Identification of a surface structure in the fourth component of human complement, C4, which becomes hidden upon activation by C1(-)s.
Treatment of complement component C4 with C1(-)s and methylamine induces a series of conformation changes such as to generate functional binding sites. A monoclonal antibody (mAb), Al 121/6, which does not inhibit the haemolytic activity of C4 was found to bind to native C4 and C4d, but not to C4b and methylamine-treated C4, unless these C4 derivatives were denatured. These results suggested that a linear epitope for mAb Al 121/6 in the C4d domain is originally located at the surface of C4 and becomes hidden as a result of conformational changes induced by C1(-)s or methylamine treatment. The hidden linear epitope was exposed again upon further cleavage of C4b into C4c and C4d. Trypsin digestion of C4d and its chemical modification with phthalic anhydride suggested that the epitope is located at the C-terminal 13 kDa region of C4d and that lysine residues are involved in the epitope. There is a single lysine residue at 1259 in the 13 kDa C-terminal side of C4d and the synthetic undecapeptide Leu1254-Asp1264 was found to inhibit the binding of C4 to mAb Al 121/6, suggesting that the epitope for mAb Al 121/6 is involved in the sequence. The N-terminal portion of the peptide is partly overlapping, with a highly hydrophobic amino acid sequence spanning residues Ala1249-Leu-Leu-His-Leu-Leu-Leu1255. The surface hydrophobicity of C4 has been reported to decrease upon treatment with C1(-)s and methylamine. So it appears that the hydrophobic sequence spanning Ala1249-Leu1255 may be hidden, together with the linear epitope, into the inner region of C4 upon treatment with C1s and methylamine.
Maeda
S
S
Faculty of Pharmaceutical Sciences, School of Medicine, Hokkaido University, Sapporo, Japan.
Takamaru
Y
Y
Fukatsu
J
J
Nagasawa
S
S
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003173:Complement C1s / Q000378:metabolism*; D003181:Complement C4 / Q000737:chemistry* / Q000378:metabolism*; D015934:Complement C4a / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D006461:Hemolysis; D006801:Humans; D008744:Methylamines / Q000494:pharmacology; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D003181:Complement C4; D000939:Epitopes; D008744:Methylamines; D015934:Complement C4a; C027451:methylamine; D003173:Complement C1s