Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin.
Restin and endostatin are C-terminal fragments of the noncollagenous domains of collagen XV and collagen XVIII exhibiting high sequence homology. Both polypeptides are distinguished by strong anti-angiogenic 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 (R176-L215 and R176-S219) of the entire molecule containing the recently identified beta1 integrin receptor binding site, which plays a major role in endothelial cell migration and angiogenesis. Two additional forms contain mucin-type O-glycosylations. The O-glycosylated variants possess an oligosaccharide unit consisting of one N-acetylgalactosamine (GalNAc), one N-acetylneuraminic acid (NANA) and two galactose residues (Gal) occurring as sialo-(V117-S311-GalNAc-Gal2-NANA) and asialoglycopeptides (V117-S311-GalNAc-Gal2). The four restin variants (R(I)-R(IV)) were identified with identical C- but different N-termini and no posttranslational modification (R(I): P66-A254, R(II): P75-A254, R(III): Y81-A254 and R(IV): A89-A254). 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.
John
Harald
H
IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, D-30625 Hannover, Germany. H.John@IPF-PharmaCeuticals.de
Radtke
Kerstin
K
Ständker
Ludger
L
Forssmann
Wolf-Georg
WG
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000302:isolation & purification*; D001665:Binding Sites; D003094:Collagen / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry; D043169:Endostatins / Q000302:isolation & purification* / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins; D009369:Neoplasms / Q000378:metabolism; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D019151:Peptide Library; D016415:Sequence Alignment
D020533:Angiogenesis Inhibitors; D004220:Disulfides; D043169:Endostatins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D019151:Peptide Library; C076744:cytoplasmic linker protein 170; D003094:Collagen