Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9.
In the activated complement system, vitronectin (complement S-protein) occupies the metastable membrane binding site of the nascent precursor complex C5b-7, so that the newly formed SC5b-7 is unable to insert into cell membranes. Some evidence also indicates that vitronectin limits on-going membrane-associated pore formation by inhibiting C9 polymerization. It has been assumed that these two stages of terminal complement complex (TCC) inhibition take place through charge interactions between the heparin-binding region of vitronectin and homologous cysteine-rich sequences of the late complement proteins C6, C7, C8 and C9. We examined SC5b-7 formation and inhibition of C9 binding in the TCC using separate haemolytic assays. The mode of action of vitronectin in these assays was compared with two 15mer peptides which span residues 348-379 of the heparin-binding region, and a heparin-affinity polypeptide, protamine sulphate. The results showed that vitronectin acts predominantly through SC5b-7 production with a lesser effect on the inhibition of C9 lytic pore formation. In contrast, protamine sulphate did not prevent C5b-7 membrane attachment, but was a potent inhibitor of C9-mediated lysis. The peptides did not inhibit C5b-7 membrane insertion and only one affected C9 binding. These data suggest that the two stages of TCC inhibition involve separate binding sites on the vitronectin molecule. The site for association with nascent C5b-7 is unknown, whereas inhibition of C9 binding and pore formation takes place through the heparin-binding region.
Milis
L
L
Department of Nephrology, Prince Henry Hospital, Sydney, Australia.
Morris
C A
CA
Sheehan
M C
MC
Charlesworth
J A
JA
Pussell
B A
BA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D003182:Complement C5; D003186:Complement C9 / Q000378:metabolism*; D015941:Complement Hemolytic Activity Assay; D003169:Complement Inactivator Proteins / Q000494:pharmacology*; D015938:Complement Membrane Attack Complex / Q000187:drug effects* / Q000378:metabolism; D003171:Complement Pathway, Classical / Q000187:drug effects; D003165:Complement System Proteins / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000494:pharmacology*; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D011479:Protamines / Q000494:pharmacology; D012756:Sheep; D019096:Vitronectin
D003182:Complement C5; D003186:Complement C9; D003169:Complement Inactivator Proteins; D015938:Complement Membrane Attack Complex; D006023:Glycoproteins; D011479:Protamines; D019096:Vitronectin; C037453:complement C5b-7 complex; D003165:Complement System Proteins