Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin

Abstract

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (~50 residues), a central protease-sensitive hinge region (~70 residues) and a C-terminal stable endostatin domain (~180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.

Structure and Function of Collagen‐Derived Endostatin Inhibitors of Angiogenesis

“…The consistent observation of NC1 trimerization and other biochemical data led to the proposal of a segmental NC1 structure (20). At the N-terminus there is an association domain of about 60 residues probably including a short telopeptide connection to the triple-helix; at the C-terminus there is the 180-residue endostatin module (Fig.…”

“…Soluble monomeric endostatins could be obtained, however, from insect cells (4), yeast (17,19), and mammalian cells (20)(21)(22) but they may show subtle differences in conformation and posttranslational modifications. Domain NC1 could be also produced in mammalian cells and was shown to assemble noncovalently into a 100-kDa homotrimer as demonstrated for mouse collagens XVIII (20) and XV (21) and the C. elegans homolog (15).…”

“…Numbers refer to approximative sequence positions within NC1. The same structure of NC1-XV differs by a shorter hinge region (∼20 residues) containing only a few protease-sensitive sites (20,21).…”

“…This identified both collagens as components of several basement membranes and of many large vessels and capillaries suggesting that endothelial, some epithelial and smooth muscle cells, as well as fibroblasts are involved in collagen XV/XVIII synthesis [see (7) for references]. Recent studies with antibodies against NC1/endostatin have added more data on C-terminal processing, ultrastructural localizations, and circulating forms (20,21,31). Tissue extracts contain substantial amounts of a 35-38-kDa NC1-XVIII fragment, accompanied by shorter (22-30-kDa) endostatin forms, which were particularly prominent in aorta, demonstrating a complete processing of collagen XVIII.…”

Endostatin expression in a pancreatic cell line is modulated by a TNFα-dependent elastase

“…In vitro studies identified secreted cathepsin L as an enzyme capable of generating endostatin, whereas the matrix metalloproteases produce larger fragments in an alternate pathway . In vivo, proteolytic processing of collagen XVIII can generate both the NC1 trimer and the 20 kDa endostatin monomer (Sasaki et al, 1998;Wen et al, 1999) since both fragments are found in tissues and serum (Standker et al, 1997;John et al, 1999). In vitro, the NC1 fragment is more sensitive to proteolytic degradation than 20 kDa endostatin (Ferreras et al, 2000).…”