J. Michael Kehoe

STRUCTURE-FUNCTION RELATIONSHIPS AMONG ANTI-GAMMA GLOBULIN ANTIBODIES*

The three principal experimental approaches to the study of the antibody combining site have been antigenic analysis, amino acid sequence studies and affinity labeling. We first attempted to delineate common antigenic properties among antibodies with similar specificities, utilizing antisera produced against isolated cold agglutinins, which, after absorption, showed the expected strong specificity for the immunizing antigen. However, most of these antisera also reacted with heterologous cold agglutinins but not with 50 other macroglobulins that lacked the cold agglutinin property.’ These studies indicated that there were a group of antigenic determinants on macroglobulins with cold agglutinin activity that could be localized to the Fab portion of the molecule. The term “cross specificity” was applied to this antigenic similarity, thus distinguishing it from the “individual antigenic specificity” which is readily observed with cold agglutinins. In related studies in a mouse system. Cohn and coworkers 2 and Potter and associates 3 have independently demonstrated a similar phenomenon. It thus seems clear that antibodies with similar specificities may share certain antigenic characteristics. A reasonable interpretation is that the similarity they share relates to the antibody combining site. Sequence studies of immunoglobulins have been carried out largely on either homogeneous immunoglobulins devoid of recognizable antibody activity or on pooled heterogeneous antibodies.-‘-‘ On the basis of these studies, antibody activity is believed to be associated with the variable regions of the heavy and light chains. It seems logical to assume that an area of such variability would provide the required diversity for the many different specificities attributed to antibodies. Within the light chain variable regions three small stretches of extreme variability, termed hypervariable regions, have been demonstrated.8-10

Phylogenetic Aspects of Immunoglobulin Variable Region Diversity

There is now widespread agreement that the antigen-binding function of immunoglobulin molecules is mediated by the variable regions of their heavy and light polypeptide chains. Studies from a number of laboratories have shown that many general features of antibodies, such as the capacity to interact with a given antigen (e.g., dinitrophenyl), susceptibility to proteolytic cleavage, and mediation of biological properties (e.g., complement fixation), are shared across a wide phylogenetic spectrum of animal species. From a broad biological standpoint, an important question arises. To what extent do these various animal species produce similar immunoglobulin proteins to fulfill the functional requirements of the humoral response system? One approach to answering this question is to study the molecular products of the humoral response in a wide variety of species, especially including amino acid sequence analyses. Unfortunately, such studies have been impeded by the difficulty of obtaining sufficient quantities of pure material from the different species, in large part because of the restriction of the availability, until recently, of homogeneous myeloma proteins to man and the mouse. However, the discovery and sequence analysis of myeloma proteins from dogs and cats (Kehoe and Capra, 1972), progress in amino acid sequence studies of murine myeloma proteins (Bourgois and Fougereau, 1970; Hood and Talmage, 1970; Rudikoff et al., 1973), the recent discovery of myeloma proteins in rats (Bazin et al., 1972; Querinjean et al., 1974), and the remarkable complete sequence analyses performed by Cebra et al. (1971) on pools of immunoglobulins from inbred guinea pigs has considerably widened the scope of the available sequence data on the characteristics of antibody proteins from various species.

Amino Acid Sequence Studies of Human IgA Myeloma Proteins

The structure of the variable region of immunoglobulin heavy chains has been a major interest of our laboratory during the past four years. Recently, we have begun the determination of the complete amino acid sequence of two IgA myeloma proteins. Other sequence studies have permitted an analysis of the distribution of heavy chain variable region subgroups among the IgA immunoglobulins.

Evolutionary divergence of immunoglobulin constant region genes.

The development of the humoral immune response over evolutionary time has resulted in the occurrence of numerous immunoglobulin gene products in animal serum. The variable region gene products which are now known to be responsible for providing the distinct combining specificities that are required for interacting with the multitude of antigens a given individual encounters in its lifetime (1) are of obvious importance in this regard. Genetic complexity also exists on another level in the humoral response, however, albeit less pronounced. This complexity is presented at the level of constant region genes. The precise extent of this diversity is not yet clear, especially in any complete phylogenetic context since extensive information concerning the question is available at present only for higher species, particularly mammals. While this information has provided much knowledge regarding the extent of constant region diversity in these higher species, and may show us the highest degree of such divergence that has yet occurred in the biological world, the data obviously can speak only for a limited spectrum of the animal kingdom.