Alloreactive monoclonal antibodies select Kd molecules with different peptide profiles.
As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies. First, we have studied the capacity of five different Kd-reactive mAbs to recognize a panel of 25 site-directed mutants of the H-2Kd molecule. Both the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indicating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab binding. Mutation of two alpha-helix-situated residues, 58 and 166, completely abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb. The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs. We have addressed this issue by comparison of the repertoire of peptides eluted from Kd molecules precipitated by different Kd-reactive mAbs. The results reveal that the two-dimensional profile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different. The use of 21 single amino acid variants of a Kd-restricted 10-mer peptide allowed us to identify the residue of the bound peptide contributing to the epitope recognized by this mAb. Thus, we have shown that at least in some instances, changes induced in the MHC molecules by the binding of distinct peptides can be recognized as alterations in serologic determinants expressed on the class I molecules.
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Mouse Immunogenetics, Unit 93 INSERM, Saint-Louis Hospital, Paris, France.
Reboul
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M
Abastado
J P
JP
Jaulin
C
C
Kourilsky
P
P
Pla
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007518:Isoantibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008805:Mice, Inbred A; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009154:Mutation / Q000276:immunology; D010455:Peptides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D007518:Isoantibodies; D010455:Peptides