Isolation and characterization of the circulating form of human endostatin

Abstract

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10^20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C-terminal fragment of collagen K K1(XVIII) was guided by MALDI-MS and the exact molecular mass determined by ESI-MS was found to be 18 494 Da. N-terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1^3 and 2^4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no antiproliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.z 1997 Federation of European Biochemical Societies.

Endogenous Inhibitors of Angiogenesis

“…Endostatin is an endogenous collagen XVIII-derived angiogenesis inhibitor identified and purified from murine hemangioendothelioma cell line (26) and later characterized in mice (27). It corresponds to a 20-kDa fragment derived from the COOHterminal NC1 domain of type XVIII collagen (26,28,29).…”

Low plasma levels of matrix metalloproteinase 9 permit increased tumor angiogenesis

“…One hundred micrograms of total plasma proteins from untreated or doxycycline-treated mice were loaded on 10% SDS ± PAGE and run under non-reducing conditions (Rivas et al, 1998;Standker et al, 1997). Gels were then transferred onto immobilon-P (Millipore) and incubated with a puri®ed mouse mAb (®nal concentration 10 mg/ml) that interact with a mouse angiostatin fragment (anti K1-3), as previously described (Pozzi et al, 2000).…”

Cysteine Cathepsins S and L Modulate Anti-angiogenic Activities of Human Endostatin

“…Matrix metalloproteinases (MMPs) have also been implicated in the release of high molecular mass endostatin forms (24 -30 kDa) (11). Despite high primary structural identity in collagen XVIII between human and mice, their hinge regions display some critical differences in their amino acid sequences (9,12). Consequently, the proteases involved in the release of human endostatin from collagen XVIII have not been clearly identified.…”