PMID: 10441114

 

    Legend: Sugar

Title : Novel glycosylated forms of human plasma endostatin and circulating endostatin-related fragments of collagen XV

Abstract :
  1. Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate
  2. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting
  3. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117) HLRPAR
  4. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII , respectively
  5. These polypetides are found to be O-glycosylated at T(125) ( residue 9 ) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues
  6. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis
  7. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation
  8. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins
  9. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate
  10. These polypeptides exhibit the N-terminal sequences P(66) HLLPPP
  11. and Y(81) EKPALH
  12. of the collagen XV NC-1 domain
  13. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa
  14. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments