Determination of the active site of CD59 with synthetic peptides.
CD59 inhibits the formation of membrane attack complex (MAC) of human complement by binding to C8 and C9 in the nascent membrane attack complex and inhibiting C9 binding to C8 in C5b-8 and C9 polymerization. Considering five disulfide bridges of CD59, we divided the molecule into two portions and synthesized the two peptides. One represented an amino-terminal half, P1-41, consisting of residues 1-41, while another represented a carboxyl-terminal half, P42-77, consisting of residues 42-77. P1-41 inhibited the MAC formation much more strongly than P42-77, indicating that the amino-terminal half contained the active site. We further synthesized P4-18 that consisted of residues 4-18 and P19-41 that consisted of residues 19-41. The activity of P4-18 was less than that of P19-41. Surprisingly, P19-41 showed higher activity than P1-41 and was comparable to urine CD59. Residues 19-41 were further divided into two portions: P20-25 which consisted of residues 20-25 and P27-38 which consisted of residues 27-38. Although their activities were significantly less than the activity of P19-41, P27-38 showed higher activity than P20-25. Residues 27-38 were further divided into three portions: P27-32 which consisted of residues 27-32, P30-34 which consisted of residues 30-34 and P33-38 which consisted of residues 33-38. When these peptides were assayed for the activities, all of them showed significant activities, even though they needed 10-fold more concentrations than P19-41. These data suggest that the portion made up of residues 27-38 is the active site constituting the binding site to C8 and C9.
Nakano
Y
Y
Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Tozaki
T
T
Kikuta
N
N
Tobe
T
T
Oda
E
E
Miura
N
N
Sakamoto
T
T
Tomita
M
M
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000652:urine; D001665:Binding Sites; D001681:Biological Assay; D018957:CD59 Antigens; D003186:Complement C9 / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000652:urine; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D013329:Structure-Activity Relationship
D015703:Antigens, CD; D018957:CD59 Antigens; D003186:Complement C9; D017931:DNA Primers; D008562:Membrane Glycoproteins; D010455:Peptides