Pilar Garin-Chesa

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium.

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 investigation.

Molecular Cloning and Characterization of EndoGlyx-1, an EMILIN-like Multisubunit Glycoprotein of Vascular Endothelium

EndoGlyx-1, the antigen identified with the monoclonal antibody H572, is a pan-endothelial human cell surface glycoprotein complex composed of four different disulfide-bonded protein species with an apparent molecular mass of approximately 500 kDa. Here, we report the purification and peptide analysis of two EndoGlyx-1 subunits, p125 and p140, and the identification of a common, full-length cDNA with an open reading frame of 2847 base pairs. The EndoGlyx-1 cDNA encodes a protein of 949 amino acids with a predicted molecular mass of 105 kDa, found as an entry for an unnamed protein with unknown function in public data bases. A short sequence tag matching the cDNA of this gene was independently discovered by serial analysis of gene expression profiling as a pan-endothelial marker, PEM87. Bioinformatic evaluation classifies EndoGlyx-1 as an EMILIN-like protein composed of a signal sequence, an N-terminal EMI domain, and a C-terminal C1q-like domain, separated from each other by a central coiled-coil-rich region. Biochemical and carbohydrate analysis revealed that p125, p140, and the two additional EndoGlyx-1 subunits, p110 and p200, are exposed on the cell surface. The three smaller subunits show a similar pattern of N-linked and O-linked carbohydrates, as shown by enzyme digestion. Because the two globular domains of EndoGlyx-1 p125/p140 show structural features shared by EMILIN-1 and Multimerin, two oligomerizing glycoproteins implicated in cell-matrix adhesion and hemostasis, it will be of interest to explore similar functions for EndoGlyx-1 in human vascular endothelium.