Biomedical subjects
Publications and source records attributed to M Z Atassi.
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These studies were undertaken to analyse the genetic control of the immune response to an oligomeric protein and the role of individual subunits in the regulation of the response. Human adult haemoglobin (Hb) was selected as a model for these studies because it is a well-characterized protein and its antigenic structure is being determined in our laboratories. Mice of various congenic strains were immunized with Hb and the lymph node cells from Hb-primed mice were challenged in vitro with Hb, and its alpha-chain and beta-chains as well as an appropriate control antigen. Lymphocyte proliferation was determined by 3H-thymidine incorporation. The data collected indicated that mice of the H-2b and H-2d haplotypes were high responders while H-2k, H-2s, H-2q and H-2j haplotype mice were low responders to Hb. Studies with H-2 recombinant strains indicated that the immune response to Hb and its subunits is determined by genes in the I-A subregion and the D end of the H-2 complex. The significance of these findings in terms of control and regulation of the overall response to native Hb are discussed.
We have previously shown that the antibody response to mammalian myoglobins is under genetic control. In the present study we examined antibodies to sperm-whale. Atlantic bottlenosed dolphin, Black Sea dolphin, horse and badger myoglobins, raised in high responder strains of mice, to ascertain whether there is genetic control of antibody affinity to mammalian myoglobins. Using antisera of varying dilutions, the binding to 125I-labelled homologous myoglobins was studied by inhibition with homologous myoglobin over a wide range of inhibitor concentration in a modified Farr essay. The results indicated that there are no large differences between high responder strains of mice in the affinity of antibodies to mammalian myoglobins.
Mice of independent haplotypes and several recombinant inbred strains were immunized with highly purified preparations of either the alpha-chain or beta-chain subunit of human adult haemoglobin. Cells from the sensitized lymph nodes were challenged in vitro with the appropriate subunit (or in some cases both chains) and cell proliferation assessed by 3H-thymidine incorporation. Mice of the H-2b and H-2d haplotypes were high responders to alpha-chain while mice of the H-2f, H-2j, H-2k, H-2r, H-2s, H-2u, and H-2v haplotype were low responders. the low responsivenesss of B10.A(4R) and B 10.MBR and high responsiveness of B10 indicated that the Ir gene(s) determining responsiveness to the alpha-chain subunit resides in the I-A subregion of the mouse major histocompatibility complex. Mice of the H-2d, H-2f, and H-2s haplotypes were high responders and H-2b, H-2j, H-2a, and H-2u haplotype mice were low responders to beta-chain. H-2k, H-2p, H-2r, and H-2v haplotype mice were intermediate responders to beta-chain. The low responsiveness of B10.S(8R) and B10.TL and the high responsivenes of B10.S(9R) mapped the Ir gene(s) to beta-chain to the I-A subregion. Data collected from challenging high responder cells with both subunit indicated that alpha-chain and beta-chain do not crossreact. These results are discussed in reference to earlier observations suggesting that the low responsiveness of some strains of mice to priming and challenging using the intact haemoglobin molecule might be due to a negative regulatory influence mediated by one of the subunits. In the absence of this influence these mice would respond normally.
Mice bearing the I-A subregion mutation bm12 were immunized and challenged with the alpha -subunit of human adult haemoglobin. Under conditions in which parental B6/Kh mice respond, B6.C-H-2bm12 mice are inhibited nearly 100% in their ability to respond to challenge to the alpha-chain of haemoglobin. D2.GD mice which express a variant Ae (E beta) polypeptide of the I-E subregion can respond as well as B10.D2 mice to both subunits (alpha- and beta-) of haemoglobin. These observations as well as other genetic mapping data confirm the I-A mapping of alpha -chain-specific Ir genes and extend the genetic fine mapping to the A beta gene within the I-A subregion or a combinatorial Ia determinant generated by an interaction of A alpha and A beta. In addition they implicate the Ia.8 specificity in determining immune responsiveness to alpha -chain determinants.
The determination of the entire antigenic structure of myoglobin has made it possible to focus attention on the molecular factors controlling and influencing immune recognition. Recently, using antisera raised against sperm-whale myoglobin (Mb) in six different host species and investigating their reactions with Mb from 15 species, we showed that the binding capacity of an antigenic site is influenced by substitutions within site residues as well as within the residues close (within 6.0 å) to the sites. Based on these effects it was possible to correlate, at least in qualitative terms, the expected effects of the substitutions in each Mb and its observed cross-reaction with antisera to sperm-whale Mb. In the present work, these correlations were tested using sperm-whale Mb antibodies raised in four inbred mouse strains and in which the amount of antibodies directed to each antigenic site was determined. The amounts of 125I-labelled antibodies that could be bound maximally be each of 12 Mb variants were determined. Because of genetic control, the response to some antigenic sites was not expressed and therefore permitted us to evaluate with a good degree of confidence the effects of amino acid replacements in various myoglobins upon the reactivity of the sites. The values of cross-reaction expected for each Mb variant from these considerations agreed well with the values found experimentally. The results confirm unambiguously that the major factors affecting the cross-reactions of proteins can be attributed to substitutions within the sites and within environmental residues of the sites.
Previously it has been shown that the immune responses to sperm-whale myoglobin are under H-2-linked Ir-gene control. More importantly the responses to the synthetic antigenic sites are each under separate genetic control. In the present studies we investigated the genetic control of the antibody response to four different myoglobins of known structure, to determine whether this genetic control is influenced by changes in the properties of the sites. The results suggest that genetic control of the responses to individual antigenic sites on a protein antigen is not only determined by the genetic constitution of the host species but also by the chemical properties of the individual sites. It appears that the H-2 subregions mapping the responses to given antigenic sites can also recognize other sites, which were previously unrecognizable in a homologous protein, if their chemical properties are suitably altered.
By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).
The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.
A comprehensive synthetic approach for the determination of continuous antigenic sites of proteins is presented. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire primary structure of the protein under study. Its application to the alpha-chain of human haemoglobin afforded, for the first time, a full profile of immunochemically active alpha-chain peptides and enabled the localization of all the major continuous antigenic sites of this haemoglobin subunit.
In recent studies, we have localized and confirmed by synthetic peptides five major antigenic sites in each of bovine and human serum albumins which are recognized by rabbit antibodies against the respective protein. In order to determine whether the antigenic sites of albumin are peculiar to the protein or are dependent on the species of the immunized host, we have studied here the specificity of the antibody response in outbred mice against bovine serum albumin. By immunoadsorbent titration studies, the synthetic antigenic sites, previously localized with rabbit antisera against bovine serum albumin, were shown to bind considerable amounts of specific mouse 125I-labelled antibodies against bovine serum albumin. The amounts of mouse anti-bovine serum albumin antibodies bound by the adsorbents of the synthetic sites were comparable to the amounts of rabbit anti-bovine serum albumin antibodies that could be bound. It was concluded that recognition of the antigenic sites of serum albumin is independent of the immunized species and is inherent in their structural and conformational uniqueness.
Intensive research in the author's laboratory had culminated in the determination and synthesis of all the antigenic sites of myoglobin in 1975 and of lysozyme in 1978. Very recently most of the antigenic sites of serum albumin were also localized and synthesized. These investigations provided the first unique insight into the molecular features responsible for the immune recognition of protein antigens and of the factors which determine and regulate the antigenicity of the sites. But moreover, these studies have charted a multi-approach chemical strategy for investigation and synthetic duplication of protein binding sites. Furthermore, the concept of 'surface-simulation' synthesis, which we introduced and developed during our determination of the antigenic structure of lysozyme, has provided a remarkable dimension of unlimited versatility for the synthetic mimicking of any type of protein binding sites. In this concept, the spatially adjacent residues of a protein binding site are linked directly via peptide bonds with appropriate spacers into a single peptide which does not exist in the protein but mimicks a surface region of it. This has proved to be a powerful concept in protein molecular recognition and has opened up many untapped avenues in investigation, duplication and perhaps manipulation of a variety of protein activities. In fact, binding sites representing other protein activities (including antibody combining sites) have or are now being mimicked synthetically in our laboratory by the concept of surface-simulation synthesis.