Identification of a Novel Family of Cell-surface Proteins Expressed in Human Vascular Endothelium
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m207410200
ISSN1083-351X
AutoresRuey‐Bing Yang, Chi Kin Domingos Ng, Scott M. Wasserman, Steven D. Colman, Suresh Shenoy, Fuad Mehraban, László G. Kömüves, James Tomlinson, James N. Topper,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoVascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1β and tumor necrosis factor-α treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis. Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1β and tumor necrosis factor-α treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis. Vascular endothelium (EC), 1The abbreviations used for: EC, endothelial cells; EGF, epidermal growth factor; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECM, extracellular matrix proteins; PDGF, platelet-derived growth factor; FL, full length; TGF-β, transforming growth factor-β; LTBP, latent TGF-β-binding protein. 1The abbreviations used for: EC, endothelial cells; EGF, epidermal growth factor; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ECM, extracellular matrix proteins; PDGF, platelet-derived growth factor; FL, full length; TGF-β, transforming growth factor-β; LTBP, latent TGF-β-binding protein. the single layer of cells located at the interface between tissue and blood, plays an essential role in the maintenance of normal vascular physiology. Dysfunction of this cell type can lead to vascular diseases such as hypertension and atherosclerosis (1Cines D.B. Pollak E.S. Buck C.A. Loscalzo J. Zimmerman G.A. McEver R.P. Pober J.S. Wick T.M. Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar). The functional phenotype of EC is dynamically responsive to a variety of physiologic and pathophysiologic stimuli that include proinflammatory cytokines, growth factors, bacterial products, as well as biomechanical forces (2Topper J.N. Gimbrone Jr., M.A. Mol. Med. Today. 1999; 5: 40-46Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 3Topper J.N. Cai J. Falb D. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10417-10422Crossref PubMed Scopus (719) Google Scholar). Many of these functions are mediated by proteins selectively expressed on the surface of ECs. For example, tissue factor expressed on the surface of EC in response to activation serves as a cofactor for factor VIIa to activate factor X and factor IX in the coagulation cascade (4Rapaport S.I. Rao L.V. Arterioscler. Thromb. 1992; 12: 1111-1121Crossref PubMed Scopus (271) Google Scholar). Conversely, the anticoagulant protein thrombomodulin is an EC surface molecule that binds thrombin, thereby activating protein C that in the presence of protein S degrades factor Va and VIIIa (5Thompson E.A. Salem H.H. J. Clin. Invest. 1986; 78: 13-17Crossref PubMed Scopus (21) Google Scholar, 6Esmon C.T. FASEB J. 1995; 9: 946-955Crossref PubMed Scopus (355) Google Scholar). E-selectin is an EC-selective adhesion molecule that is rapidly induced on inflamed EC and plays a critical role in leukocyte recruitment (7Rosen S.D. Bertozzi C.R. Curr. Opin. Cell Biol. 1994; 6: 663-673Crossref PubMed Scopus (442) Google Scholar). In addition, organ-selective EC surface molecules have been functionally identified in several tissues, and an endothelial marker responsible for tumor homing to the lungs has been identified (8Johnson R.C. Zhu D. Augustin-Voss H.G. Pauli B.U. J. Cell Biol. 1993; 121: 1423-1432Crossref PubMed Scopus (88) Google Scholar, 9Arap W. Kolonin M.G. Trepel M. Lahdenranta J. Cardo-Vila M. Giordano R.J. Mintz P.J. Ardelt P.U. Yao V.J. Vidal C.I. Chen L. Flamm A. Valtanen H. Weavind L.M. Hicks M.E. Pollock R.E. Botz G.H. Bucana C.D. Koivunen E. Cahill D. Troncoso P. Baggerly K.A. Pentz R.D. Do K.A. Logothetis C.J. Pasqualini R. Nat. Med. 2002; 8: 121-127Crossref PubMed Scopus (493) Google Scholar, 10Pasqualini R. Ruoslahti E. Nature. 1996; 380: 364-366Crossref PubMed Scopus (1014) Google Scholar, 11Rajotte D. Arap W. Hagedorn M. Koivunen E. Pasqualini R. Ruoslahti E. J. Clin. Invest. 1998; 102: 430-437Crossref PubMed Scopus (419) Google Scholar). To begin to understand the repertoire of human EC surface molecules, we have combined comprehensive library sequencing with transcriptional profiling to identify EC-selective genes (12Yang R.B. Tomlinson J.E. Conley P.B. Hart M.J. Komuves L.G. Ziman M. Lum P.Y. Roberts C. Topper J.N. Physiologist. 2002; 45: 76Google Scholar). Approximately 100,000 cDNA fragments derived from EC were sequenced, and these sequences were then merged with public data bases to obtain ∼10,000 independent gene assemblies putatively expressed in EC. To validate their endothelial expression, these genes were then represented on customized oligonucleotide microarrays (13Hughes T.R. Mao M. Jones A.R. Burchard J. Marton M.J. Shannon K.W. Lefkowitz S.M. Ziman M. Schelter J.M. Meyer M.R. Kobayashi S. Davis C. Dai H. He Y.D. Stephaniants S.B. Cavet G. Walker W.L. West A. Coffey E. Shoemaker D.D. Stoughton R. Blanchard A.P. Friend S.H. Linsley P.S. Nat. Biotechnol. 2001; 19: 342-347Crossref PubMed Scopus (1039) Google Scholar), together with all of the non-redundant human genes from public data bases (12Yang R.B. Tomlinson J.E. Conley P.B. Hart M.J. Komuves L.G. Ziman M. Lum P.Y. Roberts C. Topper J.N. Physiologist. 2002; 45: 76Google Scholar). Competitive hybridizations were performed utilizing both endothelial and non-endothelial cell types, and these analyses revealed ∼400 genes that were uniquely expressed in EC. These included the majority of genes known to be expressed in an EC-selective pattern, such as angiotensin-converting enzyme, acetylated low density lipoprotein receptor, E-selectin, Tie-2, VEGFR2 (KDR), von Willebrand factor, NOS3, CD31, endothelin, VE-cadherin, EphB4, and ephrin-B2, and many uncharacterized genes. One full-length cDNA identified by these approaches encoded a potential secreted protein harboring a signal peptide at the amino terminus followed by 10 EGF-like repeats and 1 CUB domain at the carboxyl terminus (termed SCUBE1 for Signal peptide-CUB- EGF-like domain containing protein1). Interestingly, when overexpressed, SCUBE1 protein is not only secreted but is also tethered on the cell surface. Likewise, a second human gene encoding a homologue (designated SCUBE2) was also identified and appears to be expressed in EC and displayed onto cell surface in overexpressing 293T cells. SCUBE1 and SCUBE2, when singly or coexpressed, can manifest homo- and heterotypic interactions. Furthermore, SCUBE1 and SCUBE2 expression is down-regulated in EC after IL-1β and TNF-α treatment in vitro and after LPS injection in vivo, suggesting a possible role of the SCUBE gene family in the inflammatory response. Previous work has described the apparent mouse homologues of Scube1 andScube2 (14Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (65) Google Scholar, 15Grimmond S. Larder R. Van Hateren N. Siggers P. Morse S. Hacker T. Arkell R. Greenfield A. Mech. Dev. 2001; 102: 209-211Crossref PubMed Scopus (41) Google Scholar). Based solely on their expression in a variety of embryonic tissues, it was proposed that the Scubegene family may play roles in development; however, no adult expression data were reported. Our results indicate that SCUBE1 and SCUBE2 define an emerging secreted and cell-surface protein family that is expressed in human vascular endothelium. The details of tissue preparation andin situ hybridization have been described earlier (16Komuves L.G. Hanley K. Lefebvre A.M. Man M.Q. Ng D.C. Bikle D.D. Williams M.L. Elias P.M. Auwerx J. Feingold K.R. J. Invest. Dermatol. 2000; 115: 353-360Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Komuves L.G. Feren A. Jones A.L. Fodor E. J. Histochem. Cytochem. 2000; 48: 821-830Crossref PubMed Scopus (65) Google Scholar, 18Stelnicki E.J. Komuves L.G. Kwong A.O. Holmes D. Klein P. Rozenfeld S. Lawrence H.J. Adzick N.S. Harrison M. Largman C. J. Invest. Dermatol. 1998; 110: 110-115Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Following the manufacturer's protocol, digoxigenin-labeled antisense and sense riboprobes were synthesized from DNA templates (nucleotides 2241–2529) using reagents supplied by Roche Molecular Biochemicals. Sectioning, pretreatment of the sections, and hybridization of the probes were done under strict RNase-free conditions. All reagents were prepared using diethyl pyrocarbonate-treated distilled water. 15-μm thick sections were collected on positively charged slides and dried at 55 °C overnight. The sections were deparaffinized and rehydrated in Histosolve and ethanol and rinsed in diethyl pyrocarbonate-treated distilled water. The sections were treated at room temperature with 0.2n HCl (20 min), 1.5% H2O2 (15 min), 0.3% Triton X-100 (15 min) followed by proteinase K treatment at 37 °C (30 min). The sections then were washed with triethanolamine buffer followed by acetylation with acetic anhydride. Following prehybridization in 2× SSC, containing 50% formamide at 37 °C (1 h), the sections were air-dried at room temperature. Sections were hybridized with the probes diluted in hybridization solution (2× SSC, containing 50% formamide, 10× Denhardt's, 0.001% SDS, 10 mm Tris, pH 7.4, 0.005% sodium pyrophosphate, and 500 μg/ml yeast tRNA) at 55 °C overnight. Following hybridization, the sections were washed with 4× SSC (twice for 15 min) and 2× SSC (twice for 15 min). The sections then were treated with RNase A at 37 °C (30 min) followed by stringency washes in 2× SSC at 37 °C (15 min), 0.1× SSC at 42 °C (40 min), and finally in 0.1× SSC at room temperature (twice for 15 min). The sections were washed with maleate buffer (30 min) and then blocked with 10 mm Tris buffer, pH 7.6, containing 500 mm NaCl, 4% bovine serum albumin, 0.5% cold-water fish skin gelatin, and 0.05% Tween 20. The sections were then incubated with anti-digoxigenin antibody, conjugated to peroxidase (Roche Molecular Biochemicals), for 1 h. The signal was amplified using TSA-Plus kit (PerkinElmer Life Sciences), and the signal was detected with Vector Blue substrate (Vector Laboratories, Burlingame, CA). Following incubation with substrate the sections were dehydrated in ethanol and Histosolve and coverslipped. Hybridization with the sense control probe did not result in detectable signal, indicating the specificity of hybridization. Slides were observed with an Olympus BX50 microscope (Olympus, Inc., Melville, NY), using DIC illumination. The microscope was equipped with a Nikon DXM1200 digital camera (Technical Instruments, San Francisco, Burlingame, CA). Digitized images (1280 × 1024-pixel resolution) were acquired using ACT-1 software (Nikon, Melville, NY). Images were resized, cropped, and assembled using Photoshop version 6.0 (Adobe Systems, San Jose, CA). Apart from equalizing the background intensities, no other digital modifications of the original digital images were carried out. To capture the diverse repertoire of genes expressed in EC, multiple libraries from EC under various stimuli (such as shear-stressed, proinflammatory cytokine-treated) were generated and normalized using methods described previously (19Shimkets R.A. Lowe D.G. Tai J.T. Sehl P. Jin H. Yang R. Predki P.F. Rothberg B.E. Murtha M.T. Roth M.E. Shenoy S.G. Windemuth A. Simpson J.W. Simons J.F. Daley M.P. Gold S.A. McKenna M.P. Hillan K. Went G.T. Rothberg J.M. Nat. Biotechnol. 1999; 17: 798-803Crossref PubMed Scopus (152) Google Scholar). Approximately 100,000 cDNA fragments were sequenced, and these sequences were then merged with public data bases to obtain ∼10,000 EC gene independent assemblies. To validate their endothelial expression, these genes were then represented on custom oligonucleotide microarrays (13Hughes T.R. Mao M. Jones A.R. Burchard J. Marton M.J. Shannon K.W. Lefkowitz S.M. Ziman M. Schelter J.M. Meyer M.R. Kobayashi S. Davis C. Dai H. He Y.D. Stephaniants S.B. Cavet G. Walker W.L. West A. Coffey E. Shoemaker D.D. Stoughton R. Blanchard A.P. Friend S.H. Linsley P.S. Nat. Biotechnol. 2001; 19: 342-347Crossref PubMed Scopus (1039) Google Scholar), together with all of non-overlapping human genes from public data bases (12Yang R.B. Tomlinson J.E. Conley P.B. Hart M.J. Komuves L.G. Ziman M. Lum P.Y. Roberts C. Topper J.N. Physiologist. 2002; 45: 76Google Scholar). Competitive hybridizations were performed among both endothelial and non-endothelial cells. Clustering analyses revealed ∼400 genes that were uniquely expressed in EC. One cDNA fragment encoding multiple copies of EGF-like domains was identified and subsequently mapped onto human chromosome 22q13. Based on gene prediction, two oligonucleotides (5′-CAG CGG GGC CCG CAT TGA GCA TGG GCG CGG-3′ and 5′-CCC GGT TAT TTG TAG GGC CGC AGG AAC CGA-3′) were used to amplify the entire open reading frame by PCR from a mixture of human cDNA libraries. The amplified SCUBE1 full-length cDNA was cloned into pCR2.1 (Invitrogen) and confirmed by sequencing. The clone containing full-length SCUBE2 was obtained from OriGene Technologies (Rockville, MD). Full-length sequences for SCUBE1 were deposited into GenBankTM with accession number AF525689, and the SCUBE2 sequence is the same as NM_020974 (except nucleotides 1287 to 1526 are spliced out in the clone we used). The human Northern blot was purchased from Clontech and hybridized with a radiolabeled human SCUBE1 cDNA probe (nucleotides 2077–2851) per the manufacturer's protocol. The epitope-tagged versions of SCUBE1 or SCUBE2 were constructed in the following expression vectors. The pcDNA4/Myc-His (Invitrogen) was used to add a Myc tag to the carboxyl terminus of SCUBE1 containing endogenous signal peptide. The pSecTag2 (Invitrogen) including Ig κ-chain leader sequence was used to add a Myc tag at the carboxyl terminus of SCUBE2. The pFLAG-CMV-1 (Sigma) was used to include a FLAG tag at the amino terminus of target protein. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded in 6-well plates overnight before transfection. The transfection was performed by using FuGENE 6 reagent (Roche Molecular Biochemicals). The total amount of DNA was kept constant in all transfections by supplementing empty vector DNA. Human umbilical vein endothelial cells (HUVEC) were cultured as described previously (20DiChiara M.R. Kiely J.M. Gimbrone M.A. Lee M.E. Perrella M.A. Topper J.N. J. Exp. Med. 2000; 192: 695-704Crossref PubMed Scopus (89) Google Scholar). Transfected cells were washed once with PBS and lysed for 15 min on ice in 0.5 ml of lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mmNaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 25 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin). Lysates were clarified by centrifugation at 4 °C for 15 min at 10,000 × g. Cells lysates were incubated with 1 μg of indicated antibody and 20 μl of 50% (v/v) protein A-agarose (Pierce) for 2 h with gentle rocking. After three washes with lysis buffer, precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with PBS, pH 7.5, containing 0.1% gelatin and 0.05% Tween 20 and were blotted with the indicated antibodies. After two washes, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (The Jackson Laboratories) for 1 h. After washing the membranes, the reactive bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences). Transfected cells were washed with PBS and lysed in hypotonic lysis buffer (10 mm Tris, pH 7.4, 10 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 2 mm EDTA). After incubation for 30 min on ice, cells were homogenized with 80 strokes in a tight fitting Dounce homogenizer. The lysed cells were then centrifuged at 1,000 × g (30 min, 4 °C), and the supernatant taken and further centrifuged at 100,000 × g (30 min, 4 °C) to obtain the cytosolic (S100) and membrane (P100) fractions. The P100 fraction was washed once with hypotonic lysis buffer, resuspended in 0.1 m Na2CO3, pH 12, sonicated briefly, and then incubated on ice for 30 min. Samples were centrifuged again at 100,000 × g for 30 min to give washed fractions S100′ and pellets P100′. Transfected cells were collected and suspended in PBS, 2% bovine serum albumin in a volume of 0.25 ml. A total of 1 μg of purified anti-FLAG M2 antibody and fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody (1:100 dilution, Jackson ImmunoResearch Laboratories, West Grove, PA) was added sequentially; each were incubated for 45 min on ice. FACS analyses were performed with a FACScan (BD Biosciences). LPS (Escherichia coli serotype O111:B4) was purchased from Sigma and dissolved in PBS at concentration of 1 mg/ml. LPS solution (5 mg/kg) or PBS was injected intraperitoneally into a group of three C67BL/6 mice, and at the indicated time points (3, 6, 24, and 48 h post-injection), the kidneys of these animals were harvested, washed in sterile cold PBS, and frozen in liquid nitrogen. Total RNA was prepared from cultured cells or harvested animal kidneys using TRIzol reagents (Invitrogen). First-strand cDNA synthesis using SuperScript II reverse transcriptase (Invitrogen) was prepared on 5 μg of total RNA. One-tenth of the first-strand cDNA reaction was used for each PCR as template. Alternatively, premade first-strand cDNAs from human tissues (Clontech, Palo Alto, CA) were used as PCR template. Semi-quantitative RT-PCR primers specific for human SCUBE1 and SCUBE2 were as follows: SCUBE1-f2, AGT GTT CTC CAG GCT TCT TCT; SCUBE1-r2, CAG TGC TGG TTT TTG CAG TGT; SCUBE2-f2, AGA CCC CAG AAG CTT GGA ATA; SCUBE2-r2, TCC CCT CCA CAT CTT CTG TTT. GAPDH primers were obtained from Clontech. Real time TaqMan PCR analyses were performed using Applied Biosystems PRISM 7700 Sequence Detection System. Normalization was performed using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels as controls in parallel TaqMan reactions. The 5′ and 3′ primers and a fluorescence-labeled probe were designed as follows: SCUBE1 5′ primer, AAC ACA CGG GTA CCG CCT CTT; SCUBE1 3′ primer, GTA TTG TAG TGG TGT CCG GGA GA; SCUBE1 probe, CCA GGA CTG CGA GGC CAA AGT GCA T; SCUBE2 5′ primer, CAG GAT TGT GAA ACC CGA GTT C; SCUBE2 3′ primer, CGG ATA CAT CGG TGT GTG GTG; SCUBE2 probe, TGC TCG CCT GGG CAT TTC TAC AAC A; GAPDH primer-probe was purchased from BioSource International (Camarillo, CA). The endothelial origin of a SCUBE1 cDNA fragment identified by genomic approaches was confirmed by in situ hybridization (Fig.1). It was expressed in the luminal endothelial cells of the human umbilical vessels (Fig. 1 a). Furthermore, localization of this gene to ECs was validated in cynomolgus monkey brain (Fig. 1 b), lung (Fig. 1,c and d), and kidney (Fig. 1, e andf). In addition to endothelial expression in artery and vein, this gene is also expressed in microvascular endothelial cells in a variety of tissues (Fig. 1, b–f, and data not shown). To obtain the full-length cDNA of this gene, the original cDNA fragment was mapped to human genomic sequence (www.ensembl.org) and was found to be localized on chromosome 22q13, where a human gene was predicted based on its homology to mouse Scube1 (14Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (65) Google Scholar). Two oligonucleotides, based on this gene prediction, were used to amplify the entire open reading frame from human cDNAs. This cDNA contains an open reading frame of 2964 nucleotides and encodes a polypeptide of 988 amino acids (Fig.2 a). Hydropathy (21Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16895) Google Scholar) and protein family analyses (22Sonnhammer E.L. Eddy S.R. Durbin R. Proteins. 1997; 28: 405-420Crossref PubMed Scopus (909) Google Scholar) predict one amino-terminal signal peptide (22 amino acids) followed by 10 EGF-like repeats and a CUB domain (Fig.2). There is an apparent spacer region located between the 9th and 10th EGF-like repeats (Fig. 2 b). This domain structure is identical to that of mouse Scube1, therefore, this human orthologue was designated SCUBE1 to be consistent with the literature (14Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (65) Google Scholar). Mature SCUBE1 is predicted to contain 966 amino acids with calculated molecular mass of 106 kDa. A Northern blot containing poly(A)+-enriched mRNA (2 μg) from a variety of human adult tissues was hybridized with a human SCUBE1 cDNA radiolabeled probe. The expression level of the SCUBE1 transcripts was highest in liver, kidney, lung, and small intestine, followed by brain, colon, and spleen. The expression in remaining tissues was very low or undetectable (Fig.3). Expression of SCUBE1 in several highly vascular tissues, such as liver, kidney, and lung, is consistent with the endothelial origin of SCUBE1 demonstrated by in situ hybridization (Fig. 1). The size of the primary transcript for SCUBE1 (4 kb) is consistent with both the predicted and cloned full-length cDNA (Fig. 2). Because human SCUBE1 protein has a putative signal peptide at the amino terminus and because human SCUBE1 protein contains 10 EGF-like repeats that are found in many extracellular matrix proteins (ECM) (23Prigent S.A. Lemoine N.R. Prog. Growth Factor Res. 1992; 4: 1-24Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 24Davis C.G. New Biol. 1990; 2: 410-419PubMed Google Scholar), we examined whether the SCUBE1 protein is a secretory and/or ECM protein. For this purpose, recombinant SCUBE1 protein was expressed by means of transient expression in human embryonic kidney 293T cells. The Myc epitope tag was added at the carboxyl terminus for the detection of the recombinant protein. Two days after transfection, the culture supernatants were collected, and cells were detached from dishes by EDTA treatment, and residual ECM proteins were extracted with Laemmli buffer. Samples collected from these three fractions were subjected to Western blot analyses using anti-Myc antibody. As shown in Fig.4 a, human SCUBE1 protein was detected in the conditioned cell culture medium (Medium) and in cells (Cell) but was not detected in the ECM fraction (Matrix) or in fractions from the control vector-transfected cells. These data demonstrate that the SCUBE1 protein is a secreted and cell-associated protein. Because the CUB domain was recently described in two novel members of the platelet-derived growth factor (PDGF) family that require proteolytic activation (25Li X. Ponten A. Aase K. Karlsson L. Abramsson A. Uutela M. Backstrom G. Hellstrom M. Bostrom H. Li H. Soriano P. Betsholtz C. Heldin C.H. Alitalo K. Ostman A. Eriksson U. Nat. Cell Biol. 2000; 2: 302-309Crossref PubMed Scopus (493) Google Scholar, 26Gilbertson D.G. Duff M.E. West J.W. Kelly J.D. Sheppard P.O. Hofstrand P.D. Gao Z. Shoemaker K. Bukowski T.R. Moore M. Feldhaus A.L. Humes J.M. Palmer T.E. Hart C.E. J. Biol. Chem. 2001; 276: 27406-27414Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 27Bergsten E. Uutela M. Li X. Pietras K. Ostman A. Heldin C.H. Alitalo K. Eriksson U. Nat. Cell Biol. 2001; 3: 512-516Crossref PubMed Scopus (456) Google Scholar, 28LaRochelle W.J. Jeffers M. McDonald W.F. Chillakuru R.A. Giese N.A. Lokker N.A. Sullivan C. Boldog F.L. Yang M. Vernet C. Burgess C.E. Fernandes E. Deegler L.L. Rittman B. Shimkets J. Shimkets R.A. Rothberg J.M. Lichenstein H.S. Nat. Cell Biol. 2001; 3: 517-521Crossref PubMed Scopus (309) Google Scholar), we tested whether secreted SCUBE1 could be subject to proteolytic cleavage. A dual epitope-tagged SCUBE1 with FLAG and Myc tags added to the amino and carboxyl terminus, respectively, was transiently expressed in 293T cells. Forty eight hours post-transfection, samples from conditioned medium, cells, and ECM were individually immunoblotted with anti-FLAG or anti-Myc antibodies. As shown in Fig. 4 b, the molecular weight of detected SCUBE1 protein is identical either by anti-FLAG or anti-Myc antibodies. These results suggest that the secreted SCUBE1 protein does not undergo further proteolytic processing. Consistent with this finding, the dual-tagged SCUBE1 protein immunoprecipitated by FLAG antibody from cell culture conditioned medium (Medium) showed the identical molecular size when blotted either with anti-FLAG or Myc antibodies separately (Fig. 4 c). The molecular size of expressed SCUBE1 in 293T cells is about 130 kDa, slightly larger than the predicted size of the full-length SCUBE1 protein. Because human SCUBE1 possesses six putative N-linked glycosylation motifs (Fig. 2 a), we hypothesized that SCUBE1 is subject to post-translational modification by glycosylation. Thus, we examined whether tunicamycin, an inhibitor of N-glycosylation, affected the molecular size of the protein. As shown in Fig.5, tunicamycin treatment of cells resulted in a reduction in the molecular size of the precursor form of the SCUBE1 protein (lanes 2 and 5), indicating that the majority of SCUBE1 is glycosylated when expressed in 293T cells. To determine the contribution of the six putativeN-linked glycosylation sites in the SCUBE1 protein, we compared the molecular size of the carboxyl-terminal deletion mutants (D1 and D2) with or without tunicamycin treatment. As shown in Fig. 5, the precursor form of mutant D1 (in which the carboxyl-terminal CUB domain is deleted) was detected at 110 kDa without tunicamycin (lane 3), whereas the protein size was shifted to a faster migrating band by treatment with tunicamycin (lane 6), indicating that the mutant D1 is glycosylated. However, tunicamycin treatment did not change the apparent molecular size of the precursor form of mutant D2 (lanes 4 and 7) (in which five of six putative N-linked glycosylation motifs in the protein are deleted) (Fig. 2 b). Taken together, these data demonstrated that SCUBE1 is N-glycosylated at multiple sites within the carboxyl-terminal region. Because families of secreted growth factors or cytokines are often capable of forming dimeric or higher ordered complexes (29Davies D.R. Wlodawer A. FASEB J. 1995; 9: 50-56Crossref PubMed Scopus (46) Google Scholar, 30Sun P.D. Davies D.R. Annu. Rev. Biophys. Biomol. Struct. 1995; 24: 269-291Crossref PubMed Google Scholar), and because SCUBE1 is a secretory protein, we hypothesized that oligomeric forms of SCUBE1 protein may exist. We constructed cDNAs encoding FLAG- or Myc-tagged SCUBE1 proteins, and we examined their association by co-immunoprecipitation assays from both singly and co-transfected 293T cells (Fig. 6 a). Lysates of th
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