The First Draft of the Endostatin Interaction Network

Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds to heparin/heparan sulfate and to a number of proteins, but its molecular mechanisms of action are not fully elucidated. We have used surface plasmon resonance (SPR) arrays to identify new partners of endostatin, and to give further insights on its molecular mechanism of action. New partners of endostatin include glycosaminoglycans (chondroitin and dermatan sulfate), matricellular proteins (thrombospondin-1 and SPARC), collagens (I, IV, and VI), the amyloid peptide Aβ-(1–42), and transglutaminase-2. The biological functions of the endostatin network involve a number of extracellular proteins containing epidermal growth factor and epidermal growth factor-like domains, and able to bind calcium. Depending on the trigger event, and on the availability of its members in a given tissue at a given time, the endostatin network might be involved either in the control of angiogenesis, and tumor growth, or in neurogenesis and neurodegenerative diseases.Endostatin is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It inhibits angiogenesis and tumor growth (1–3). The effect of endostatin depends on its concentration (4, 5), on the length of exposure (6), on the type of endothelial cells (7), and on the growth factor inducing cell proliferation (fibroblast growth factor 2 or VEGF)³ (8, 9).Endostatin binds to several membrane proteins including α5β1 and αvβ3 integrins (10, 11), heparan sulfate proteoglycans (glypican-1 and -4) (12), and KDR/Flk1/VEGFR2 (13). We have previously characterized the binding of endostatin to heparan sulfate chains (9), and of endostatin to integrins (11). Furthermore, we have shown that α5β1, αvβ3, and αvβ5 integrins bind to heparin/heparan sulfate (11).The broad molecular targets of endostatin suggest that multiple signaling systems are involved in mediation of its antiangiogenic action. Endostatin is a broad spectrum angiogenesis inhibitor that suppresses angiogenesis by blocking general mechanisms that govern endothelial cell growth (14), and initiates a complex network of signaling at the gene level (15). However, its molecular mechanism of action is still a matter of debate.An integrative view of the endostatin interaction network, including interactions between endostatin partners, is necessary to provide a clear understanding of how all these molecules work together to regulate angiogenesis, and tumor growth. This global approach places individual proteins into a functional context, and takes into account the fact that a single molecule such as endostatin can affect a wide range of other cell components. Indeed, most proteins and other components carry out their functions within a complex network of interactions and this approach based on protein-protein interaction networks has been developed for several years to give new clues on biological processes (16).This study was thus designed to identify additional extracellular partners of endostatin in an attempt to obtain new insights into its mechanisms of action, and the biological processes in which it participates. For this purpose, we have developed protein and glycosaminoglycan arrays using an automated surface plasmon resonance (SPR) platform. Proteins and glycosaminoglycans selected for SPR analysis were present in the same tissues or structures, such as basement membranes (17), brain (18), cartilage (19), or they were involved in the same physio-pathological processes (angiogenesis, neuro-degenerative diseases) as endostatin, and they were available as full-length molecules. Collagens I and VI, for example, have been selected because the α1 and α2 chains of collagen VI were determined to be potential pan-endothelial markers as was the α1 chain of collagen XVIII containing endostatin (20), and because the genes coding for the α1 and α2 chains of collagen I, and the α3 chain of collagen VI are up-regulated in angiogenic vessels and elevated in tumor endothelium (20). Some proteins and glycosaminoglycans were also included to serve as positive controls for well known interactions with the potential partners of endostatin. We report that endostatin binds to other endogenous angiogenesis inhibitor, the matricellular proteins thrombospondin-1 and SPARC, and to several collagens (I, IV, and VI). Other interacting partners of endostatin are transglutaminase-2, the amyloid peptide Aβ-(1–42), chondroitin, and dermatan sulfate.