1H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: Assignment of histidine resonances in the CH3 domain and identification of IgG3 protein carrying G3m(st) allotypes

Abstract A 1 H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. 1 H NMR data were collected for the Fc and pFc' fragments obtained from human monoclonal IgG1 and IgG3 and also from rabbit IgG. The C2-H proton signal of His-435 in the C H 3 domain of IgG1 was assigned by comparing the spectra of the Fc fragment of IgG1 with that of IgG3[G3m(g)] where there is a substitution of histidine by arginine at position 435. Chemical shifts and linewidths of the His-435 signal are quite different for the Fc and pFc' fragments. We suggest that His-435 is involved in the interdomain C H 2-C H 3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the C H 3 domain were made on the basis of our previous 1 H NMR results on the human light chain. NMR measurements clearly show that IgG3 Jir, which was isolated from a Japanese patient with cryoglobulinemia, has histidine at position 435 as in the case of IgG1. We also confirmed that IgG3 Jir reacts strongly with protein A. In marked contrast, IgG3[G3m(g)] does not bind protein A. These results show that binding of protein A to the Fc region is not subclass-specific and the existence of His-435 is a necessary condition for the protein A binding. It has recently been demonstrated that IgG3 proteins carrying G3m(st) allotypes. which are relatively common in Mongoloid populations but quite rare in Caucasians, bind protein A strongly. We confirmed that, as expected, IgG3 Jir carries G3m(st) allotypes. The pH titration curve of His-435 observed for IgG3 Jir is quite different from that for IgG1. This result makes it possible to identify by 1 H NMR IgG3 proteins carrying G3m(st) allotypes. In the case of the pFc' fragments, His-435 gives identical titration curves for IgG1 and IgG3[G3m(st)]. This is consistent with the fact that no serological distinction can be made between the pFc' fragments obtained from these two types of proteins. We suggest that G3m(st)-specific antiserum differentiates IgG1 and IgG3[G3m(st)] by recognizing the difference in the way in which the C H 2 and C H 3 domains make contact with each other.