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

Abstract

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-coilrich 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.The EndoGlyx-1 antigen was identified in a survey of normal and neoplastic tissues conducted at the Ludwig Institute for Cancer Research in pursuit of new antigenic markers of vascular endothelium. A peculiar feature of the monoclonal antibody (mAb) 1 H572, the probe first used to discover EndoGlyx-1 (1), is its distinctive reactivity with human tissues. In an extensive immunohistochemical survey of normal human fetal and adult tissues as well as human cancer tissues, H572 immunostaining was found exclusively on blood vessel endothelium. Notably, these included capillaries, veins, arterioles, and muscular arteries, but interestingly no immunoreactivity was observed in the sinusoidal endothelial cells of the spleen and liver. In neoplastic tissues, H572 immunostaining was consistently found in tumor capillaries, including "hot spots" of neoangiogenesis in certain tumors (2). The endothelial staining pattern revealed a uniform cell surface and cytoplasmic distribution of the antigen, in some cases with an accentuated staining at the abluminal side of the endothelial cell layer. All nonendothelial cell types in normal and tumor tissues were unreactive with mAb H572. The expression of the antigen on cultured human tumor cell lines and normal cells in vitro was also studied in detail. Thus, a host of cultured transformed cells of mesenchymal, neuroectodermal, and epithelial derivation as well as normal lymphocytes, hematopoetic cells, and platelets we...

MULTIMERIN2 impairs tumor angiogenesis and growth by interfering with VEGF-A/VEGFR2 pathway

“…Alternatively, endogenous expression was downregulated by specific siRNA sequences, construct 4 giving the highest downregulation, which was optimal at 72 h post transduction (Supplementary Figure 1b and Figure 2d). In accordance with the fact that MMRN2 is located in tight contact with the endothelium (Christian et al, 2001), ECs adhered to MMRN2 even though the interaction was weaker compared with the adhesion to type I collagen, and was Ca 2 þ and Mg 2 þ independent (Figures 1b and c). Furthermore, EC adhesion on MMRN2 was not affected by a number of anti-integrin blocking antibodies, including anti-a 1 , anti-a 4 , anti-a v and anti-b 1 (data not shown).…”

“…In order to analyze its putative effects on ECs behavior we have either used purified recombinant MMRN2 (Figure 1a, left) or induced its overexpression in ECs through adenoviral transduction (Figure 1a, bottom, Supplementary Figure 1a, bottom). As previously demonstrated (Christian et al, 2001), the purified recombinant molecule barely entered a 4-12% polyacrylamide gel under non-reducing conditions (Figure 1a, right). Alternatively, endogenous expression was downregulated by specific siRNA sequences, construct 4 giving the highest downregulation, which was optimal at 72 h post transduction (Supplementary Figure 1b and Figure 2d).…”

“…Our investigation was driven by the hypothesis that its specific localization along the blood vessels and the intimate contact with ECs (Christian et al, 2001) could affect their behavior and hence play an important role in the regulation of angiogenesis.…”

“…The molecule displays a panendothelial expression pattern both in normal and tumoral vasculature, including hot spots of neovascularization in some tumors (Sanz-Moncasi et al, 1994;Christian et al, 2001;Huber et al, 2006;Koperek et al, 2007). It is specifically deposited along the blood vessels in tight juxtaposition with ECs and is also present in the luminal side of the vessels (Christian et al, 2001), however, its function has thus far remained obscure. Here, we show evidence that MMRN2 contributes to the maintenance of blood vessels' homeostasis, and that its overexpression affects EC migration and tumor angiogenesis.…”

“…MULTIMERIN2 (MMRN2), also known as EndoGlyx-1, is an ECM member of the EDEN (EMI Domain ENdowed) protein family (Braghetta et al, 2004) associated with a high molecular weight glycoprotein complex (Christian et al, 2001). The molecule displays a panendothelial expression pattern both in normal and tumoral vasculature, including hot spots of neovascularization in some tumors (Sanz-Moncasi et al, 1994;Christian et al, 2001;Huber et al, 2006;Koperek et al, 2007).…”