Identification and characterization of novel endogenous proteolytic forrns of the human anglogenesis inhibitors restin and endostatin

Restin and endostatin are C-terminal fragments of the nonco. Ilagenous domains of collagen XV and collagen XVIll exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic genic activity in vivo restricting the growth of solid tumors and metastasis. They are therefore currently being tested in clinical trials as anti-cancer drugs. We present the identification of new endogenous variants of both angiogenesis inhibitors isolated from a human hemofiltrate peptide library. Using an immunological screening approach with time-resolved rare earth metal fluorometry, immunoreactive compounds were purified chromatographically and characterized by mass spectrometry. We discovered four novel proteolytic products of restin as well as four variants of endostatin. Two endostatin products were characterized as short internal fragments (R-176-L-215 and R-176-S-219) of the entire molecule containing the recently identified beta(1) integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-lycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GaINAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V-117-S-311-GalNAc-Gal(2)-NANA) and asialoglycopeptides (V-117-S-311-GalNAc-Gal(2)). The four restin variants (R-1-R-IV) were identified with identical C- but different N-termini and no posttranslational modification (R-1: P-66-A(254), R-11: P75-A(254), R-111: Y-81-A(254) and R-IV: A(89)-A(254)). Following a differential peptide mass fingerprint approach by reflector mode MALDI-TOFMS, the disulfide patterns of these circulating restins were determined as Cys1-Cys4 and Cys2-Cys3. These endogenous circulating collagen fragments will help to understand the physiological processing of the therapeutic proteins. (C) 2004 Elsevier B.V. All rights reserved.