M. Zouhair Atassi

Precise determination of protein antigenic structures has unravelled the molecular immune recognition of proteins and provided a prototype for synthetic mimicking of other protein binding sites

SummaryIntensive research in the authors 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.Intensive research in the authors 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.

Use of immunoadsorbents for the study of antibody binding to sperm whale myoglobin and its synthetic antigenic sites

Conditions for preparing immunoadsorbents of sperm-whale myoglobin and its five synthetic antigenic sites and for desorption of radiolabeled antibodies from the immunoadsorbents were studied. In immunoadsorbent titration studies, the sum of the amounts of antibodies bound in the plateau (maximum binding) by the adsorbents of the five sites accounted quantitatively for the entire (100%) antibody response to sperm-whale myoglobin.

Genetic control of the immune response to myoglobin. IV. Mouse antibodies in outbred and congenic strains against sperm-whale myoglobin recognize the same antigenic sites that are recognized by antibodies raised in other species

The determination of the entire antigenic structure of sperm-whale myoglobin (Mb) was initially performed with antisera raised in rabbits and goats. Subsequently, we demonstrated that the synthetic antigenic sites were effective in stimulating in vitro mouse T-cell proliferation and that this proliferative response was under genetic control, with each antigenic site being controlled by a unique Ir gene. To determine unambiguously whether T-cells recognize the same molecular features as do B-cells, it was necessary to establish whether mouse antibodies are directed against these same sites. In the present work, using immunoadsorbent titration studies, these synthetic antigenic sites were shown to bind mouse 125I-labelled antibodies against Mb. With each of three outbred mice and four congenic strains, the total amounts of antibodies bound by the five sites accounted quantitatively for the entire antibody response against Mb. Moreover, with the four congenic strains, only the sites that were active in T-cell proliferation bound significant amounts of antibodies. It was concluded that (at least for Mb) the molecular features recognized by B-cells are also recognized by T-cells. These studies also confirm that the antigeniclty of the sites Is Independent of the immunized species and is inherent in their three-dimensional locations.

Immunochemistry of serum albumin. X. five major antigenic sites of human serum albumin are extrapolated from bovine albumin and confirmed by synthetic peptides

Abstract In a recent report from this laboratory, we have predicted and confirmed by synthesis the locations of five major antigenic sites of bovine serum albumin. In view of the high structural similarity between bovine and human serum albumins, analogous regions of human serum albumin are predicted here to comprise antigenic sites in this protein. Inimunochemlcal studies with antlsera to human albumin and the synthetic antigenic sites of bovine albumin verified this prediction and also identified the major structural locations responsible for the immunochemical cross-reaction between these two albumins.

Antibodies with specificities to preselected protein regions evoked by free synthetic peptides representing protein antigenic sites or other surface locations: Demonstration with myoglobin

Previous studies have resulted in the determination of the entire structure of myoglobin. The present work was carried out to investigate the antigenicity of the synthetic antigenic sites and other surface peptides of Mb in their free form (i.e. without coupling to a carrier). Site 1 (peptide 15-22), site 2 (peptide 56-62), site 3 (peptide 94-100), site 4 (peptide 113-120), site 5 (peptide 145-151) and two surface peptides, peptide 1-6 and peptide 121-127, were injected in complete Freunds adjuvant into Balb/c mice. Radioimmune antibody binding studies showed that immunization with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to Mb and to the immunizing peptide. The advantage and significance of these findings are discussed.

Immune recognition of serum albumin—XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin

Abstract Recently, this laboratory reported an autoreactivity of myoglobin (Mb)‡ antisera with the Mb of the species in which the antisera are raised. Also, animals injected with autologous Mb mounted an autoimmune antibody and T-lymphocyte proliferative response against this protein. This posed the possibility that autoimmune recognition might be a general phenomenon not confined only to sequestered proteins such as Mb. Using RSA, we have demonstrated unambiguously that RSA cross-reacted with rabbit antisera to bovine (BSA) or to human (HSA) albumin. In exchange experiments, 125I-labelled RSA was bound by the IgG in the immune complexes isolated from rabbit antisera to BSA or HSA. Also, 125I-antibodies were bound by RSA-adsorbents. Both binding activities were inhibited specifically by RSA. RSA was isolated from three rabbits and each rabbit was immunized with its own RSA. In each rabbit autoantibodies were found by exchange between immune complexes and the rabbits own [125I]-RSA. Also, 125I-antibodies from each rabbit were bound by adsorbents of the rabbits own RSA. Inhibition studies, data on preimmune sera and on antisera against other proteins confirmed the specificity of the binding. The findings confirm the universality of autoimmune recognition and lend support to our previous suggestion that antigenic sites are ‘structurally-inherent’ in the protein.

Production of Monoclonal Antibodies to Surface Regions that are Non-Immunogenic in a Protein Using Free Synthetic Peptide as Immunogens: Demonstration with Sperm-Whale Myoglobin

Two synthetic peptides corresponding to surface regions of sperm whale myoglobin that are not antigenic in the native molecule were used in their free form (i.e. not coupled to a carrier) to immunize separate groups of Balb/cByJ mice. The synthetic peptides corresponded to regions 1-6 and 121-127. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing peptide. Monoclonal antibodies to each of the two surface regions were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were IgM (kappa). They expressed the same isotype as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection and that monoclonal antibodies with preselected submolecular binding specificities to non-antigenic surface regions in a protein molecule can be produced by the techniques of somatic cell hybridization when the corresponding free synthetic peptides are used as immunogens.

Antibodies to Sperm-Whale Myoglobin Evoked by Free Synthetic Peptides of an Antigenic Site

Previous studies from this laboratory have resulted in the determination of the entire antigenic structure of myoglobin (Mb). The present work was carried out to investigate the antigenicity of the synthetic antigenic sites of Mb in their free form (i.e. without coupling to a carrier) and the effect of peptide size on antigenicity. Site 5 (peptide 145-151) and the longer peptides 145-153, 143-153 and 132-153, each of which carries within it the region of site 5, were synthesized. Immunization of three different mouse strains with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to intact Mb. The advantages and significance of these findings are discussed.Previous studies from this laboratory have resulted in the determination of the entire antigenic structure of myoglobin (Mb). The present work was carried out to investigate the antigenicity of the synthetic antigenic sites of Mb in their free form (i.e. without coupling to a carrier) and the effect of peptide size on antigenicity. Site 5 (peptide 145-151) and the longer peptides 145-153, 143-153 and 132-153, each of which carries within it the region of site 5, were synthesized. Immunization of three different mouse strains with each of these peptides, in their free form, resulted in the formation of antibodies that bound specifically to intact Mb. The advantages and significance of these findings are discussed.

GENETIC CONTROL OF THE IMMUNE RESPONSE TO HEN'S EGG‐WHITE LYSOZYME IN MICE: I. ANTIBODY AND T‐LYMPHOCYTE PROLIFERATIVE RESPONSES TO THE NATIVE PROTEIN*

We have initiated studies to determine whether the antibody and T‐lymphocyte proliferative responses to lysozyme and its antigenic sites is genetically controlled in mice. Mice of the H‐2f, H‐2k and H‐2p were high responders, while haplotypes H‐2b, H‐2d, H‐2r and H‐2s were low responders. Studies with recombinants indicated that the immune response is controlled by two H‐2I region loci, one being in the I‐A subregion and the other may be in the I‐C subregions.

Preparation of T-lymphocyte lines and clones with specificities to preselected protein sites by in vitro passage with free synthetic peptides: Demonstration with myoglobin sites

Recently, this laboratory has demonstrated that antibodies to preselected regions of a protein can be obtained by immunization with free small synthetic peptides (6-7 residues) without conjugation to a carrier. In the present work, we report the use of free synthetic peptides representing myoglobin (Mb) antigenic sites to prepare T-cell lines and clones of preselected specificities. Lymph node cells from mice primed in vivo with sperm-whale Mb were periodically passaged in vitro with synthetic peptide. After several passages, the peptide-driven long term T-cell cultures responded to the intact protein and exclusively to the peptide that was used to drive the cells. From these cultures, T-cell clones were prepared that responded only to the driving peptide and to the whole protein. The ability to prepare T-cell lines and T-cell clones with preselected submolecular specificities to a protein by driving cultures with desired synthetic peptides affords an important and simple tool for basic immunological investigations and for clinical applications.