OB-BP1/Siglec-6
1999; Elsevier BV; Volume: 274; Issue: 32 Linguagem: Inglês
10.1074/jbc.274.32.22729
ISSN1083-351X
AutoresNeela Patel, Els C.M. Brinkman-Van der Linden, S. Altmann, Kurt Gish, Sriram Balasubramanian, Jackie C. Timans, David M. Peterson, Marcum P. Bell, J. Fernando Bazán, Ajit Varki, Robert A. Kastelein,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoWe report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K d 91 nm), the other two showed weak binding with K d values in the 1–2 μmrange. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acα2–6GalNAcα (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels. We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K d 91 nm), the other two showed weak binding with K d values in the 1–2 μmrange. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acα2–6GalNAcα (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels. Sialic acid-bindingimmunoglobulin superfamily memberlectins (Siglecs), 1The abbreviations used are: Siglec, sialic acid-binding lectin of the immunoglobulin superfamily; Ig, immunoglobulin; SAP, SLAM-associated protein; SLAM, signaling lymphocyte activation molecule; Sn, sialoadhesin; MAG, myelin-associated glycoprotein; Ob-R, leptin receptor; rhOB-F, recombinant FLAG-tagged human leptin; rhOB, recombinant human leptin; rhGM-CSF, recombinant human granulocyte macrophage-colony stimulating factor; rhIL-10, recombinant human interleukin-10; PBL, peripheral blood leukocytes; IGF-I, insulin-like growth factor I; IGF-II, insulin-like growth factor II; IGFBP, insulin-like growth factor-binding protein; PBS, phosphate-buffered saline; SH2, Src homology domain 2; FACS, fluorescence-activated cell sorter a recently designated family of cell surface molecules (1Crocker P.R. Clark E.A. Filbin M. Gordon S. Jones Y. Kehrl J.H. Kelm S. Le Douarin N. Powell L. Roder J. Schnaar R.L. Sgroi D.C. Stamenkovic K. Schauer R. Schachner M. Van den Berg T.K. Van der Merwe P.A. Watt S.M. Varki A. Glycobiology. 1998; 8: vCrossref PubMed Google Scholar), are a subset of the I-type lectins (2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), which in turn belong to the larger immunoglobulin superfamily. Siglecs share conserved cysteine residues which form two characteristic disulfide bonds: an intra-β-sheet bond within the NH2-terminal V-set immunoglobulin (Ig) domain, the other between the V-set domain and the proximal C2-set domain (3Kelm S. Schauer R. Crocker P.R. Glycoconjugate J. 1996; 13: 913-926Crossref PubMed Scopus (108) Google Scholar). Family members each have a single V-set NH2-terminal Ig domain followed by a variable number of C2-set Ig domains, as many as 16 for sialoadhesin (4Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar), or as few as one for CD33 (5Simmons D. Seed B. J. Immunol. 1988; 141: 2797-2800PubMed Google Scholar). While the family members are notable for structural similarities within their extracellular domains, overall primary amino acid sequence identities among them is relatively low (∼30%). In contrast to the majority of immunoglobulin superfamily members which recognize protein ligands, Siglec family members have all been shown to bind to specific sialylated glycans (1Crocker P.R. Clark E.A. Filbin M. Gordon S. Jones Y. Kehrl J.H. Kelm S. Le Douarin N. Powell L. Roder J. Schnaar R.L. Sgroi D.C. Stamenkovic K. Schauer R. Schachner M. Van den Berg T.K. Van der Merwe P.A. Watt S.M. Varki A. Glycobiology. 1998; 8: vCrossref PubMed Google Scholar, 2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 3Kelm S. Schauer R. Crocker P.R. Glycoconjugate J. 1996; 13: 913-926Crossref PubMed Scopus (108) Google Scholar). Three Siglec family members appear to be tightly restricted in expression to specific populations within the hematopoietic lineage: sialoadhesin/Siglec-1 (Sn) to macrophages (6van der Berg T.K. Breve J.J. Damoiseaux J.G. Dopp E.A. Kelm S. Crocker P.R. Dijkstra C.D. Kraal G. J. Exp. Med. 1992; 176: 647-655Crossref PubMed Scopus (139) Google Scholar), CD33/Siglec-3 to cells of the myelomonocytic lineage (7Andrews R.G. Torok-Storb B. Bernstein I.D. Blood. 1983; 62: 124-132Crossref PubMed Google Scholar), and CD22/Siglec-2 to B cells (8Erickson L.D. Tygrett L.T. Bhatia S.K. Grabstein K.H. Waldschmidt T.J. Int. Immunol. 1996; 8: 1121-1129Crossref PubMed Scopus (68) Google Scholar). Likewise myelin-associated glycoprotein (MAG)/Siglec-4 is only expressed on oligodendrocytes in the central nervous system and on Schwann cells in the peripheral nervous system (9Griffiths I.R. Mitchell L.S. McPhilemy K. Morrison S. Kyriakides E. Barrie J.A. J. Neurocytol. 1989; 18: 345-352Crossref PubMed Scopus (86) Google Scholar, 10Higgins G.A. Schmale H. Bloom F.E. Wilson M.C. Milner R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2074-2078Crossref PubMed Scopus (19) Google Scholar). For Sn and MAG, specific cell populations which bear the cognate "ligand" have been identified: Sn preferentially interacts with cells of the granulocytic lineage (11Crocker P.R. Freeman S. Gordon S. Kelm S. J. Clin. Invest. 1995; 95: 635-643Crossref PubMed Google Scholar), and MAG with neuronal processes (12Johnson P.W. Abramow-Newerly W. Seilheimer B. Sadoul R. Tropak M.B. Arquint M. Dunn R.J. Schachner M. Roder J.C. Neuron. 1989; 3: 377-385Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 13Sadoul R. Fahrig T. Bartsch U. Schachner M. J. Neurosci. Res. 1990; 25: 1-13Crossref PubMed Scopus (70) Google Scholar). Additionally, for each Siglec family member, certain sialic acid ligand preferences have been determined (2Powell L.D. Varki A. J. Biol. Chem. 1995; 270: 14243-14246Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 3Kelm S. Schauer R. Crocker P.R. Glycoconjugate J. 1996; 13: 913-926Crossref PubMed Scopus (108) Google Scholar, 14Kelm S. Brossmer R. Isecke R. Gross H.J. Strenge K. Schauer R. Eur. J. Biochem. 1998; 255: 663-672Crossref PubMed Scopus (143) Google Scholar). However, the identities of the individual glycoconjugates which carry the sialic acid determinants remain to be conclusively identified. By virtue of the restricted distribution of Siglecs, these lectins are thought to mediate highly specific cell-cell recognition events. Siglecs are also capable of transducing intracellular signals. For example, ligation of MAG results in activation of the Fyn tyrosine kinase (15Umemori H. Sato S. Yagi T. Aizawa S. Yamamoto T. Nature. 1994; 367: 572-576Crossref PubMed Scopus (351) Google Scholar) and contributes to the initiation and maintenance of myelination by the Schwann cells (16Fruttiger M. Montag D. Schachner M. Martini R. Eur. J. Neurosci. 1995; 7: 511-515Crossref PubMed Scopus (214) Google Scholar). Recent knockout studies of CD22 demonstrate that it is a dual modulator of signal transduction by B lymphocyte antigen receptors, acting as a negative regulator by activation of phosphatases and also a positive regulator by activation of kinases (17Sato S. Miller A.S. Inaoki M. Bock C.B. Jansen P.J. Tang M.L. Tedder T.F. Immunity. 1996; 5: 551-562Abstract Full Text PDF PubMed Scopus (391) Google Scholar). In the course of experiments intended to identify leptin-binding proteins, we cloned a novel Siglec family member, OB-BP1 from the TF-1 human erythroleukemic cell line. We initially undertook experiments to survey hematopoietic cell lines for leptin binding, based on structure prediction and fold recognition algorithms which revealed an unmistakable structural link between leptin and the diverse family of hematopoietic cytokines that have a unique four α-helical bundle fold (18Madej T. Boguski M.S. Bryant S.H. FEBS Lett. 1995; 373: 13-18Crossref PubMed Scopus (241) Google Scholar, 19Rock F.L. Altmann S.W. van Heek M. Kastelein R.A. Bazan J.F. Horm. Metab. Res. 1996; 28: 649-652Crossref PubMed Scopus (54) Google Scholar). We discovered OB-BP1 by FACS-based expression screening of a TF-1 cDNA library for leptin-binding cell surface molecules. Conventional cross-hybridization screening of the TF-1 cDNA library with an OB-BP1 probe led to the identification of a related molecule OB-BP2, which is identical to the recently reported Siglec-5 (20Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar). OB-BP1 and 2 display no similarity to the previously reported leptin receptor (Ob-R) (21Cioffi J.A. Shafer A.W. Zupancic T.J. Smith-Gbur J. Mikhail A. Platika D. Snodgrass H.R. Nat. Med. 1996; 2: 585-589Crossref PubMed Scopus (629) Google Scholar, 22Tartaglia L.A. Dembski M. Weng X. Deng N. Culpepper J. Devos R. Richards G.J. Campfield L.A. Clark F.T. Deeds J. Muir C. Sanker S. Moriarty A. Moore K.J. Smutko J.S. Mays G.G. Woolf E.A. Monroe C.A. Tepper R.I. Cell. 1995; 83: 1263-1271Abstract Full Text PDF PubMed Scopus (3231) Google Scholar). The expression of each is restricted to a limited set of tissue and cell types. We further assessed putative interactions between these molecules and leptin by Biacore studies and found that only OB-BP1 bound with relatively high affinity. We also show a specific interaction of OB-BP1 with a sialic acid containing ligand, allowing its designation as Siglec-6. Unless otherwise indicated all cell lines were obtained from ATCC. HEL, HL60, Daudi, human EBV-transformed lymphoblastoid B cells (gift from Dr. Peter Parham, Stanford University), Jurkat, MOLT-4, THP-1, U937, a mouse pro-B line, Ba/F3 (23Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Abstract Full Text PDF PubMed Scopus (584) Google Scholar), and a human erythroleukemic line, TF-1 (24Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (715) Google Scholar) were maintained in RPMI 1640/10% fetal bovine serum with specific growth factor supplements for Ba/F3, 10 ng/ml mIL-3, and for TF-1, 10 ng/ml hGM-CSF. BOSC23 cells were maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum supplemented with GPT selection reagent (Specialty Media). Two days prior to use for transfection, BOSC23 cells were passaged into media lacking GPT. Recombinant FLAG-tagged human leptin (rhOB-F) was expressed in Escherichia coli and purified (25Altmann S.W. Timans J.C. Rock F.L. Bazan J.F. Kastelein R.A. Protein Expression Purif. 1995; 6: 722-726Crossref PubMed Scopus (22) Google Scholar). For analysis and sorting, cells were washed in FACS buffer (Hanks' buffered salt solution, 1 mmCaCl2, 3% fetal bovine serum, 0.05% sodium azide), then incubated at 1 × 106 cells/100 μl in the presence of 10 μg/ml rhOB-F (approximately 0.63 μm) in buffer for 1 h on ice. Following a buffer wash, cells were incubated at 1 × 106 cells/100 μl in 5 μg/ml biotinylated anti-FLAG M1 antibody in buffer for 15 min on ice. Anti-FLAG M1 antibody was purchased from Kodak/IBI. After a single buffer wash, cells were incubated at 1 × 106 cells/100 μl in a 1:25 dilution of streptavidin-phycoerythrin (Becton Dickinson). Cells were washed once, resuspended in HBBS, 1 mmCaCl2, 0.05% sodium azide, 1 μg/ml propidium iodide, and sorted (Becton Dickinson FacSort) or analyzed by flow cytometry (Becton Dickinson FacScan). A TF-1 cDNA library was constructed with oligo(dT) priming and cloned into theEcoRI/NotI sites of the retroviral vector pBabe-X-puro, in which the multiple cloning site from pBabe-X was substituted for that of pBabe-puro (26Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (224) Google Scholar). The library was packaged in BOSC23 cells and then introduced into Ba/F3 cells by retroviral infection (26Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (224) Google Scholar). For expression cloning, three rounds of sorting were necessary to isolate a homogeneous population positive for rhOB-F staining. Genomic DNA was extracted from the third sort of the Ba/F3 cells using the Easy DNA kit (Invitrogen). Polymerase chain reaction was performed with Vent polymerase (New England Biolabs) using nested primers designed from the retroviral vector (26Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (224) Google Scholar). Another related molecule was cloned from the TF-1 library in pBabe-X-puro by colony hybridization using the OB-BP1 cDNA as a probe. After the initial submission of these sequences to GenBank other reports indicated that OB-BP1 (GenBank accession number U71382) is identical to CD33L (GenBank accession number D86358) (27Takei Y. Sasaki S. Fujiwara T. Takahashi E. Muto T. Nakamura Y. Cytogenet. Cell Genet. 1997; 78: 295-300Crossref PubMed Scopus (38) Google Scholar) and OB-BP2 (GenBank accession numberU71383) is identical to Siglec-5 (20Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar). For surface plasmon resonance studies, soluble forms containing the extracellular domains of OB-BP1, OB-BP2, and CD33 (OB-BP1ec, OB-BP2ec, and CD33ec) were expressed as fusions to a His6 and FLAG epitope tag in Sf9 insect cells. Gene constructs were made by amplifying the region of interest by polymerase chain reaction and were subcloned into the EcoRI and BglII sites of the pHF-His-FLAG vector (28Koch-Nolte F. Petersen D. Balasubramanian S. Haag F. Kahlke D. Willer T. Kastelein R. Bazan F. Thiele H.G. J. Biol. Chem. 1996; 271: 7686-7693Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The extracellular domain of CD33 was cloned by polymerase chain reaction from a monocyte cDNA library. All constructs were verified by sequencing of the entire inserted DNA fragment. Baculoviral supernatants were assayed by Western blot analysis with anti-FLAG M2 antibody (Kodak/IBI) to confirm the size of the FLAG-tagged fusion protein. In experiments on the BIAcore, baculoviral supernatants were flowed directly across an anti-FLAG M2 antibody-conjugated chip to bind the FLAG-tagged protein to the chip without further purification, as attempts to purify OB-BP1ec by anti-FLAG M2 affinity chromatography inactivated the binding activity. All affinity and kinetic measurements were carried out on a BIAcore instrument (Pharmacia Biosensor) using surface plasmon resonance. Experiments were performed at 25 °C in HBS buffer (10 mm Hepes pH 7.4, 120 mm NaCl, 3 mm EDTA) with 0.05% P-20 surfactant to minimize nonspecific interactions. Anti-FLAG M2 antibody was immobilized at pH 5 on a CM-5 sensor chip (Biosensor). Baculoviral supernatants containing the FLAG-tagged binding proteins (OB-BP1ec, OB-BP2ec, and CD33ec) were then injected into the cell until approximately 1000 resonance units of protein were bound. The FLAG-tagged proteins dissociate slowly from the M2 surface (K d ∼ 10−4-10−5 s−1). The dissociation was continued for 1 h to obtain a relatively stable baseline for subsequent binding experiments. Various putative ligands such as recombinant human leptin (rhOB) (29Fawzi A.B. Zhang H. van Heek M. Graziano M.P. Horm. Metab. Res. 1996; 28: 694-697Crossref PubMed Scopus (19) Google Scholar), recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), or recombinant human interleukin-10 (rhIL-10) were then injected at concentrations ranging from 0.1 to 10 μm. Due to protein concentration and injection time limitations, true equilibrium was not attained. Thus the equilibrium dissociation constant (K d) was measured kinetically from the ratio of the dissociation and association rate constants (k off/k on). The rate constants were obtained under pseudo-first order rate conditions by fitting the dissociation and association phases to single exponentials using the BIAevaluation 2.1 program (Biosensor). Since the fitting errors were quite low, the experiments were repeated at least three times and the run-to-run variation was used as a measure of the experimental errors. The standard deviation from these runs was typically about 20%. The error in the K d was then calculated to be about 25%. Between runs, the M2 antibody surface was regenerated with a 2-min injection of 10 mm HCl. Multiple tissue RNA blots of human poly(A)+ RNA (CLONTECH) were hybridized to probes specific for either OB-BP1 (Siglec-6) or OB-BP2/Siglec-5. Hybridization was performed in ExpressHyb (CLONTECH) at 68 °C overnight. The final wash was performed in 0.1 × SSC, 0.1% SDS at 65 °C for 30 min. Signals on the blots were detected by a PhosphorImager analysis (Storm 860, Molecular Dynamics). A specific monoclonal antibody (10F10) directed against OB-BP1 was raised by conventional procedures (data not shown). The antibody reacts with the extracellular domain of the molecule, and does not cross-react with OB-BP2/Siglec-5 or CD33/Siglec-3 (data not shown). The endogenous peroxidase activities of freshly cut brain and placenta sections were blocked with 0.03% H2O2 in PBS for 20 min at room temperature. Each subsequent step was preceded by washing 3 × with PBS. The sections were blocked with 10% goat serum, 1% bovine serum albumin in PBS and stained with 10F10 supernatant (1:1 in blocking buffer) for 1 h at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad; 1:50 in blocking-buffer with 5% normal human serum) for 30 min at room temperature and subsequent development with 3-amino-9-ethylcarbazole (Vector). Various cell lines (HEL, HL60, Daudi, human EBV-transformed lymphoblastoid B cells, Jurkat, MOLT-4, THP-1, and U937) were analyzed for the expression of OB-BP1. Cells (1 × 106) were incubated with 10F10 supernatant (1:1 in PBS, 1% bovine serum albumin) for 1 h at 4 °C, followed by incubation with 100 × diluted fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Pierce) for analysis by flow cytometry (Beckton-Dickinson FacScan). Peripheral blood mononuclear cells and neutrophils were isolated from human blood obtained from healthy volunteers using Mono-Poly Resolving Medium Ficoll-Hypaque (ICN) and analyzed for expression of OB-BP1. To analyze which subsets of peripheral blood mononuclear cells express OB-BP1 the staining with the 10F10 supernatant was followed by staining with the following subset-specific antibodies: tri-color-conjugated anti-human CD19 and CD14 (CalTag), cytochrome-conjugated anti-human CD4 and CD56, and phycoerythrin-conjugated CD8 and CD3 (Pharmingen). Cells were blocked with PBS, 1% bovine serum albumin, 5% mouse serum for 30 min before incubation with these antibodies. P3 × 63Ag8 (mouse-IgG1 secreting myeloma; ATCC) supernatant was used as isotype control for the 10F10 supernatant. The extracellular domains of OB-BP1 were cloned into a FLAG-human Fc expression vector (pEDdc). COS-7 cells were transiently transfected at 60–70% confluency using LipofectAMINE Reagent (Life Technologies), in serum-free OptiMEM medium. After 5 h the medium was diluted 2 times with 10% fetal calf serum containing OptiMEM medium and the next day the medium was changed for OptiMEM with 2% fetal calf serum. The COS-7 cell supernatants were collected 5–7 days after transfection. The fusion protein (OB-BP1/FLAG/human IgG-Fc) was purified on Protein A-Sepharose. Microtiter wells (Nunc) were coated overnight at 4 °C with Protein A (200 ng/well) in 50 mmcarbonate/bicarbonate buffer, pH 9.5. Wells were blocked with enzyme-linked immunosorbent assay buffer (20 mm HEPES, 1% bovine serum albumin, 125 mm NaCl, 1 mm EDTA, pH 7.45) for 1 h and incubated with OB-BP1-Fc (500 ng/well) for 2 h. Between incubations (all at room temperature) wells were washed 3 times with enzyme-linked immunosorbent assay buffer. Biotin-conjugated polyacrylamide substituted with Neu5Acα2–6Galβ1–4Glc, Neu5Acα2–3Galβ1–4Glc, Neu5Acα2–6GalNAcα (sialyl-Tn), and GalNAcα (Tn) (Glycotech) were added for 2 h at various concentrations ranging from 100 ng to 2 μg/well, followed by incubation with alkaline phosphatase-conjugated streptavidin (Life Technologies; 1:1000) for 1 h and development with 100 μl/well of p-nitrophenyl phosphate Liquid Substrate System (Sigma). Plates were read-out at 405 nm. We employed a FACS-based expression cloning strategy to identify molecules which bound rhOB-F (recombinant FLAG-tagged human leptin). Based on the helical fold of leptin, the NH2-terminal FLAG-tag octapeptide should not interfere with the native folding of the molecule. Initially, a panel of hematopoietic cell lines was tested for binding to rhOB-F. Cells were incubated with purified rhOB-F, then with biotinylated anti-FLAG M1 antibody, and finally with a streptavidin-phycoerythrin conjugate for detection. Biotinylation of the second step reagent allowed for signal amplification, thus increasing the sensitivity of our assay. The human erythroleukemic cell line TF-1 exhibited strong staining (Fig.1 A), whereas the mouse pro-B cell line Ba/F3 did not (Fig. 1 B). The strong staining of TF-1 cells could be competed with non-FLAG-tagged rhOB (recombinant human leptin) but not with other 4 α-helix bundle cytokines such as rhGM-CSF and rhIL-10 (data not shown). We therefore constructed a TF-1 cDNA library in the retroviral vector pBabe-X-puro (26Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (224) Google Scholar), introduced the library into Ba/F3 cells by infection, and performed three successive rounds of sorting to select for Ba/F3 cells which displayed rhOB-F binding (Fig. 1 C). The integrated TF-1 cDNA was rescued from genomic DNA of the third sort population by nested polymerase chain reaction with retroviral vector primers. The identity of the candidate cDNA was verified by re-introducing this clone (clone 1–2) into uninfected Ba/F3 cells, followed by FACS analysis to detect rhOB-F binding. The clone conferred a rhOB-F binding phenotype on the Ba/F3 cells (Fig. 1 D), indicating that we had cloned a leptin-binding molecule, hereafter referred to as OB-BP1. The original TF-1 cDNA library was then screened by colony hybridization with a full-length OB-BP1 cDNA probe. From this screen we identified a clone with an open reading frame encoding a protein similar but not identical to that encoded by OB-BP1. The full-length cDNA for this molecule was introduced into Ba/F3 cells, and when assessed by FACS analysis also bound rhOB-F, albeit with a significantly lower staining intensity than observed on OB-BP1-infected Ba/F3 cells (data not shown). The cDNA for OB-BP1 (1711 nucleotides) was sequenced and found to encode a type I membrane protein. An open reading frame of 441 was deduced for OB-BP1 and displayed 59% sequence identity to OB-BP2. In Fig. 2, approximate domain boundaries are indicated for signal sequences, extracellular domains, as well as transmembrane and intracellular regions. For the surface plasmon resonance experiments described below, we expressed soluble forms of each molecule in insect cells which consisted of the entire 315-amino acid extracellular domain for OB-BP1ec and of a comparable region consisting of Ig domains 1–3 (332 amino acid residues) for OB-BP2ec. A BLAST search of the nonredundant GenBank data base using the nucleotide sequences of either OB-BP1 or OB-BP2 revealed a striking similarity of both OB-BP1 and OB-BP2 to CD33 (5Simmons D. Seed B. J. Immunol. 1988; 141: 2797-2800PubMed Google Scholar), a human leukocyte antigen of undetermined function which is expressed exclusively on cells of myelomonocytic lineage (7Andrews R.G. Torok-Storb B. Bernstein I.D. Blood. 1983; 62: 124-132Crossref PubMed Google Scholar, 30Pierelli L. Teofili L. Menichella G. Rumi C. Paoloni A. Iovino S. Puggioni P.L. Leone G. Bizzi B. Br. J. Haematol. 1993; 84: 24-30Crossref PubMed Scopus (44) Google Scholar). Following submission of our data to GenBank on 19 September 1996 (GenBank accession numbers U71382and U71383), identical sequences appeared entitled CD33L identical to OB-BP1 (GenBank accession D86358) (27Takei Y. Sasaki S. Fujiwara T. Takahashi E. Muto T. Nakamura Y. Cytogenet. Cell Genet. 1997; 78: 295-300Crossref PubMed Scopus (38) Google Scholar) and Siglec-5 (identical to OB-BP2) (20Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar). The former was picked up by a group performing random cloning of cDNAs capable of protein expression in vitro(27Takei Y. Sasaki S. Fujiwara T. Takahashi E. Muto T. Nakamura Y. Cytogenet. Cell Genet. 1997; 78: 295-300Crossref PubMed Scopus (38) Google Scholar) and the latter by a group using a commercial EST data base to specifically search for new Siglec family members (20Cornish A.L. Freeman S. Forbes G. Ni J. Zhang M. Cepeda M. Gentz R. Augustus M. Carter K.C. Crocker P.R. Blood. 1998; 92: 2123-2132Crossref PubMed Google Scholar). Data base searching with the amino acid sequences also detected similarities of the OB-BPs to other Siglec family members including MAG (31Sato S. Fujita N. Kurihara T. Kuwano R. Sakimura K. Takahashi Y. Miyatake T. Biochem. Biophys. Res. Commun. 1989; 163: 1473-1480Crossref PubMed Scopus (45) Google Scholar), Sn (4Crocker P.R. Mucklow S. Bouckson V. McWilliam A. Willis A.C. Gordon S. Milon G. Kelm S. Bradfield P. EMBO J. 1994; 13: 4490-4503Crossref PubMed Scopus (225) Google Scholar), and CD22 (32Wilson G.L. Fox C.H. Fauci A.S. Kehrl J.H. J. Exp. Med. 1991; 173: 137-146Crossref PubMed Scopus (127) Google Scholar). At the primary sequence level, however, OB-BP1 and OB-BP2/Siglec-5 most closely resemble CD33 (overall sequence identity 63 and 56%, respectively) (see Fig. 2). The extracellular region of OB-BP1 is composed of Ig domains typical of Siglec family members (33Williams A.F. Davis S.J. He Q. Barclay A.N. Cold Spring Harbor Symp. Quant. Biol. 1989; LIV: 637-647Crossref Google Scholar): an NH2-terminal V-set type followed by multiple C2-set Ig domains. OB-BP1 contains a total of 3 Ig domains while OB-BP2/Siglec-5 contains a total of 4 Ig domains. As with the other family members, the V-set domain of OB-BP1 and OB-BP2/Siglec-5 contains a pattern of conserved cysteines predicted
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