1H NMR studies of the Fc region of human IgG1 and IgG3 immunoglobulins: Assignment of histidine resonances in the C H3 domain and identification of IgG3 protein carrying G3m(st) allotypes

Abstract

A 1H NMR study of the Fc region of human IgG1 and IgG3 immunoglobulins is presented. 1H 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 H3 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 H2-C H3 contact. Assignments of the C2-H proton signals of His-429 and His-433 in the C H3 domain were made on the basis of our previous 1H 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 1H 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 H2 and C H3 domains make contact with each other.