Analysis of C5b-8 binding sites in the C9 molecule using monoclonal antibodies: participation of two separate epitopes of C9 in C5b-8 binding.
C5b-8 binding sites in C9 were examined using mAbs raised against C9. Among 16 mAbs, two, designated P40 and X197, blocked C9-mediated EAC1-8 lysis. C9 pretreated with the mAbs failed to bind to EAC1-8 at 4 degrees C. In addition, the mAbs became inaccessible to the C9 that had been incorporated into EAC1-8 at 4 degrees C. These findings suggest that C9 binding to EAC1-8, but not its membrane spanning or polymerization, is blocked by mAbs. By immunoblotting analysis using alpha-thrombin proteolytic fragments derived from C9 [a N-terminal fragment of mol. wt 25,000 (C9a) and a C-terminal one of mol. wt 37,000 (C9b)] and tryptic fragments of C9 (mol. wts 53,000 (C9a') and 20,000 (C9b')), the epitopes of P40 and X197 were mapped to the N-terminal and C-terminal regions of C9b, respectively. Both P40 and X197 bound to the C9 polymerized with Zn2+ in the fluid phase, whereas X197 but not P40 reacted with the membrane attack complex (MAC) formed on membranes. The results suggest that two distinct epitopes are involved in C9 binding to EAC1-8, and behave in a different manner for globular C9 bound to EAC1-8 at 4 degrees C, C9 assembled in MAC, or poly-C9 induced by Zn2+. These mAbs may be useful in clarifying the conformational states of C9 and in analyzing the molecular interaction between C9 and its inhibitors.
Hatanaka
M
M
Department of Clinical Pathology, Osaka Medical College, Takatsuki, Japan.
Seya
T
T
Yoden
A
A
Fukamoto
K
K
Semba
T
T
Inai
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D003186:Complement C9 / Q000276:immunology / Q000378:metabolism*; D003165:Complement System Proteins / Q000378:metabolism*; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011108:Polymers; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D003186:Complement C9; D000939:Epitopes; D010446:Peptide Fragments; D011108:Polymers; C042295:complement C5b-8 complex; D003165:Complement System Proteins