237Abstract Full Text PDF PubMed Google Scholar, 22Belcourt D.R. Okawara Y. Fryer J.N. Bennett H.P. J. Leukocyte Biol. 1995; 57: 94-100Crossref PubMed Scopus (20) Google Scholar, 37Couto M.A. Harwig S.S. Cullor J.S. Hughes J.P. Lehrer R.I. Infect. Immun. 1992; 60: 3065-3071Crossref PubMed Google Scholar). In general, granulin gene sequences that encode for functional peptides are progranulin genes. There are a number of published granulin-like sequences identified in lower vertebrates as well as invertebrates (e.g. zebrafish, GenBank™ accession numbers AF273479 and AF273480). Although a number of granulin genes have been identified in lower vertebrates and invertebrates, many of the peptides encoded by these genes have yet to be functionally characterized. We report on a unique granulin-like gene of the goldfish. Northern blot, real-time PCR, and RT-PCR 3The abbreviations used are: RT, reverse transcription; PKM, primary kidney macrophage; MAF, macrophage-activating factor; Grn, granulin; rgfGrn, recombinant goldfish granulin; IGF-1, insulin-like growth factor 1; Epi, epithelin. analyses revealed that this granulin gene was expressed exclusively in the hematopoietic tissues of the goldfish. Recombinant goldfish granulin induced dose-dependent proliferation of primary goldfish macrophages in vitro, which was abrogated by an affinity-purified anti-granulin IgG. Our findings indicate that granulin was present in macrophage culture supernatants and that it promoted growth of cells at discrete stages of myeloid differentiation pathway. Fish—Goldfish (Carassius auratus) were purchased from Mt. Parnell Fisheries Inc. (Mercersburg, PA) and maintained at the Aquatic Facility of the Department of Biological Sciences, University of Alberta. The fish were kept at 20 °C in a flow-through water system and fed to satiation daily with trout pellets. The fish were acclimated to this environment for at least 3 weeks prior to use in experiments. Isolation of Primary Macrophages from Goldfish and RNA Isolation—Isolation of goldfish kidney leukocytes, and the generation of primary kidney macrophages (PKM) and peripheral blood mononuclear cells were performed as previously described (38Neumann N.F. Barreda D. Belosevic M. Dev. Comp. Immunol. 1998; 22: 417-432Crossref PubMed Scopus (89) Google Scholar, 39Neumann N.F. Barreda D.R. Belosevic M. Fish Shellfish Immunol. 2000; 10: 1-20Crossref PubMed Scopus (112) Google Scholar, 40Neumann N.F. Fagan D. Belosevic M. Dev. Comp. Immunol. 1995; 19: 473-482Crossref PubMed Scopus (162) Google Scholar, 41Neumann N.F. Stafford J.L. Belosevic M. Fish Shellfish Immunol. 2000; 10: 167-186Crossref PubMed Scopus (43) Google Scholar, 42Stafford J.L. Ellestad K.K. Magor K.E. Belosevic M. Magor B.G. Dev. Comp. Immunol. 2003; 27: 685-698Crossref PubMed Scopus (105) Google Scholar). The kinetics of PKM growth in culture were similar to those reported for mammalian macrophages derived from bone marrow cultures in the presence of conditioned medium from the L-929 fibroblast cell line (43Belosevic M. Davis C.E. Meltzer M.S. Nacy C.A. J. Immunol. 1988; 141: 890-896PubMed Google Scholar). Three distinct macrophage subpopulations are a feature of PKM cultures: the early progenitors, the monocytes, and mature macrophages (44Barreda D.R. Belosevic M. Fish Shellfish Immunol. 2001; 11: 169-185Crossref PubMed Scopus (29) Google Scholar, 45Barreda D.R. Neumann N.F. Belosevic M. Dev. Comp. Immunol. 2000; 24: 395-406Crossref PubMed Scopus (34) Google Scholar). PKM cultures were incubated at 20 °C until the cells were at a stage of active proliferation (proliferative phase) or nonproliferation (senescence phase), typically 6 and 10 days post-cultivation, respectively. PKM from the proliferative and senescence phases were isolated, flash-frozen using liquid nitrogen, and stored at –80 °C until used. The mRNA for the two macrophage subpopulations was isolated using TRIzol™ reagent (Invitrogen) and the Oligotex mRNA isolation kit (Qiagen) according to the manufacturers' specifications. Generation of Macrophage-activating Factor (MAF) Supernatants—MAF supernatants were prepared using protocols described previously (41Neumann N.F. Stafford J.L. Belosevic M. Fish Shellfish Immunol. 2000; 10: 167-186Crossref PubMed Scopus (43) Google Scholar). These supernatants contain a complex mixture of factors that have been functionally characterized and shown to induce antimicrobial responses of goldfish macrophages (41Neumann N.F. Stafford J.L. Belosevic M. Fish Shellfish Immunol. 2000; 10: 167-186Crossref PubMed Scopus (43) Google Scholar, 42Stafford J.L. Ellestad K.K. Magor K.E. Belosevic M. Magor B.G. Dev. Comp. Immunol. 2003; 27: 685-698Crossref PubMed Scopus (105) Google Scholar). Construction of cDNA Libraries of Primary Kidney Macrophages of Goldfish—Complementary DNA libraries were constructed from 2 μg of proliferative or senescence phase PKM poly (A)+ RNA by directional ligation of PKM cDNA into λ ZAP bacteriophage using a ZAP cDNA synthesis kit, and the ZAP-cDNA Gigapack III Gold cloning kit (Stratagene) as described previously (46Barreda D.R. Hanington P.C. Walsh C.K. Wong P. Belosevic M. Dev. Comp. Immunol. 2004; 28: 727-746Crossref PubMed Scopus (44) Google Scholar). Non-amplified PKM proliferative and senescence phase cDNA libraries were screened using standard procedures described previously (46Barreda D.R. Hanington P.C. Walsh C.K. Wong P. Belosevic M. Dev. Comp. Immunol. 2004; 28: 727-746Crossref PubMed Scopus (44) Google Scholar). Following the tertiary PCR-based screen, individual clones were PCR-amplified, sequenced, confirmed to encode for a single-sized insert, and stored individually at 4 °C in 500 μl of SM buffer (50 mm Tris-HCl, 100 mm NaCl, 8 mm MgSO4, pH 7.5) and chloroform. DNA Sequencing and Analysis—The PCR-amplified clone inserts corresponding to each of the confirmed granulin positive clones were purified using the QIAquick PCR purification kit (Qiagen) and sequenced using a DYEnamic™ ET terminator cycle sequencing kit (Amersham Biosciences) and a PE-Applied Biosystems 377 automated sequencer. Sequences were analyzed using Genetool™ (Biotools) and subsequent gene annotations were conducted using BLAST programs (www.ncbi.nlm.nih.gov/BLAST/). Conserved motifs were identified, and predictions were based on analytical tools provided in the ExPASy proteomics server (www.expasy.org) (47Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic. Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3534) Google Scholar). Sequence alignments were performed using ClustalX, version 1.83. Real-time PCR Analysis of Granulin Expression—Real-time PCR analysis was carried out using the Applied Biosystems 7500 Fast real-time PCR system. The relative expression of goldfish granulin in relation to β-actin was assessed using primers generated with Primer Express software (Applied Biosystems). The primers used for expression analysis of goldfish granulin were: 5′-TTGATGTTACTCATGGCAGCTCTT-3′ and 5′-GGGCCTGAGAGATCCATCATT-3′. The primers used for expression analysis of goldfish β-actin were: 5′-GCACGCGACTGACACTGAAG-3′ and 5′-GAAGGCCGCTCCGAGGTA-3′. Analysis of the relative tissue expression data from five fish was carried out using the 7500 Fast software (Applied Biosystems). RT-PCR Analysis of Goldfish Granulin Expression in Macrophages—Cultured PKM were sorted into early progenitor, monocyte, and mature macrophage subpopulations using a FACSCalibur flow cytometer (BD Biosciences) as described previously (39Neumann N.F. Barreda D.R. Belosevic M. Fish Shellfish Immunol. 2000; 10: 1-20Crossref PubMed Scopus (112) Google Scholar, 44Barreda D.R. Belosevic M. Fish Shellfish Immunol. 2001; 11: 169-185Crossref PubMed Scopus (29) Google Scholar, 45Barreda D.R. Neumann N.F. Belosevic M. Dev. Comp. Immunol. 2000; 24: 395-406Crossref PubMed Scopus (34) Google Scholar), and the RNA was isolated immediately after sorting. First-strand synthesis was done using an oligo(dT) primer (Stratagene, La Jolla, CA) with 2.5 μg of total RNA according to manufacturer's protocols. The primers used to amplify goldfish granulin by RT-PCR were: sense 5′-AAGATGGTTCCAGTGTTGATGTTAC-3′, antisense 5′-ACCCCACTGGCCGGCTGCTGT-3′. Northern Blot Analysis—Twenty five μg of total RNA was subjected to electrophoresis on a 1.5% agarose, 20% formaldehyde gel and transferred overnight to Genescreen Plus nylon membranes (PerkinElmer Life Sciences). Blots were screened using 200 ng of a goldfish granulin probe created using RT-PCR. The probe was singly labeled using [α-32P]dCTP and purified using QIAquick gel extraction columns (Qiagen). Hybridization with the probe was allowed to proceed overnight at 42 °C, and then unbound probe was removed by three consecutive washes of 2× SSC, 0.1% SDS for 5 min each and three times with 0.1× SSC, 0.1% SDS for 20 min. Blots were then exposed to Kodak X-Omat film and stored at –80 °C for 24 h before being developed. Prokaryotic Expression of Goldfish Granulin—Goldfish granulin was expressed using a prokaryotic protein expression system. PCR amplification of the protein expression construct insert was performed as follows. 7 μl of the granulin clone template was added to 76 μl of double-distilled H2O, dNTPs (0.2 μl each of dATP, dCTP, dGTP, dTTP in 100 mm solutions), 10× PCR buffer (10 μl of 100 mm Tris-HCl, pH 8.3, 500 mm KCl, 15 mm MgCl2, 0.01% (w/v) gelatin), and expression primers (2.4 μl of each 20 μm solution (sense, 5′-CACCCTCATGGCAGCTCTTGTAG-3′; antisense, 5′-ACGGGGGTTGTTTACTTAC-3′) and a 15:1 ratio of Taq:Pfu DNA polymerases (1 μl of 5-unit solution). PCR amplification was conducted in an Eppendorf Mastercycler Gradient™ thermal cycler. Amplification was confirmed by agarose gel electrophoresis. The granulin amplicon was cloned into the pET SUMO TA expression vector (Invitrogen) and transformed into chemically competent TOP10 Escherichia coli (Invitrogen) according to the manufacturer's specifications. Cells were plated onto LB-ampicillin (100 μg/ml) plates and incubated overnight at 37 °C. Randomly selected colonies were amplified by PCR, and positive clones were grown overnight in 5 ml of LB medium containing 100 μg/ml ampicillin. Plasmids were isolated using a QIAprep Spin Miniprep kit (Qiagen). Once positive clones were isolated, restriction digests followed by gel electrophoresis verified the presence of insert and vector DNA. Plasmids were sequenced, as described above, to confirm that inserts were ligated into the expression vector in the proper orientation and in-frame. Sequence data were analyzed using Genetool (Biotools). Production of Recombinant Granulin—Plasmid DNA containing the granulin expression construct was transformed into BL21 Star™(DE3) One Shot® E. coli (Invitrogen) for recombinant protein expression. 10 ng of plasmid DNA was transformed into the bacteria, which was then grown overnight at 37 °C in LB medium containing 50 μg/ml kanamycin. Induction of recombinant protein expression was performed in a pilot expression experiment by the addition of isopropyl-β-d-thiogalactopyranoside according to the manufacturer's protocols. The expression of recombinant granulin was evaluated at 1, 2, 4, and 6 h post-induction with isopropyl-β-d-thiogalactopyranoside. Individual samples were then analyzed by SDS-PAGE and Western blotting for the presence of recombinant protein expression. For large scale expression and purification of the target proteins, 50 ml of LB medium containing 100 μg/ml carbenicillin was grown overnight at 30 °C with shaking to an A600 of ∼1.0 to 2.0. Ten milliliters of this culture was then inoculated into 250 ml of LB (100 μg/ml carbenicillin), and a total of four flasks were prepared (1 liter total). Cultures were incubated until mid-log phase of growth was achieved followed by the induction of target protein expression with 0.1 mm isopropyl-β-d-thiogalactopyranoside. Cultures were then grown for 2 h prior to the purification of the recombinant molecules. Recombinant granulin was engineered to contain a N-terminal His6 tag to facilitate subsequent detection and purification. Bacteria were removed by centrifugation at 2000 × g, and supernatants were collected. Granulin was purified from culture supernatants using MagneHIS beads (Promega) according to the manufacturer's specifications. Purified proteins were eluted in a solution containing 100 mm HEPES and 500 mm imidazole and then were dialyzed overnight against 1× phosphate-buffered saline. Protein samples were then filter-sterilized in preparation for immunodetection and analysis of biological activity. Total protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce) according to the manufacturer's protocols. Immunodetection of Recombinant Goldfish Granulin (rgfGrn)—rgfGrn was used as a source of antigen for rabbit immunizations. The primary immunization was performed by combining an equal volume of purified recombinant granulin (100 μg), with 750 μl of Freund's complete adjuvant. Booster injections were done exactly as the primary immunizations but substituted with Freund's incomplete adjuvant. The IgG fraction was purified by precipitation using saturated ammonium sulfate, solubilization of precipitate in phosphate-buffered saline, and purification using a HiTrap protein A HP column (Amersham Biosciences) according to the manufacturer's protocol. Fractions containing IgG were pooled and filter-sterilized (0.22 μm filter, Millipore). The specificity of the antibody was determined by immunoblot using as a target rgfGrn under native and denaturing conditions. rgfGrn and native goldfish granulin (4× concentrated macrophage culture supernatants) were detected by immunoblot analysis using an anti-His6 monoclonal antibody (Invitrogen) or with affinity-purified rabbit anti-goldfish rgfGrn IgG. Briefly, proteins were separated by SDS-PAGE under reducing conditions using 12.5% polyacrylamide gels, transferred to 0.2-μm nitrocellulose membranes (Bio-Rad), and incubated overnight at 4 °C in the presence of the primary antibody. Membranes were subsequently washed, incubated with a horseradish peroxidase-conjugated monoclonal antibody, and developed using the ECL Advance™ Western blotting detection kit (Amersham Biosciences) according to the manufacturer's specifications. Induction of Proliferation of Macrophages by Recombinant Granulin—PKM cultures were established, and distinct differentiation stages were sorted by fluorescence-activated cell sorting and seeded at a density of 1 × 104 cells well–1 in 96-well culture plates (Falcon). Cells were seeded in 50 μl of complete culture medium and treated with 5, 50, 100, 250, and 500 ng of recombinant goldfish granulin suspended in 50 μl of incomplete cell culture medium and incubated for 52 h at 20 °C. Fifteen μl of bromodeoxyuridine labeling reagent (BrdUrd, Roche Applied Science) per well was added, and cells were incubated for an additional 24 h at 20 °C. The reaction was developed according to the manufacturer's specifications, and optical densities were determined at 450 nm using a microplate spectrophotometer (Biotek). The colorimetric reaction was directly proportional to the number of proliferating PKM in culture (data not shown). The induction of macrophage proliferation by rgfGrn was determined after the addition of different amounts (1, 10, 50, 100, 300, 500 ng) of anti-rgfGrn to cultures. The most common transcript identified in differential cross-screening of proliferative and senescence phase goldfish macrophage cDNA libraries was granulin (Fig. 1). Thirty-one partial granulin-like transcripts were identified, and all exhibited higher expression in proliferating macrophages. All of the transcripts were sequenced and found to be identical. The fully sequenced cDNA transcript of goldfish granulin is 947 nucleotides in length with an open reading frame of 477 nucleotides. The predicted protein is 159 amino acids long and had 18 conserved cysteine residues, 12 of which represent a full granulin cysteine motif common for all known granulin proteins. The remaining 6 cysteine residues make up one-half of this motif (Fig. 2). The granulin sequence has been submitted to GenBank™ (accession number DQ369750).FIGURE 2cDNA sequence of goldfish granulin with the predicted amino acid translation of the open reading frame. The cysteine residues composing the 1.5-granulin cysteine motif are underlined (GenBank™ accession number DQ369750).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The predicted goldfish granulin protein possessed conserved amino acids found in granulins spanning the metazoans. Granulins have been identified in mammals, fish, insects, bivalves, and nematodes. The amino acid sequence alignment of goldfish granulin and other known fish granulins of carp, zebrafish, and goldfish intestine show highly conserved cysteine-rich motifs (Fig. 3B). Goldfish granulin was most similar to carp granulin 3, with an amino acid identity of 56%. Of all the granulins analyzed, goldfish granulin shared the highest identity with other fish granulins (Fig. 3A), a finding that was supported by phylogenetic analysis that grouped the goldfish granulin in close proximity to carp granulins 2 and 3 (Fig. 4). Phylogenetic analysis also suggested that all fish granulins share distinct features that separate them from the granulins of mammals. Although the granulin proteins identified in carp and from goldfish intestine have no corresponding mRNA transcript sequences, zebrafish granulins 1 and 2 and zebrafish hybrid granulin had transcript organization similar to that of goldfish granulin.FIGURE 4Phylogenetic tree of selected granulin peptides. Goldfish granulin groups closely to carp granulins 3 and 2, which are closely associated with the zebrafish granulins 1 and 2 and hybrid as well as with carp granulin-1 and the goldfish granulin identified from intestinal exudates. All of the progranulin peptides from Xenopus, human, mouse, and rat are closely grouped, and all are out-grouped by granulin-like peptides identified in C. elegans. The tree was bootstrapped 10,000 times to ensure accuracy. Abbreviations are the same as for Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The expression of goldfish granulin transcript was analyzed by Northern blot, RT-PCR, and real-time PCR. Analysis of transcript expression in the heart, brain, spleen, kidney, gill, liver, and intestine revealed that goldfish granulin was expressed primarily in the kidney and the spleen (Fig. 5). Real-time PCR and RT-PCR analyses of granulin expression were also done using non-activated and activated macrophages and sorted goldfish macrophage subpopulations. Goldfish granulin expr" @default.
• W2007375089 created "2016-06-24" @default.
• W2007375089 creator A5019288725 @default.
• W2007375089 creator A5028299110 @default.
• W2007375089 creator A5072239752 @default.
• W2007375089 date "2006-04-01" @default.
• W2007375089 modified "2023-10-02" @default.
• W2007375089 title "A Novel Hematopoietic Granulin Induces Proliferation of Goldfish (Carassius auratus L.) Macrophages" @default.
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