Molecular Cloning and Characterization of Endosialin, a C-type Lectin-like Cell Surface Receptor of Tumor Endothelium
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m009604200
ISSN1083-351X
AutoresSven Christian, Horst Ahorn, Andreas Koehler, Frank Eisenhaber, Hans-Peter Rodi, Pilar Garin‐Chesa, John E. Park, Wolfgang J. Rettig, Martin Lenter,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoEndosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive toO-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigationAJ295846. Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive toO-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigationAJ295846. monoclonal antibody base pair(s) epidermal growth factor expressed sequence tag O-sialoglycoprotein endopeptidase polyacrylamide gel electrophoresis high performance liquid chromatography 4-morpholinepropanesulfonic acid rapid amplification of cDNA ends serial analysis of gene expression tumor endothelial marker 1 The endosialin antigen was identified in a survey of normal and neoplastic human tissues conducted at the Ludwig Institute for Cancer Research in pursuit of new targets for antibody-based cancer therapies (37Old L.J. Sci. Am. 1996; 275: 102-109Crossref Scopus (63) Google Scholar). The hallmark of monoclonal antibody (mAb)1 FB5, the probe used to discover endosialin (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), is its distinctive pattern of reactivity with human tissues. Thus, in a detailed study of biopsy and surgical specimens representing diverse cancer types, FB5 immunostaining was found primarily in tumor blood vessels and not in malignant tumor cells. Significantly, the antigen was not observed in all cancer samples examined, and even in cancers showing FB5-immunoreactive endothelial cells, the antigen was frequently detected with a heterogeneous pattern in the tumor vascular bed. Such a mixed pattern might be expected for a molecule involved in the reorganization of blood vessels in tissues such as cancers, in which areas of stable blood supply and histology are juxtaposed to regions of necrosis, hypoxia, excessive growth, tissue invasion, and remodeling. The normal tissues examined were unreactive with mAb FB5, including the blood vessel endothelium present in the respective organs.The expression of the FB5 antigen by cultured normal and tumor cells was also investigated (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), revealing that the standard test cells for normal endothelial differentiation markers, cultured human umbilical vein endothelial cells, and microvascular endothelial cells derived from bone marrow and dermis are antigen-negative. Human umbilical vein endothelial cell cultures stimulated with a range of mediators known to induce the expression of endothelial activation antigens (2Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1148) Google Scholar, 3Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar) maintain their FB5-negative phenotype (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar). Among a host of cultured epithelial, neuroectodermal, mesenchymal, and hematopoietic cell types tested, most were FB5 antigen-negative. Notable exceptions are short term cultures of normal fibroblasts and neuroblastoma cell lines, which consistently express the antigen in tissue culture (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), allowing the chromosomal assignment of an FB5 coding gene to human chromosome 11q13-qter, based on the serologic analysis of mouse-human neuroblastoma cell hybrids. The reason why cultured fibroblasts and neuroblastomas are FB5 positive in vitro, yet the corresponding cells in uncultured tissue sections are FB5 negative, is not known. Presumably, some of these findings reflect cell type-restricted, adaptive changes of the cell surface phenotype triggered by tissue culture factors (38Rettig W.J. Old L.J. Annu. Rev. Immunol. 1989; 7: 481-511Crossref PubMed Scopus (41) Google Scholar); the mechanism underlying such induction and its relationship to FB5 antigen induction in tumor blood vessels are not known.Methods are not generally available to examine the biochemical nature of antigens with restricted expression in tumor endothelium, because of to three properties of these cells: (i) they constitute a very minor fraction in most tumor tissues; (ii) they are not readily purified in sufficient numbers for direct protein analysis; and (iii) no cell culture model of tumor endothelium exists that faithfully reproduces its distinctive in vivo phenotype. With no source for antigen characterization in tumor endothelium, the target molecule for mAb FB5 was studied in cultured neuroblastoma and sarcoma cells and fibroblast cultures. In these cell types, the antigen is a unique 165-kDa glycoprotein, comprised of a core protein that migrates as a 95-kDa species on SDS gels and carries the mAb FB5-defined epitope, and abundant, highly sialylated O-linked carbohydrate moieties. There has been no proof that the same protein carries the FB5 epitope in tumor endothelium, but in recognition of the characteristic biochemical properties and the tumor endothelial expression pattern the molecule was designated endosialin.In the absence of more detailed information about the molecular structure of endosialin, no clues were available regarding its potential function in cancer. Therefore, considering the keen interest in angiogenesis as a determinant of cancer progression and metastasis (4Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6018) Google Scholar) and as a target for novel cancer therapies (5Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21919) Google Scholar), this study aimed to clone the endosialin gene and provide the requisite probes and structural information to explore endosialin function in suitable model systems and in human cancers.DISCUSSIONWe have cloned the gene coding for the human endosialin core protein based on several lines of evidence. First, transfection of endosialin cDNA into HeLa-S3 cells leads to induction of cell surface reactivity with the cognate mAb FB5. Secondly, mAb FB5 detects the expected 165-kDa sialoglycoprotein in the transfectants, with a core protein migrating as a 95-kDa species on SDS gels. Third, the endosialin gene maps to chromosome 11q13, refining the previous assignment to 11q13-qter based on serologic analysis of somatic cell hybrids with mAb FB5. Finally, the amino acid sequence provides the requisite attachment sites for abundant O-glycosylation of endosialin.The present study is not the only evidence linking endosialin induction to vascular endothelial cells in human cancer. Rather, an independent line of investigation, aimed at dissecting comprehensive gene expression profiles in cancers with the serial analysis of gene expression (SAGE) method, has implicated endosialin-specific ESTs in tumor angiogenesis. St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) employed SAGE to survey about 32,500 unique gene transcripts for differential expression in purified tumor endothelium versus endothelium of nonmalignant tissue. The study identified 46 transcripts elevated at least 10-fold in tumor endothelium, and the highest and most consistent up-regulation was noted for a hitherto uncharacterized gene, deposited as ESTs of unknown function in data bases and designated tumor endothelial marker 1 (TEM1) by St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar). We show here that TEM1 is the endosialin gene.In their study, St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) confirmed the SAGE data and showed by in situ RNA hybridization that TEM1/endosialin expression in vivo is selective for tumor endothelium, with no detection in the malignant epithelial cells of colorectal and other cancer, a range of normal organs, and a panel of cultured tumor cell lines. Nevertheless, TEM1/endosialin mRNA was not unique to tumor blood vessels because endothelial cells associated with wound healing and corpus luteum formation also showed gene expression by in situ RNA hybridization.Taking together the endosialin expression data generated with mAb FB5 (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar) and the RNA expression data derived from SAGE and in situ RNA hybridization studies (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar), a consistent picture of endosialin induction in tumor endothelium emerges. Such a close agreement between epitope and protein/mRNA expression is instructive for a molecule like endosialin, which falls into the group of sialomucin-type glycoproteins. There is precedent for sialomucins and other glycoconjugates for which mAb-defined epitope expression and protein/mRNA distribution sharply diverge. Several levels of complexity have been described (26Hakomori S. Curr. Opin. Immunol. 1991; 3: 646-653Crossref PubMed Scopus (218) Google Scholar, 27Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (301) Google Scholar, 28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 29Puri K.D. Finger E.B. Gaudernack G. Springer T.A. J. Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar), including (i) epitopes on core proteins that are masked by glycosylation or specific sialylation, (ii) epitopes on carbohydrate moieties shared by several unrelated glycoproteins or glycolipids, and (iii) epitopes on distinct splice variants or processing variants of the same core protein. In the case of endosialin, the observed cotyping for the FB5 epitope and TEM1/endosialin mRNA suggests that mAb FB5 binds to an invariant epitope on the 95-kDa core protein. Nevertheless, this conclusion needs to be confirmed for each cell type under investigation, because epitope masking and mimicry can show exquisite cell type selectivity.A persistent question in the exploration of tumor angiogenesis is the extent of phenotypic and functional heterogeneity that may distinguish subsets of tumor endothelial cells in a macroscopic tumor lesion with regard to their derivation from pre-existing blood vessels or new vessel sprouting, and their function and state of activation in response to varying milieus of tumor-derived, paracrine angiogenesis inducers and inhibitors. Generally, the capillary bed of a locally advanced or metastatic tumor mass will extend into areas of relatively stable blood supply and tissue architecture and closely juxtaposed or intermingled regions of hypoxia, necrosis, excessive proliferation, tissue invasion, fibroblastic remodeling, or inflammatory cell infiltration. Placed in this context of malignant tissue disruption, markers of tumor angiogenesis might be expected to display a heterogeneous pattern of expression as a rule rather than exception. Considering the marked heterogeneity in FB5 staining in tumor vessels (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), it may be surprising that the report on TEM1/endosialin mRNAin situ hybridization (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) does not address this aspect. One explanation would suggest that endosialin mRNA is uniformly expressed in tumor endothelium and that only subsets of endothelial cells accumulate the endosialin protein or protein with the FB5 epitope; however, as outlined above, there is currently no evidence supporting such a mechanism. Alternatively, it may be argued that mRNA in situ hybridization is the more sensitive detection method for endosialin and that FB5 immunostaining marks tumor endothelium with the highest levels of protein/epitope density rather than a distinct tumor endothelial cell type. Finally, there may simply be differences in the design of the two available studies on endosialin expression.Our analysis shows that the N-terminal extracellular portion of endosialin is likely to consist of five domains with globular structure, and in this region homology to thrombomodulin and the complement receptor C1qRp, with 39 and 33% amino acid sequence identity, respectively, is observed. Importantly, a pattern of cysteine residues is highly conserved among the three proteins and provides a scaffold of anchoring points for disulfide bridges, presumably imposing similar three-dimensional structures on each protein. Thrombomodulin exhibits a C-type lectin domain, six EGF-like repeats, and a serine/threonine-rich region in the extracellular domain. Unlike endosialin, thrombomodulin is expressed on a variety of normal cell types and on normal vascular endothelium serves as a receptor for thrombin, modulating the coagulation cascade and triggering the thrombin-activable fibrinolysis inhibitor (30Kokame K. Zheng X. Sadler J.E. J. Biol. Chem. 1998; 273: 12135-12139Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The thrombin interaction depends on the EGF repeats 5 and 6 of thrombomodulin (31Wood M.J. Sampoli Benitez B.A. Komives E.A. Nat. Struct. Biol. 2000; 7: 200-204Crossref PubMed Scopus (43) Google Scholar,32Wang W. Nagashima M. Schneider M. Morser J. Nesheim M. J. Biol. Chem. 2000; 275: 22942-22947Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which are not present in endosialin, making it unlikely that the two molecules overlap in this particular function. The complement receptor C1qRp is also expressed on a wider variety of cell types than endosialin, including macrophages, neutrophilic granulocytes, and normal vascular endothelium. The extracellular portion of C1qRp is composed of a C-type lectin domain, a tandem of five EGF-like repeats, and a mucin-like region. The molecule binds the complement factor C1q, mannose-binding lectin, and pulmonary surfactant protein A (22Nepomuceno R.R. Henschen-Edman A.H. Burgess W.H. Tenner A.J. Immunity. 1997; 6: 119-129Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). On myeloid cells, C1qRp mediates phagocytosis of antibody-coated particles, but the function of C1qRp on normal endothelial cells is largely unknown (33Hess K.L. Tudor K.S. Johnson J.D. Osati-Ashtiani F. Askew D.S. Cook-Mills J.M. Exp. Cell Res. 1997; 236: 404-411Crossref PubMed Scopus (58) Google Scholar).Predictions about the membrane-proximal extracellular domain focus on fewer structural features. Thus, the high content of proline, threonine, and serine residues indicates likely attachment points for more than 30 O-linked carbohydrate side chains consistent with the sialomucin-like features of endosialin (28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Certain sialomucins have been implicated in sequestering growth factors to the plasma membrane and presenting these factors to endothelial cell surface receptors (34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 35Goretzki L. Burg M.A. Grako K.A. Stallcup W.B. J. Biol. Chem. 1999; 274: 16831-16837Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar); endosialin may be a candidate for such a function unique to tumor endothelium.Apart from further biochemical investigations that will benefit from the availability of mAb FB5 and recombinant endosialin protein, study of defined alterations in endosialin gene expression and structure are now possible, including genetic manipulations in mice and rats, which harbor a close homologue of endosialin. In conclusion, key experiments are now within reach to explore the endosialin system in human physiology and disease and to extend these investigations into experimentally more amenable rodent models. A better understanding of endosialin may even enable new approaches to cancer detection and treatment. The endosialin antigen was identified in a survey of normal and neoplastic human tissues conducted at the Ludwig Institute for Cancer Research in pursuit of new targets for antibody-based cancer therapies (37Old L.J. Sci. Am. 1996; 275: 102-109Crossref Scopus (63) Google Scholar). The hallmark of monoclonal antibody (mAb)1 FB5, the probe used to discover endosialin (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), is its distinctive pattern of reactivity with human tissues. Thus, in a detailed study of biopsy and surgical specimens representing diverse cancer types, FB5 immunostaining was found primarily in tumor blood vessels and not in malignant tumor cells. Significantly, the antigen was not observed in all cancer samples examined, and even in cancers showing FB5-immunoreactive endothelial cells, the antigen was frequently detected with a heterogeneous pattern in the tumor vascular bed. Such a mixed pattern might be expected for a molecule involved in the reorganization of blood vessels in tissues such as cancers, in which areas of stable blood supply and histology are juxtaposed to regions of necrosis, hypoxia, excessive growth, tissue invasion, and remodeling. The normal tissues examined were unreactive with mAb FB5, including the blood vessel endothelium present in the respective organs. The expression of the FB5 antigen by cultured normal and tumor cells was also investigated (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), revealing that the standard test cells for normal endothelial differentiation markers, cultured human umbilical vein endothelial cells, and microvascular endothelial cells derived from bone marrow and dermis are antigen-negative. Human umbilical vein endothelial cell cultures stimulated with a range of mediators known to induce the expression of endothelial activation antigens (2Lasky L.A. Science. 1992; 258: 964-969Crossref PubMed Scopus (1148) Google Scholar, 3Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar) maintain their FB5-negative phenotype (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar). Among a host of cultured epithelial, neuroectodermal, mesenchymal, and hematopoietic cell types tested, most were FB5 antigen-negative. Notable exceptions are short term cultures of normal fibroblasts and neuroblastoma cell lines, which consistently express the antigen in tissue culture (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), allowing the chromosomal assignment of an FB5 coding gene to human chromosome 11q13-qter, based on the serologic analysis of mouse-human neuroblastoma cell hybrids. The reason why cultured fibroblasts and neuroblastomas are FB5 positive in vitro, yet the corresponding cells in uncultured tissue sections are FB5 negative, is not known. Presumably, some of these findings reflect cell type-restricted, adaptive changes of the cell surface phenotype triggered by tissue culture factors (38Rettig W.J. Old L.J. Annu. Rev. Immunol. 1989; 7: 481-511Crossref PubMed Scopus (41) Google Scholar); the mechanism underlying such induction and its relationship to FB5 antigen induction in tumor blood vessels are not known. Methods are not generally available to examine the biochemical nature of antigens with restricted expression in tumor endothelium, because of to three properties of these cells: (i) they constitute a very minor fraction in most tumor tissues; (ii) they are not readily purified in sufficient numbers for direct protein analysis; and (iii) no cell culture model of tumor endothelium exists that faithfully reproduces its distinctive in vivo phenotype. With no source for antigen characterization in tumor endothelium, the target molecule for mAb FB5 was studied in cultured neuroblastoma and sarcoma cells and fibroblast cultures. In these cell types, the antigen is a unique 165-kDa glycoprotein, comprised of a core protein that migrates as a 95-kDa species on SDS gels and carries the mAb FB5-defined epitope, and abundant, highly sialylated O-linked carbohydrate moieties. There has been no proof that the same protein carries the FB5 epitope in tumor endothelium, but in recognition of the characteristic biochemical properties and the tumor endothelial expression pattern the molecule was designated endosialin. In the absence of more detailed information about the molecular structure of endosialin, no clues were available regarding its potential function in cancer. Therefore, considering the keen interest in angiogenesis as a determinant of cancer progression and metastasis (4Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6018) Google Scholar) and as a target for novel cancer therapies (5Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (21919) Google Scholar), this study aimed to clone the endosialin gene and provide the requisite probes and structural information to explore endosialin function in suitable model systems and in human cancers. DISCUSSIONWe have cloned the gene coding for the human endosialin core protein based on several lines of evidence. First, transfection of endosialin cDNA into HeLa-S3 cells leads to induction of cell surface reactivity with the cognate mAb FB5. Secondly, mAb FB5 detects the expected 165-kDa sialoglycoprotein in the transfectants, with a core protein migrating as a 95-kDa species on SDS gels. Third, the endosialin gene maps to chromosome 11q13, refining the previous assignment to 11q13-qter based on serologic analysis of somatic cell hybrids with mAb FB5. Finally, the amino acid sequence provides the requisite attachment sites for abundant O-glycosylation of endosialin.The present study is not the only evidence linking endosialin induction to vascular endothelial cells in human cancer. Rather, an independent line of investigation, aimed at dissecting comprehensive gene expression profiles in cancers with the serial analysis of gene expression (SAGE) method, has implicated endosialin-specific ESTs in tumor angiogenesis. St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) employed SAGE to survey about 32,500 unique gene transcripts for differential expression in purified tumor endothelium versus endothelium of nonmalignant tissue. The study identified 46 transcripts elevated at least 10-fold in tumor endothelium, and the highest and most consistent up-regulation was noted for a hitherto uncharacterized gene, deposited as ESTs of unknown function in data bases and designated tumor endothelial marker 1 (TEM1) by St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar). We show here that TEM1 is the endosialin gene.In their study, St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) confirmed the SAGE data and showed by in situ RNA hybridization that TEM1/endosialin expression in vivo is selective for tumor endothelium, with no detection in the malignant epithelial cells of colorectal and other cancer, a range of normal organs, and a panel of cultured tumor cell lines. Nevertheless, TEM1/endosialin mRNA was not unique to tumor blood vessels because endothelial cells associated with wound healing and corpus luteum formation also showed gene expression by in situ RNA hybridization.Taking together the endosialin expression data generated with mAb FB5 (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar) and the RNA expression data derived from SAGE and in situ RNA hybridization studies (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar), a consistent picture of endosialin induction in tumor endothelium emerges. Such a close agreement between epitope and protein/mRNA expression is instructive for a molecule like endosialin, which falls into the group of sialomucin-type glycoproteins. There is precedent for sialomucins and other glycoconjugates for which mAb-defined epitope expression and protein/mRNA distribution sharply diverge. Several levels of complexity have been described (26Hakomori S. Curr. Opin. Immunol. 1991; 3: 646-653Crossref PubMed Scopus (218) Google Scholar, 27Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (301) Google Scholar, 28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 29Puri K.D. Finger E.B. Gaudernack G. Springer T.A. J. Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar), including (i) epitopes on core proteins that are masked by glycosylation or specific sialylation, (ii) epitopes on carbohydrate moieties shared by several unrelated glycoproteins or glycolipids, and (iii) epitopes on distinct splice variants or processing variants of the same core protein. In the case of endosialin, the observed cotyping for the FB5 epitope and TEM1/endosialin mRNA suggests that mAb FB5 binds to an invariant epitope on the 95-kDa core protein. Nevertheless, this conclusion needs to be confirmed for each cell type under investigation, because epitope masking and mimicry can show exquisite cell type selectivity.A persistent question in the exploration of tumor angiogenesis is the extent of phenotypic and functional heterogeneity that may distinguish subsets of tumor endothelial cells in a macroscopic tumor lesion with regard to their derivation from pre-existing blood vessels or new vessel sprouting, and their function and state of activation in response to varying milieus of tumor-derived, paracrine angiogenesis inducers and inhibitors. Generally, the capillary bed of a locally advanced or metastatic tumor mass will extend into areas of relatively stable blood supply and tissue architecture and closely juxtaposed or intermingled regions of hypoxia, necrosis, excessive proliferation, tissue invasion, fibroblastic remodeling, or inflammatory cell infiltration. Placed in this context of malignant tissue disruption, markers of tumor angiogenesis might be expected to display a heterogeneous pattern of expression as a rule rather than exception. Considering the marked heterogeneity in FB5 staining in tumor vessels (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), it may be surprising that the report on TEM1/endosialin mRNAin situ hybridization (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) does not address this aspect. One explanation would suggest that endosialin mRNA is uniformly expressed in tumor endothelium and that only subsets of endothelial cells accumulate the endosialin protein or protein with the FB5 epitope; however, as outlined above, there is currently no evidence supporting such a mechanism. Alternatively, it may be argued that mRNA in situ hybridization is the more sensitive detection method for endosialin and that FB5 immunostaining marks tumor endothelium with the highest levels of protein/epitope density rather than a distinct tumor endothelial cell type. Finally, there may simply be differences in the design of the two available studies on endosialin expression.Our analysis shows that the N-terminal extracellular portion of endosialin is likely to consist of five domains with globular structure, and in this region homology to thrombomodulin and the complement receptor C1qRp, with 39 and 33% amino acid sequence identity, respectively, is observed. Importantly, a pattern of cysteine residues is highly conserved among the three proteins and provides a scaffold of anchoring points for disulfide bridges, presumably imposing similar three-dimensional structures on each protein. Thrombomodulin exhibits a C-type lectin domain, six EGF-like repeats, and a serine/threonine-rich region in the extracellular domain. Unlike endosialin, thrombomodulin is expressed on a variety of normal cell types and on normal vascular endothelium serves as a receptor for thrombin, modulating the coagulation cascade and triggering the thrombin-activable fibrinolysis inhibitor (30Kokame K. Zheng X. Sadler J.E. J. Biol. Chem. 1998; 273: 12135-12139Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The thrombin interaction depends on the EGF repeats 5 and 6 of thrombomodulin (31Wood M.J. Sampoli Benitez B.A. Komives E.A. Nat. Struct. Biol. 2000; 7: 200-204Crossref PubMed Scopus (43) Google Scholar,32Wang W. Nagashima M. Schneider M. Morser J. Nesheim M. J. Biol. Chem. 2000; 275: 22942-22947Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which are not present in endosialin, making it unlikely that the two molecules overlap in this particular function. The complement receptor C1qRp is also expressed on a wider variety of cell types than endosialin, including macrophages, neutrophilic granulocytes, and normal vascular endothelium. The extracellular portion of C1qRp is composed of a C-type lectin domain, a tandem of five EGF-like repeats, and a mucin-like region. The molecule binds the complement factor C1q, mannose-binding lectin, and pulmonary surfactant protein A (22Nepomuceno R.R. Henschen-Edman A.H. Burgess W.H. Tenner A.J. Immunity. 1997; 6: 119-129Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). On myeloid cells, C1qRp mediates phagocytosis of antibody-coated particles, but the function of C1qRp on normal endothelial cells is largely unknown (33Hess K.L. Tudor K.S. Johnson J.D. Osati-Ashtiani F. Askew D.S. Cook-Mills J.M. Exp. Cell Res. 1997; 236: 404-411Crossref PubMed Scopus (58) Google Scholar).Predictions about the membrane-proximal extracellular domain focus on fewer structural features. Thus, the high content of proline, threonine, and serine residues indicates likely attachment points for more than 30 O-linked carbohydrate side chains consistent with the sialomucin-like features of endosialin (28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Certain sialomucins have been implicated in sequestering growth factors to the plasma membrane and presenting these factors to endothelial cell surface receptors (34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 35Goretzki L. Burg M.A. Grako K.A. Stallcup W.B. J. Biol. Chem. 1999; 274: 16831-16837Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar); endosialin may be a candidate for such a function unique to tumor endothelium.Apart from further biochemical investigations that will benefit from the availability of mAb FB5 and recombinant endosialin protein, study of defined alterations in endosialin gene expression and structure are now possible, including genetic manipulations in mice and rats, which harbor a close homologue of endosialin. In conclusion, key experiments are now within reach to explore the endosialin system in human physiology and disease and to extend these investigations into experimentally more amenable rodent models. A better understanding of endosialin may even enable new approaches to cancer detection and treatment. We have cloned the gene coding for the human endosialin core protein based on several lines of evidence. First, transfection of endosialin cDNA into HeLa-S3 cells leads to induction of cell surface reactivity with the cognate mAb FB5. Secondly, mAb FB5 detects the expected 165-kDa sialoglycoprotein in the transfectants, with a core protein migrating as a 95-kDa species on SDS gels. Third, the endosialin gene maps to chromosome 11q13, refining the previous assignment to 11q13-qter based on serologic analysis of somatic cell hybrids with mAb FB5. Finally, the amino acid sequence provides the requisite attachment sites for abundant O-glycosylation of endosialin. The present study is not the only evidence linking endosialin induction to vascular endothelial cells in human cancer. Rather, an independent line of investigation, aimed at dissecting comprehensive gene expression profiles in cancers with the serial analysis of gene expression (SAGE) method, has implicated endosialin-specific ESTs in tumor angiogenesis. St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) employed SAGE to survey about 32,500 unique gene transcripts for differential expression in purified tumor endothelium versus endothelium of nonmalignant tissue. The study identified 46 transcripts elevated at least 10-fold in tumor endothelium, and the highest and most consistent up-regulation was noted for a hitherto uncharacterized gene, deposited as ESTs of unknown function in data bases and designated tumor endothelial marker 1 (TEM1) by St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar). We show here that TEM1 is the endosialin gene. In their study, St. Croix et al. (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) confirmed the SAGE data and showed by in situ RNA hybridization that TEM1/endosialin expression in vivo is selective for tumor endothelium, with no detection in the malignant epithelial cells of colorectal and other cancer, a range of normal organs, and a panel of cultured tumor cell lines. Nevertheless, TEM1/endosialin mRNA was not unique to tumor blood vessels because endothelial cells associated with wound healing and corpus luteum formation also showed gene expression by in situ RNA hybridization. Taking together the endosialin expression data generated with mAb FB5 (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar) and the RNA expression data derived from SAGE and in situ RNA hybridization studies (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar), a consistent picture of endosialin induction in tumor endothelium emerges. Such a close agreement between epitope and protein/mRNA expression is instructive for a molecule like endosialin, which falls into the group of sialomucin-type glycoproteins. There is precedent for sialomucins and other glycoconjugates for which mAb-defined epitope expression and protein/mRNA distribution sharply diverge. Several levels of complexity have been described (26Hakomori S. Curr. Opin. Immunol. 1991; 3: 646-653Crossref PubMed Scopus (218) Google Scholar, 27Girling A. Bartkova J. Burchell J. Gendler S. Gillett C. Taylor-Papadimitriou J. Int. J. Cancer. 1989; 43: 1072-1076Crossref PubMed Scopus (301) Google Scholar, 28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 29Puri K.D. Finger E.B. Gaudernack G. Springer T.A. J. Cell Biol. 1995; 131: 261-270Crossref PubMed Scopus (143) Google Scholar), including (i) epitopes on core proteins that are masked by glycosylation or specific sialylation, (ii) epitopes on carbohydrate moieties shared by several unrelated glycoproteins or glycolipids, and (iii) epitopes on distinct splice variants or processing variants of the same core protein. In the case of endosialin, the observed cotyping for the FB5 epitope and TEM1/endosialin mRNA suggests that mAb FB5 binds to an invariant epitope on the 95-kDa core protein. Nevertheless, this conclusion needs to be confirmed for each cell type under investigation, because epitope masking and mimicry can show exquisite cell type selectivity. A persistent question in the exploration of tumor angiogenesis is the extent of phenotypic and functional heterogeneity that may distinguish subsets of tumor endothelial cells in a macroscopic tumor lesion with regard to their derivation from pre-existing blood vessels or new vessel sprouting, and their function and state of activation in response to varying milieus of tumor-derived, paracrine angiogenesis inducers and inhibitors. Generally, the capillary bed of a locally advanced or metastatic tumor mass will extend into areas of relatively stable blood supply and tissue architecture and closely juxtaposed or intermingled regions of hypoxia, necrosis, excessive proliferation, tissue invasion, fibroblastic remodeling, or inflammatory cell infiltration. Placed in this context of malignant tissue disruption, markers of tumor angiogenesis might be expected to display a heterogeneous pattern of expression as a rule rather than exception. Considering the marked heterogeneity in FB5 staining in tumor vessels (1Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Jaffe E.A. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10832-10836Crossref PubMed Scopus (204) Google Scholar), it may be surprising that the report on TEM1/endosialin mRNAin situ hybridization (25St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar) does not address this aspect. One explanation would suggest that endosialin mRNA is uniformly expressed in tumor endothelium and that only subsets of endothelial cells accumulate the endosialin protein or protein with the FB5 epitope; however, as outlined above, there is currently no evidence supporting such a mechanism. Alternatively, it may be argued that mRNA in situ hybridization is the more sensitive detection method for endosialin and that FB5 immunostaining marks tumor endothelium with the highest levels of protein/epitope density rather than a distinct tumor endothelial cell type. Finally, there may simply be differences in the design of the two available studies on endosialin expression. Our analysis shows that the N-terminal extracellular portion of endosialin is likely to consist of five domains with globular structure, and in this region homology to thrombomodulin and the complement receptor C1qRp, with 39 and 33% amino acid sequence identity, respectively, is observed. Importantly, a pattern of cysteine residues is highly conserved among the three proteins and provides a scaffold of anchoring points for disulfide bridges, presumably imposing similar three-dimensional structures on each protein. Thrombomodulin exhibits a C-type lectin domain, six EGF-like repeats, and a serine/threonine-rich region in the extracellular domain. Unlike endosialin, thrombomodulin is expressed on a variety of normal cell types and on normal vascular endothelium serves as a receptor for thrombin, modulating the coagulation cascade and triggering the thrombin-activable fibrinolysis inhibitor (30Kokame K. Zheng X. Sadler J.E. J. Biol. Chem. 1998; 273: 12135-12139Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The thrombin interaction depends on the EGF repeats 5 and 6 of thrombomodulin (31Wood M.J. Sampoli Benitez B.A. Komives E.A. Nat. Struct. Biol. 2000; 7: 200-204Crossref PubMed Scopus (43) Google Scholar,32Wang W. Nagashima M. Schneider M. Morser J. Nesheim M. J. Biol. Chem. 2000; 275: 22942-22947Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which are not present in endosialin, making it unlikely that the two molecules overlap in this particular function. The complement receptor C1qRp is also expressed on a wider variety of cell types than endosialin, including macrophages, neutrophilic granulocytes, and normal vascular endothelium. The extracellular portion of C1qRp is composed of a C-type lectin domain, a tandem of five EGF-like repeats, and a mucin-like region. The molecule binds the complement factor C1q, mannose-binding lectin, and pulmonary surfactant protein A (22Nepomuceno R.R. Henschen-Edman A.H. Burgess W.H. Tenner A.J. Immunity. 1997; 6: 119-129Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). On myeloid cells, C1qRp mediates phagocytosis of antibody-coated particles, but the function of C1qRp on normal endothelial cells is largely unknown (33Hess K.L. Tudor K.S. Johnson J.D. Osati-Ashtiani F. Askew D.S. Cook-Mills J.M. Exp. Cell Res. 1997; 236: 404-411Crossref PubMed Scopus (58) Google Scholar). Predictions about the membrane-proximal extracellular domain focus on fewer structural features. Thus, the high content of proline, threonine, and serine residues indicates likely attachment points for more than 30 O-linked carbohydrate side chains consistent with the sialomucin-like features of endosialin (28Morgan S.M. Samulowitz U. Darley L. Simmons D.L. Vestweber D. Blood. 1999; 93: 165-175Crossref PubMed Google Scholar, 34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Certain sialomucins have been implicated in sequestering growth factors to the plasma membrane and presenting these factors to endothelial cell surface receptors (34Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 35Goretzki L. Burg M.A. Grako K.A. Stallcup W.B. J. Biol. Chem. 1999; 274: 16831-16837Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar); endosialin may be a candidate for such a function unique to tumor endothelium. Apart from further biochemical investigations that will benefit from the availability of mAb FB5 and recombinant endosialin protein, study of defined alterations in endosialin gene expression and structure are now possible, including genetic manipulations in mice and rats, which harbor a close homologue of endosialin. In conclusion, key experiments are now within reach to explore the endosialin system in human physiology and disease and to extend these investigations into experimentally more amenable rodent models. A better understanding of endosialin may even enable new approaches to cancer detection and treatment. The expert technical assistance of Claudia Eiberle, Elfriede Müller, Werner Rust, and Kai Zuckschwerdt is gratefully acknowledged. Prof. Junichi Sakamoto kindly provided cell line MF-SH. Dr. Elisabeth Stockert kindly supplied purified mAb FB5. We thank Dr. Lloyd J. Old for valuable contributions and suggestions. Sven Christian's contribution was in partial fulfillment of his Ph.D. thesis; he acknowledges the support of Prof. Klaus Pfizenmaier throughout the work.
Referência(s)