Characterization of a Novel Drosophila melanogasterGalectin
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m112105200
ISSN1083-351X
AutoresKaren E. Pace, Tim Lebestky, Thomas Hummel, Pascal Arnoux, Kent Kwan, Linda G. Baum,
Tópico(s)Invertebrate Immune Response Mechanisms
ResumoWe have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding β-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound β-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophilagalectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophilagalectin had a unique and specific tissue distribution.Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system. We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding β-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound β-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophilagalectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophilagalectin had a unique and specific tissue distribution.Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system. Drosophila melanogaster galectin rapid amplification of cDNA ends gene-specific primer expressed sequence tag dithiothreitol enhanced chemiluminescence carbohydrate recognition domain Many biological processes have been elucidated using Drosophila melanogaster as a model system. However, little is known about lectin-ligand interactions in Drosophila. Of the few Drosophila lectins that have been identified, a subset has been shown to be vital in embryogenesis and to function in innate immunity (1.Franc N.C. White K. Microbes Infect. 2000; 2: 243-250Crossref PubMed Scopus (40) Google Scholar, 2.Vilmos P. Kurucz E. Immunol. Lett. 1998; 62: 59-66Crossref PubMed Scopus (199) Google Scholar, 3.Haq S. Kubo T. Shoichiro K. Kobayashi A. Natori S. J. Biol. Chem. 1996; 271: 20213-20218Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 4.Tiemeyer M. Goodman C.S. Development. 1996; 122: 925-936PubMed Google Scholar, 5.Leshko-Lindsay L. Corces V.G. Development. 1997; 124: 169-180Crossref PubMed Google Scholar). In embryonic Drosophila, two lectins, gliolectin and a selectin homologue, have been identified and determined to play a role in embryogenesis (4.Tiemeyer M. Goodman C.S. Development. 1996; 122: 925-936PubMed Google Scholar, 5.Leshko-Lindsay L. Corces V.G. Development. 1997; 124: 169-180Crossref PubMed Google Scholar). Gliolectin mediates cell-cell interactions that may be required for the formation of axonal commissures during nervous system development (4.Tiemeyer M. Goodman C.S. Development. 1996; 122: 925-936PubMed Google Scholar). Mutations in the selectin homologue lead to profound defects in eye and mechanosensory bristle development (5.Leshko-Lindsay L. Corces V.G. Development. 1997; 124: 169-180Crossref PubMed Google Scholar). Stage-specific regulation of the expression of specific glycoconjugates also occurs during Drosophila development, further suggesting important developmental roles for lectins (6.Seppo A. 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The facile genetic analysis that is possible in Drosophila and the apparently small number of putative galectins in the Drosophila genome would simplify the examination of in vivo functions of galectins. We have cloned and characterized the first identified Drosophila galectin (Dmgal),1 and we have examined its distribution during embryogenesis and in the immune system. To obtain a complete cDNA sequence, 3′-rapid amplification of cDNA ends (3′-RACE) was performed. 3′- and 5′-RACE-Ready cDNA was synthesized from larval poly(A)+ RNA (CLONTECH, Palo Alto, CA) using the SMART RACE cDNA Amplification kit according to the manufacturer's instructions (CLONTECH). For 5′- and 3′-RACE, a gene-specific primer (GSP1) was designed against a region of the expressed sequence tag (EST) (LP06039.5prime (15.Cooper D.N.W. Barondes S.H. Glycobiology. 1999; 9: 979-984Crossref PubMed Scopus (284) Google Scholar)) encoding a highly conserved amino acid sequence critical to saccharide binding. Touchdown PCR was performed with GSP1:GAGATGTGGCGCTCCACATTAATCCA according to manufacturer's instructions with the addition of a final cycle at 72 °C for 7 min to create poly(A) tails necessary for TOPO TA cloning. The PCR product was subcloned into the pCR4 TOPO vector (Invitrogen, Carlsbad, CA) and was sequenced by the Davis Sequencing Facility (Davis, CA). To ensure that the entire sequence was obtained, three additional GSPs were designed 174, 255, and 320 bp, respectively, downstream of GSP1. 3′-RACE was performed, the products subcloned into the pCR4 TOPO vector, and sequenced as described above. To generate full-length cDNA, long distance PCR was performed with primers designed from the extreme 5′ and 3′ ends of the cDNA and 5′-RACE-Ready cDNA as a template, using the Advantage cDNA PCR kit according to the manufacturer's instructions (CLONTECH). A final 12-min cycle at 72 °C was added to create poly(A) tails. The entire cDNA sequence was subcloned directly into the pTrcHis2 TOPO expression vector (Invitrogen) and sequenced to ensure orientation and completeness. Dmgal cDNA lacking a stop codon was subcloned into the pTrcHis2 TOPO expression vector to express the recombinant Drosophila protein with a C-terminal Myc-His tag. TOP10 One Shot cells (Invitrogen) were transformed with the expression vector and were induced to express recombinant protein with 1 mmisopropyl-1-thio-β-d-galactopyranoside for 4 h. The cells were harvested by centrifugation at 8000 rpm for 10 min. The cell pellet was lysed with B-PER (Bio-Rad) containing 4 mmdithiothreitol (DTT, Sigma) for 10 min at 4 °C, followed by centrifugation at 10,000 rpm for 20 min. The bacterial supernatant was loaded directly on a β-lactosyl-Sepharose affinity column prepared as described previously (29.Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811Crossref PubMed Google Scholar) or a control fucosyl-Sepharose (Sigma) affinity column. The recombinant protein was not purified over a nickel column prior to carbohydrate affinity chromatography because this type of purification has been shown be ineffective for isolating active galectin-1. 2J. C. LeFebvre and L. G. Baum, unpublished observations. The carbohydrate affinity column was washed extensively with Ca2+,Mg2+-free phosphate-buffered saline, 4 mm DTT, 0.02% sodium azide (wash buffer). Bound proteins were eluted with 0.1 m β-lactose or 0.1 mfucose dissolved in wash buffer. Eluted fractions were separated by 10% SDS-PAGE and transferred to nitrocellulose for Western blotting. Wild type embryonic, third instar larval, and adult Drosophila were Dounce-homogenized in phosphate-buffered saline, 4 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 0.1 mβ-lactose (30.Hirabayashi J. Satoh M. Kasai K. J. Biol. Chem. 1992; 267: 15485-15490Abstract Full Text PDF PubMed Google Scholar). The homogenate was rotated for 30 min at 4 °C, and soluble proteins were collected following centrifugation at 10,000 × g for 10 min at 4 °C. A protein assay (Bio-Rad) was performed, and known quantities of homogenate were separated by 10% SDS-PAGE and transferred to nitrocellulose for Western blotting. To purify native Dmgal, 8.8 mg of protein homogenate from third instar larvae and adult Drosophila was obtained as described above. The protein homogenate was loaded directly onto a β-lactosyl column, washed with wash buffer, and eluted with 0.1 m β-lactose. Eluted proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose for Western blotting. To evaluate the binding of Dmgal to β-lactosyl-Sepharose, the adult protein homogenate prior to purification over the lactose column, the unbound fraction, and the bound fraction were analyzed by 10% SDS-PAGE followed by Coomassie Blue staining, Western blotting, and protein assay (Bio-Rad). Northern blot hybridization was performed with 2.5 μg of poly(A)+ RNA from embryonic, larval, and adult Drosophila (CLONTECH) and Dmgal cDNA as a probe. The Dmgal probe was labeled with [α-32P]dCTP using a random primer labeling kit (Amersham Biosciences). Hybridization was performed in Rapid-Hyb hybridization buffer (Amersham Biosciences) for 1.5 h at 68 °C. Stringency washes were performed at 25, 42, and 68 °C using standard procedures and checked by autoradiography between each stringency wash. Similar results were obtained for each stringency wash. Western blotting was performed using standard immunoblotting techniques followed by enhanced chemiluminescence for protein visualization. Antibody concentrations were as follows: rabbit anti-human galectin-1 polyclonal antiserum (1/1000), horseradish-peroxidase-conjugated anti-His antibody (1/4000; Invitrogen), horseradish peroxidase-conjugated goat anti-rabbit antibody (1/6000; Bio-Rad). Full-length digoxigenin-labeled RNA probes were prepared from the pSPUTK vector (Stratagene, La Jolla, CA) containing full-length Dmgal cDNA. The vector was linearized upstream of the insert with NheI and downstream of the insert with HpaI. Sense and antisense digoxigenin-labeled RNAs were produced using the SP6 and T7 RNA polymerases according to the manufacturer's directions (Roche Molecular Biochemicals). The probes were base-hydrolyzed for 30 min to generate ∼200-base pair fragments. An overnight collection of Drosophila embryos was dechorionated and fixed with 10% paraformaldehyde. In situ hybridization was performed using standard procedures and visualized with alkaline-phosphatase-conjugated anti-digoxigenin antibody (1/2000; Roche Molecular Biochemicals). Smears of circulating hemocytes from third instar hemolymph were formed by cutting larvae with tweezers and spreading the hemolymph on a glass slide. The smears were fixed, permeabilized, and immunostained exactly as described previously (31.Dong X. Tsuda L. Zavitz K.H. Lin M. Zipursky S.L. Genes Dev. 1999; 13: 954-965Crossref PubMed Scopus (76) Google Scholar). Polyclonal rabbit anti-human galectin-1 antibody was used at a concentration of 1/500 and goat anti-rabbit-fluorescein (Jackson ImmunoResearch, West Grove, PA) was used at a concentration of 1/100. Images were collected on a Olympus Flowview confocal microscope with the ×100 objective and analyzed with Fluoview Image Analysis software (version 2.1.39) A partial EST with amino acid sequence similarity to the carbohydrate recognition domain of galectin family members was identified (15.Cooper D.N.W. Barondes S.H. Glycobiology. 1999; 9: 979-984Crossref PubMed Scopus (284) Google Scholar). To isolate the entire cDNA, 5′- and 3′-RACE were performed using gene-specific primers directed against the segments that showed greatest similarity to galectin, and Drosophila larval poly(A)+ RNA as a template. The Dmgal gene is located on chromosome 2L, 21A5 (32.Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. et al.Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4854) Google Scholar). The deduced amino acid sequence encoded by the complete cDNA is shown in Fig. 2. The amino acid sequence contained important elements that are required for a protein to be defined as a galectin family member (33.Barondes S.H. Castronovo V. Cooper D.N.W. Cummings R.D. Hirabayashi J. Hughes C. Kasai K.-I. Leffler H. Liu F.-T. Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1111) Google Scholar). Specifically, Dmgal contained two domains that had sequence similarity to the canonical carbohydrate recognition domains (CRD) of galectin family members (Fig. 1). Both CRDs contained the conserved sequence motifs H-NPR and WG-ER that are important for the binding of galectins to β-galactoside sugars (Fig. 1) (34.Bournes Y. Bolgiano B. Liao D.-I. Strecker G. Cantau P. Herzberg O. Feizi T. Cambillau C. Nat. Struct. Biol. 1994; 1: 863-870Crossref PubMed Scopus (221) Google Scholar), in contrast to the mammalian tandem repeat galectin, galectin-12, in which only one CRD contained these sequence motifs (18.Yang R.-Y. Hsu D.K. Yu L. Ni J. Liu F.-T. J. Biol. Chem. 2001; 276: 20252-20260Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The two CRDs were connected by a unique peptide link that had some sequence similarity to the peptide link of galectin-9. This structural organization classifies Dmgal into the group of tandem repeat-type galectins that contain two CRDs connected by a unique peptide link (35.Hirabayashi J. Kasai K.-I. Glycobiology. 1993; 3: 297-304Crossref PubMed Scopus (469) Google Scholar). Fig. 1 depicts the Dmgal CRD organization that was derived following comparison of Dmgal with known mammalian galectin sequences and structures and by sequence comparison between each CRD. Other galectins that are classified in the tandem repeat family are galectins-4, -6, -8, -9, and -12 and the Caenorhabditis elegans 32-kDa galectin (18.Yang R.-Y. Hsu D.K. Yu L. Ni J. Liu F.-T. J. Biol. Chem. 2001; 276: 20252-20260Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 35.Hirabayashi J. Kasai K.-I. Glycobiology. 1993; 3: 297-304Crossref PubMed Scopus (469) Google Scholar, 36.Oda Y. Herrman J. Gitt M.A. Turck C.W. Burlingame A.L. Barondes S. Leffler H. J. Biol. Chem. 1993; 268: 5929-5939Abstract Full Text PDF PubMed Google Scholar, 37.Hadari Y.R. Paz K. Dekel R. Mestrovic T. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 3447-3453Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 38.Wada K. Kanwar Y.S. J. Biol. Chem. 1997; 272: 6078-6086Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 39.Gitt M.A. Wiser M.F. Leffler H. Herrmann J. Xia Y.R. Massa S.M. Cooper D.N. Lusis A.J. Barondes S.H. J. Biol. Chem. 1995; 270: 5032-5038Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Examination of the CRDs of Dmgal suggested that each domain may differ with respect to the ability to bind sugar ligands. In the N-terminal CRD (CRD I), there was an Arg to Val substitution at amino acid 206. In most galectin family members this Arg stabilizes galectin-carbohydrate interactions (40.Lobsanov Y.D. Gitt M.A. Leffler H. Barondes S.H. Rini J.M. J. Biol. Chem. 1993; 268: 27034-27038Abstract Full Text PDF PubMed Google Scholar). However, in other galectins this Arg is substituted with Lys (galectin-4, Xenopus galectin), Ile (galectin-8), and His (sponge galectin I), and these galectins retain the ability to bind β-galactoside sugars (36.Oda Y. Herrman J. Gitt M.A. Turck C.W. Burlingame A.L. Barondes S. Leffler H. J. Biol. Chem. 1993; 268: 5929-5939Abstract Full Text PDF PubMed Google Scholar, 37.Hadari Y.R. Paz K. Dekel R. Mestrovic T. Accili D. Zick Y. J. Biol. Chem. 1995; 270: 3447-3453Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 41.Marschal P. Herrmann J. Leffler H. Barondes S.H. Cooper D.N.W. J. Biol. Chem. 1992; 267: 12942-12949Abstract Full Text PDF PubMed Google Scholar, 42.Pfeifer K. Haasemann M. Gamulin V. Bretting H. Fahrenholz F. Müller W.E.G. Glycobiology. 1993; 3: 179-184Crossref PubMed Scopus (177) Google Scholar). In CRD II there is a Val to Cys substitution at amino acid 406. This amino acid is also substituted with Gln and Ile in Conger eel Lec1 and C. elegans 32-kDaa galectin, respectively. Interestingly, CRD I had the highest sequence similarity to galectin-4, -5, and -9, and CRD II had the highest sequence similarity to galectin-4 (36.Oda Y. Herrman J. Gitt M.A. Turck C.W. Burlingame A.L. Barondes S. Leffler H. J. Biol. Chem. 1993; 268: 5929-5939Abstract Full Text PDF PubMed Google Scholar, 38.Wada K. Kanwar Y.S. J. Biol. Chem. 1997; 272: 6078-6086Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar,39.Gitt M.A. Wiser M.F. Leffler H. Herrmann J. Xia Y.R. Massa S.M. Cooper D.N. Lusis A.J. Barondes S.H. J. Biol. Chem. 1995; 270: 5032-5038Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar); differences between CRD similarity are also seen in the tandem repeat galectin-12 (18.Yang R.-Y. Hsu D.K. Yu L. Ni J. Liu F.-T. J. Biol. Chem. 2001; 276: 20252-20260Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Characteristic of galectin family members, the amino acid sequence of Dmgal did not contain a classical secretion signal peptide or a transmembrane domain (33.Barondes S.H. Castronovo V. Cooper D.N.W. Cummings R.D. Hirabayashi J. Hughes C. Kasai K.-I. Leffler H. Liu F.-T. Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1111) Google Scholar). However, the sequence did contain a 120-amino acid N-terminal domain that is not found in other galectins and shows no significant sequence identity with any other known protein (Fig. 1). This domain is likely to adopt a secondary helical structure (43.Geourjon C. Deléage G. Protein Eng. 1994; 7: 157-164Crossref PubMed Scopus (310) Google Scholar). Characteristic of galectins, Dmgal did not contain a Ca2+ binding domain. Dmgal showed significant amino acid sequence similarity to galectin family members from various species (identity 23–35%). The alignment of Dmgal with galectins from selected species and the computation of sequence identities and similarity groups were generated using Genedoc (version 2.6.001) and ClustalX (version 1.8) (35.Hirabayashi J. Kasai K.-I. Glycobiology. 1993; 3: 297-304Crossref PubMed Scopus (469) Google Scholar, 36.Oda Y. Herrman J. Gitt M.A. Turck C.W. Burlingame A.L. Barondes S. Leffler H. J. Biol. Chem. 1993; 268: 5929-5939Abstract Full Text PDF PubMed Google Scholar, 44.Rechreche H. Mallo G.V. Montalto G. Dagorn J.C. Iovanna J.L. Eur. J. Biochem. 1997; 248: 225-230Crossref PubMed Scopus (71) Google Scholar, 45.Levi G. Teichberg V.I. J. Biol. Chem. 1981; 256: 5735-5740Abstract Full Text PDF PubMed Google Scholar, 46.Ahmed H. Pohl J. Fink N.E. Strobel F. Vasta G.R. J. Biol. Chem. 1996; 271: 3308-33094Google Scholar) (Fig. 2). Dmgal had a great deal of sequence similarity with human galectin-4 and murine galectin-9, which are also tandem repeat galectins (38.Wada K. Kanwar Y.S. J. Biol. Chem. 1997; 272: 6078-6086Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 44.Rechreche H. Mallo G.V. Montalto G. Dagorn J.C. Iovanna J.L. Eur. J. Biochem. 1997; 248: 225-230Crossref PubMed Scopus (71) Google Scholar). Interestingly, the similarity with these two mammalian tandem repeat galectins was slightly greater than that with the C. elegans tandem repeat galectin, demonstrating the strong conservation across vertebrate and invertebrate species. A BLAST search of the Berkeley Drosophila Genome Project with the putative Dmgal amino acid sequence resulted in seven matches with a smallest sum probability value less than 0.5. One of the sequences was identical to the Dmgal sequence (GenBankTMaccession number AE003590) However, five of the remaining six sequences lacked some of the amino acids considered critical for binding β-galactoside sugars (GenBankTM accession numbersAE003590, AE003799, AE003588, AE003713, and AE003583) (34.Bournes Y. Bolgiano B. Liao D.-I. Strecker G. Cantau P. Herzberg O. Feizi T. Cambillau C. Nat. Struct. Biol. 1994; 1: 863-870Crossref PubMed Scopus (221) Google Scholar). Only one sequence (GenBankTM accession number AE003514) contained amino acids involved in β-galactoside sugar recognition. However, more studies are necessary to determine whether this is a true galectin family member capable of binding β-galactoside sugars. The defining feature of galectin family members is the ability to bind β-galactoside sugars (33.Barondes S.H. Castronovo V. Cooper D.N.W. Cummings R.D. Hirabayashi J. Hughes C. Kasai K.-I. Leffler H. Liu F.-T. Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1111) Google Scholar). We expressed recombinant Dmgal with a C-terminal His tag and asked if the protein would specifically bind to a β-lactose affinity column. The His-tagged Dmgal was not purified over a nickel column prior to carbohydrate affinity chromatography because this type of purification has been shown to be ineffective for isolating active galectin.2 Therefore, the entire bacterial supernatant was loaded directly on a β-lactose or fucose affinity column, and bound protein was eluted with β-lactose or fucose, respectively, and analyzed by immunoblotting with anti-His antibody. A single band of 58 kDa, corresponding to the predicted molecular weight of Dmgal, eluted from the β-lactose column but not the fucose column (Fig. 3A). This demonstrated that Dmgal bound specifically to β-galactoside sugars and confirmed its identity as a galectin family member. Because all galectin family members share structural similarities within their CRDs, we reasoned that a polyclonal rabbit antibody specific for human galectin-1 might cross-react with Dmgal. The anti-His blot was stripped and reprobed with polyclonal rabbit anti-human galectin-1. As shown in Fig. 3A, the anti-human galectin-1 cross-reacted with Dmgal, providing further evidence for strong structural similarities among galectins from different species. Because the anti-human galectin-1 antibody bound Dmgal, we used this antibody as a reagent to determine the stage or stages of Drosophila development where Dmgal was expressed. 5 μg of total protein homogenate from embryonic, third instar larval and adult flies was separated by 10% SDS-PAGE and immunoblotted with anti-human galectin-1 antibody. As shown in Fig. 3B, a prominent 58-kDa band was expressed at all three stages. In addition, the Mr of native Dmgal matched the Mr of the predicted amino acid sequence. This suggests that Dmgal is not glycosylated, consistent with synthesis of galectin family members within the cytosol (47.Harrison F.L. Wilson T.J. J. Cell Sci.
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