A Novel Secreted, Cell-surface Glycoprotein Containing Multiple Epidermal Growth Factor-like Repeats and One CUB Domain Is Highly Expressed in Primary Osteoblasts and Bones
2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês
10.1074/jbc.m405912200
ISSN1083-351X
AutoresBo-Tsung Wu, Yueh-Hsing Su, Ming-Tzu Tsai, Scott M. Wasserman, James N. Topper, Ruey‐Bing Yang,
Tópico(s)Cancer-related gene regulation
ResumoWe have previously identified a novel family of secreted, cell-surface proteins expressed in human vascular endothelium. To date, two family members have been described, sharing a characteristic domain structure including an amino-terminal signal peptide followed by multiple copies of epidermal growth factor (EGF)-like repeats, a spacer region, and one CUB domain at the carboxyl terminus. Thus, this family was termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Here we described the identification and characterization of one additional member of the SCUBE family named SCUBE3 in humans, sharing an overall 60% protein identity and a similar domain structure with other family members. Real-time quantitative reverse transcriptase-PCR and Northern blot analyses revealed that this gene is highly expressed in primary osteoblasts and the long bones (humerus and femur), followed by a low level of expression in human umbilical vein endothelial cells and in heart. When overexpressed in human embryonic kidney 293T cells, the recombinant hSCUBE3 protein is a secreted glycoprotein that can form oligomers tethered to the cell surface. Interestingly, the secreted hSCUBE3 protein can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. The SCUBE3 gene is mapped to human chromosome 6p21.3, a region that has been linked with the locus for a rare form of metabolic bone disease, Paget's disease of bone 1. Together, this novel secreted, cell-surface osteoblast protein may act locally and/or distantly through a proteolytic mechanism, and may play an important role in bone cell biology. We have previously identified a novel family of secreted, cell-surface proteins expressed in human vascular endothelium. To date, two family members have been described, sharing a characteristic domain structure including an amino-terminal signal peptide followed by multiple copies of epidermal growth factor (EGF)-like repeats, a spacer region, and one CUB domain at the carboxyl terminus. Thus, this family was termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Here we described the identification and characterization of one additional member of the SCUBE family named SCUBE3 in humans, sharing an overall 60% protein identity and a similar domain structure with other family members. Real-time quantitative reverse transcriptase-PCR and Northern blot analyses revealed that this gene is highly expressed in primary osteoblasts and the long bones (humerus and femur), followed by a low level of expression in human umbilical vein endothelial cells and in heart. When overexpressed in human embryonic kidney 293T cells, the recombinant hSCUBE3 protein is a secreted glycoprotein that can form oligomers tethered to the cell surface. Interestingly, the secreted hSCUBE3 protein can be further proteolytically processed by a serum-associated protease to release the EGF-like repeats from the CUB domain. The SCUBE3 gene is mapped to human chromosome 6p21.3, a region that has been linked with the locus for a rare form of metabolic bone disease, Paget's disease of bone 1. Together, this novel secreted, cell-surface osteoblast protein may act locally and/or distantly through a proteolytic mechanism, and may play an important role in bone cell biology. We have previously utilized a combination of high throughput sequencing and genome-scale microarray expression profiling to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells (1Yang R.B. Ng C.K. Wasserman S.M. Komuves L.G. Gerritsen M.E. Topper J.N. J. Biol. Chem. 2003; 278: 33232-33238Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). One gene family identified by this approach encodes potential secreted proteins harboring a signal peptide at the amino terminus followed by multiple copies of EGF 1The abbreviations used are: EGF, epidermal growth factor; RT, reverse transcriptase; TAMRA, 6-carboxy-N,N,N′,N′-tetramethylrhodamine; h, human; m, murine; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; ECM, extracellular matrix; PDB, Paget's disease of bone. 1The abbreviations used are: EGF, epidermal growth factor; RT, reverse transcriptase; TAMRA, 6-carboxy-N,N,N′,N′-tetramethylrhodamine; h, human; m, murine; HUVEC, human umbilical vein endothelial cells; FBS, fetal bovine serum; ECM, extracellular matrix; PDB, Paget's disease of bone.-like repeats and one CUB domain at the carboxyl terminus (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Thus, these genes were termed SCUBE for signal peptide-CUB-EGF-like domain containing proteins. Expression of the first gene member SCUBE1 is highly enriched in endothelial cells, whereas the second gene member SCUBE2 is expressed in endothelial cells as well as other cell types (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The recombinant SCUBE protein, when overexpressed in human embryonic kidney 293T (HEK-293T) cells, is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Both SCUBE1 and SCUBE2 are rapidly down-regulated in endothelial cells after interleukin-1β and tumor necrosis factor-α treatment in vitro and after lipopolysaccharide injection in vivo, suggesting a possible role for the SCUBE gene family in the inflammatory response (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In the present study, we identified and characterized the third member of the SCUBE gene family, named SCUBE3. Its unique expression in bones and osteoblasts suggests that SCUBE3 may play a critical role in bone cell biology.EXPERIMENTAL PROCEDURESReagents—Human primary cultured cells, osteoblasts, human umbilical vein endothelial cells (HUVEC), and coronary smooth muscle cells, were purchased from Cambrex Bio Science (Walkersville, MD). TaqMan Universal PCR master mixture was from PE Applied Biosystems (Foster City, CA).Long Bone RNA Extraction and Analysis—The long bones (humeri and femurs) from one 2-month-old male C57Bl6 mouse were dissected free of surrounding tissue, cut into small pieces in 2 ml of TRIzol reagent (Invitrogen) and homogenized with a Polytron probe (Brinkmann Instruments) for 30 s. Total RNAs were prepared using TRIzol reagent according to the manufacturer's instructions. 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 as template for each real-time quantitative RT-PCR analysis as described below.Real-time Quantitative RT-PCR (TaqMan) Analysis—hSCUBE3 mRNA expression was measured by real-time quantitative RT-PCR on a panel of cDNAs from a variety of human primary cells or normal tissues. Probes were designed by PrimerExpress software (PE Applied Biosystems) based on the sequence of the hSCUBE3 gene. The hSCUBE3 gene-specific probe was labeled using FAM (6-carboxyfluorescein) and the β2-microglobulin reference probe was labeled with a different fluorescent dye (VIC) at the 5′ end. TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) is linked at the 3′ end as quenchers. The differential labeling of the target gene and internal reference gene thus enabled measurement in the same well. Normalization was performed using β2-microglobulin mRNA levels as controls in the same reaction. TaqMan experiments were carried out in an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems) using TaqMan reagents. The thermal cycler conditions were as follows: hold for 2 min at 50 °C and 10 min at 95 °C, followed by two-step PCR for 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Primers used for human (h) SCUBE3 analysis were as follows: forward, 5′-CAAAGTCCAGTGCTCCCCAG and reverse, 5′-GACGGAAGTCGGGCTGATAG; TaqMan probe, 5′-FAM-ACACCAGCATCCACCGCTGTATTCG-TAMRA. Primers used for mouse (m) SCUBE3 analysis were as follows: forward, 5′-CCAGAAGAAGGAATGAACTGCA and reverse, 5′-GCAATGCCCCCCTTGG; TaqMan probe, 5′-FAM-TGCCCACATTTGCCGGGAGACTTAMRA primers used for β2-microglobulin analysis were as follows: forward, 5′-GTCTCGCTCCGTGGCCTTA and reverse, 5′-TGAATCTTTGGAGTACGCTGGATA; TaqMan probe, 5′-VIC-TGCTCGCGCTACTCTCTCTTTCTGGC-TAMRA. Primers used for GAPDH analysis were as follows: forward, 5′-TGAAGGTCGGAGTCAACGG and reverse, 5′-AGAGTTAAAAGCAGCCCTGGTG; TaqMan probe, 5′-FAM-TTTGGTCGTATTGGGCGCCTGG-TAMRA.Full-length Cloning and Expression Plasmids—On the basis of gene prediction and a public sequence information (GenBank™ accession number AF452494), the entire open reading frame of hSCUBE3 was amplified by PCR via a mixture of Advantage 2 (Clontech) and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) from cDNAs derived from human primary osteoblasts. The resulting cDNA was cloned into pGEM-T Easy vector (Promega) and confirmed by sequencing. The following epitope-tagged expression constructs of hSCUBE3 were prepared by cloning of the PCR fragment encoding the mature hSCUBE3 (amino acids 23–993) into the mammalian expression vectors. The FLAG tag (DYKDDDDK) was added at the amino terminus followed by the mature hSCUBE3 (amino acids 22–993) into the pFLAG-CMV-1 expression vector (Sigma). The pSecTag2 vector (Invitrogen) was used to add a Myc tag (EQKLISEEDL) at the carboxyl terminus of hSCUBE3. The GenBank™ accession numbers for hSCUBE3 and mSCUBE3 are AY639608 and AY639609, respectively.Northern Blot Analysis—Total RNAs (10 μg) from human osteoblasts, HUVEC, and human small intestine were separated on 1.2% denaturing formaldehyde-agarose gel, transferred to nylon members, and hybridized overnight with a radiolabeled 525-bp hSCUBE3 cDNA probe (amino acids 356–531). In addition, one human tissue Northern blot (containing 1 μg of poly(A)-enriched mRNA from a variety of human tissues) was purchased from Clontech and hybridized with a radiolabeled hSCUBE3 cDNA probe according to the manufacturer's instructions. After hybridization overnight, the blots were washed once at room temperature (25 °C) and twice at 65 °C with 0.1 × SSC (15 mm NaCl, 1.5 mm sodium citrate) and 0.1% SDS (each wash for 1 h). Autoradiography was performed at –80 °C for 1 day. The blot was then stripped and hybridized with a β-actin probe as a positive control.Cell Culture and Transfection—HEK-293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded in 6-well plates overnight before transfection. The transfection was performed with FuGENE 6 reagent (Roche Diagnostics) or by the calcium-phosphate-mediated method. We consistently observed greater than 90% of transfection efficiency in HEK-293T cells, accessed by the transfection of green fluorescent protein vector (Clontech) as reporter and examined by flow cytometry. The total amount of DNA was kept constant in all transfections by supplementing empty vector DNA. HUVEC were cultured as previously described (3DiChiara 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 (90) Google Scholar).Immunoprecipitation and Western Blot Analysis—Transfected cells were washed once with phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 25 mm sodium pyrophosphate, 1 mm β-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 the 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 phosphate-buffered saline (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 (Jackson ImmunoResearch Laboratories) for 1 h. After washing the membranes, the reactive bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences).Flow Cytometry Analysis—Transfected cells were collected and suspended in phosphate-buffered saline containing 2% FBS 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) were added sequentially; each were incubated for 45 min on ice. FACS analyses were performed with a FACScan (Clontech).RESULTSIdentification and Expression Profile of Human SCUBE3— We have previously identified a family of cell-surface proteins expressed in human endothelial cells (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). To date, two gene members have been described sharing a characteristic domain structure including an amino-terminal signal peptide, followed by multiple copies of EGF-like repeats, a spacer region, and one CUB domain at the carboxyl terminus (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Thus, this family was termed SCUBE.To further understand the biology of this gene family, we investigated whether or not additional SCUBE family member(s) exist in the public sequence data bases. After searching a public genome data base, one predicted gene (GenBank™ accession XM_094924), homologous to SCUBE1 and SCUBE2, was identified on human chromosome 6p21. Subsequently, two independent cDNAs representing this predicted gene were described in the GenBank™ data base under accession numbers AF452494 and BC052263, respectively. In this report, this gene is referred to as SCUBE3 to be consistent with the literature and the order of its discovery, related to prior family members, SCUBE1 and SCUBE2 (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 4Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (66) Google Scholar, 5Grimmond S. Larder R. Van Hateren N. Siggers P. Morse S. Hacker T. Arkell R. Greenfield A. Mech. Dev. 2001; 102: 209-211Crossref PubMed Scopus (42) Google Scholar).We then examined the expression profile of human SCUBE3 (hSCUBE3) by real-time quantitative RT-PCR (TaqMan) analysis. Interestingly, when a panel of cDNAs derived from a variety of human primary cells and tissues was assessed for hSCUBE3 expression by TaqMan analysis, we found that hSCUBE3 mRNA is highly enriched in primary cultured osteoblasts, followed by primary HUVEC and coronary smooth muscle cells (Fig. 1A, top). On the other hand, hSCUBE3 transcript levels were found to be absent or quite low in a variety of normal human tissues (Fig. 1A, bottom). Because osteoblasts are the prominent cells in bone tissues, we then determined whether or not SCUBE3 mRNA is also highly expressed in skeletons. Quantitative RT-PCR analysis was used to measure the SCUBE3 mRNA levels in the long bones (i.e. humerus and femur) and a number of non-osseous tissues from an adult mouse. As shown in Fig. 1B, we found that expression of SCUBE3 is indeed highly enriched in native bone tissue, but low in all other non-bone tissues.Full-length Cloning and Sequence Analysis of hSCUBE3— To obtain the full-length cDNA of this gene, one pair of oligonucleotides based on the public sequences was used to amplify the entire open reading frame by PCR from cDNAs derived from human primary osteoblasts because hSCUBE3 is highly expressed in this cell type. This cDNA was confirmed by sequencing and shown to be identical to the sequence of GenBank™ accession number AF452494 that contains an open reading frame of 2979 nucleotides encoding a polypeptide of 993 amino acids (see Supplemental Materials, Fig. S1). Hydrophobicity (6Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17007) Google Scholar) and protein family (7Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2005) Google Scholar) analyses predict one 22-residue amino-terminal signal peptide, followed by nine copies of EGF-like repeats, a spacer region, and one CUB domain (Fig. 2). Notably, there are five potential sites for N-glycosylation located within the spacer region, between the multiple EGF-like repeats and the CUB domain (Supplemental Materials, Fig. S1). This domain organization is similar to that of two prior family members, SCUBE1 and SCUBE2 (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 4Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (66) Google Scholar, 5Grimmond S. Larder R. Van Hateren N. Siggers P. Morse S. Hacker T. Arkell R. Greenfield A. Mech. Dev. 2001; 102: 209-211Crossref PubMed Scopus (42) Google Scholar), and appears unique to the SCUBE protein family. Mature SCUBE3 protein is predicted (after cleavage of the signal peptide) to contain 971 residues with calculated molecular mass of 107 kDa.Fig. 2Domain structure of human SCUBE3. The region marked with the solid box indicates the putative signal peptide. The graphic shows the domain organization of human SCUBE3 protein. An apparent "spacer region" is located between the 9th EGF-like repeat and the CUB domain. Two deletion constructs are also depicted. SP, signal peptide; FL, full-length; D1 (amino acids 1–993), deletion mutant 1 (amino acids 1–803); D2, deletion mutant 2 (amino acids 1–529); E, EGF-like repeats; CUB, CUB domain.View Large Image Figure ViewerDownload (PPT)SCUBE3 shares an overall 66 and 60% homology with that of SCUBE1 and SCUBE2, respectively (see Supplemental Materials, Figs. S2 and S3). The CUB domain and the very carboxyl-terminal sequences are the most conserved regions sharing 82–90% identity in this family (Supplemental Materials, Fig. S3), followed by the amino-terminal nine copies of EGF-like repeats sharing 66–77% homology (Supplemental Materials, Fig. S2). The spacer regions are the most divergent and are only about 46–50% homologous among the family members (Supplemental Materials, Fig. S3).Northern Blot Analysis for the Expression of the hSCUBE3 Transcript—To further validate the expression of SCUBE3, Northern blots containing total RNAs (10 μg) from human primary cells or poly(A)-enriched mRNA (1 μg) from a variety of human adult tissues was hybridized with a hSCUBE3 cDNA radiolabeled probe, respectively. As shown in Fig. 3A, the expression level of hSCUBE3 was highest in human primary osteoblasts with a predominant transcript at the size of ∼8.0 kb, followed by a low expression level in HUVEC with an mRNA species of about 4.0 kb. On the other hand, the tissue Northern blot analysis showed that the expression of hSCUBE3 is only barely detectable in heart, but not in all other tissues examined (Fig. 3B).Fig. 3Northern blot analysis for the expression of human SCUBE3 transcripts.A, enriched expression of SCUBE3 in human osteoblasts. Ten micrograms of total RNAs isolated from human primary osteoblasts, HUVEC, and small intestine were hybridized with the SCUBE3 cDNA radiolabeled probe. The SCUBE3 probe identified one mRNA species of approximately 8.0 kb in human osteoblasts (arrowhead) and a weak transcript size of 4.0 kb in HUVEC (arrow). The lower panel shows the quantity of 28 S RNAs as loading controls. B, Northern blot analysis of poly(A)+ mRNA from a variety of human tissues for SCUBE3. One microgram of poly(A)-enriched mRNA from various human adult tissues was hybridized with the SCUBE3 radiolabeled probe. An mRNA species of ∼4.0 kb was identified in heart (arrow), but not in all other tissues examined.View Large Image Figure ViewerDownload (PPT)Secretion, N-Glycosylation, and Cell-surface Expression of Recombinant hSCUBE3 Protein in HEK-293T Cells—Because hSCUBE3 possesses a putative signal peptide indicative for a secreted protein and because multiple EGF-like repeats are found in a number of extracellular matrix proteins (ECM) (8Prigent S.A. Lemoine N.R. Prog. Growth Factor Res. 1992; 4: 1-24Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 9Davis C.G. New Biol. 1990; 2: 410-419PubMed Google Scholar), we investigated whether the hSCUBE3 protein is a secretory and/or ECM protein. To this end, the recombinant hSCUBE3 protein was expressed by means of a transient expression system in HEK-293T cells. The FLAG epitope was added immediately after the signal peptide cleavage site (i.e. at the amino terminus of the processed, mature protein) for the detection of the recombinant protein (Flag.hSCUBE3). Two days after transfection and under serum-free conditions, the culture supernatants were collected, and cells were detached from dishes by EDTA treatment, and residual ECM proteins were extracted with Laemmli buffer. Samples from these three fractions were subjected to Western blot analyses using anti-FLAG antibody. As shown in Fig. 4A, like hSCUBE1, the hSCUBE3 protein was detected in the serum-free conditioned culture medium (Medium) and in cell lysates (Cell), but was not detected in the ECM fraction (Matrix) or in fractions derived from the control vector-transfected cells. These data demonstrate that the hSCUBE3 protein is a secreted and cell-associated protein.Fig. 4Secretion, glycosylation, and cell-surface expression of SCUBE3 in HEK-293T cells.A, SCUBE3 is a secreted and cell-associated protein. HEK-293T cells were transfected with empty vector or expression vectors encoding FLAG-tagged human SCUBE3 (Flag.hSCUBE3) or SCUBE1 (Flag.hSCUBE1) as positive control (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Forty-eight post-transfection, the serum-free conditioned medium was collected, and cells were detached with phosphate-buffered saline/EDTA. The extracellular matrix on the culture dish was extracted with Laemmli sample buffer. Samples from the serum-free conditioned culture medium (Medium), cell lysates (Cell), and the extracellular matrix (Matrix) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Recombinant SCUBE proteins were detected by Western blotting with anti-FLAG M2 antibody. B, N-glycosylation of human SCUBE3. HEK-293T cells were transfected with empty vector or expression plasmid encoding the Flag.hSCUBE3 protein. Transfected cells were cultured in the absence (–) or presence (+) of tunicamycin for 24 h. Cell lysates from each culture were analyzed by Western blotting with anti-FLAG antibody. C, cell-surface expression of human SCUBE3 protein. HEK-293T cells were transfected with empty vector or expression vector encoding Flag.hSCUBE3 or Flag.hSCUBE1 proteins, respectively. Forty-eight hours post-transfection, transfected cells were detached and stained with anti-FLAG antibody and subjected to flow cytometry analysis as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT)The molecular mass of the recombinant SCUBE3 protein expressed in HEK-293T cells is approximately 130 kDa, slightly larger than the predicted size of mature hSCUBE3 (Fig. S1). Because hSCUBE3 has five putative N-linked glycosylation sites (Fig. S1), it is likely that the recombinant hSCUBE3 is subject to a post-translational modification by glycosylation. Thus, we examined whether tunicamycin, an inhibitor of N-linked glycosylation, affected the molecular mass of the expressed protein. As shown in Fig. 4B, tunicamycin treatment of cells results in a reduction of molecular size to that of the predicted, precursor form of hSCUBE3 at about 110 kDa. These results clearly demonstrate that the majority of hSCUBE3 is N-glycosylated when expressed in HEK-293T cells.Because the majority of expressed hSCUBE3 protein appears cell-associated (Fig. 4A) and because hSCUBE1 is a membrane-anchored protein (2Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), we next determined whether or not hSCUBE3 is capable of targeting to the cell surface by means of flow cytometric analysis. Expression plasmid encoding the Flag.hSCUBE3 was transfected into HEK-293T cells. Forty-eight hours post-transfection, cells were collected and incubated with anti-FLAG M2 antibody followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody, then analyzed by flow cytometry. As shown in Fig. 4C, expression of the Flag.hSCUBE3 protein resulted in a shift of a population of fluorescein isothiocyanate-labeled cells by fluorescence-activated cell sorter analysis, suggesting that hSCUBE3, like hSCUBE1, is targeted to the cell surface.Homo- and Hetero-oligomerization of hSCUBE3—We have previously shown that SCUBE1 and SCUBE2 are capable of forming a homomeric or heteromeric complex when overexpressed in HEK-293 cells. To ascertain whether or not this newly identified family member, SCUBE3, can form the oligomeric complexes, the differentially epitope-tagged SCUBE3 and SCUBE1 expression plasmids were singly or co-transfected into HEK-293T cells. Two days after transfection, cell lysates were immunoprecipitated followed by Western blot analysis to determine the protein associations. When SCUBE3.Myc is coexpressed with either Flag.SCUBE1 or Flag.SCUBE3, immunoprecipitation with anti-Myc antibody results in the coimmunoprecipitation of either protein, respectively (Fig. 5). Likewise, the reciprocal immunoprecipitation of anti-FLAG antibody followed by Western blotting with anti-Myc antibody confirmed the formation of homo- and hetero-oligomeric complexes between SCUBE3 and SCUBE1, at least in HEK-293T cells.Fig. 5Homo- and hetero-oligomerization of human SCUBE3 in transfected HEK-293T cells. HEK-293T cells were transfected with Myc-tagged SCUBE3 (hSCUBE3.Myc) singly or co-transfected with the Flag.hSCUBE1 or Flag.hSCUBE3 as indicated. The total amount of DNA was kept constant in all transfections by supplementing the empty vector DNA. Two days later, detergent lysates of each transfection were subjected to immunoprecipitate (IP) by FLAG or Myc antibodies, followed by immunoblotting (WB) using FLAG and Myc antibodies, respectively, to determine the associated proteins. Cell lysates were also immunoblotted with anti-FLAG or Myc antibodies to examine the protein expression levels (bottom two panels).View Large Image Figure ViewerDownload (PPT)Proteolytic Processing of the Secreted SCUBE3 Protein—Because two novel CUB domain-containing platelet-derived growth factor family members, platelet-derived growth factor-C and -D (10Bergsten 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 (459) Google Scholar, 11Gilbertson 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. 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