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M Z Atassi

Publications and source records attributed to M Z Atassi.

At least 127 records · Page 7Linked to original sources
PubMed1985

T-cell dependency of the antibody response to free small synthetic peptides of a protein: demonstration with an antigenic site of myoglobin.

Recent studies from this laboratory have found that synthetic peptides of proteins, as small as 6 residues, when administered in their free form (i.e. without coupling to any carrier) elicit the formation of antibodies with submolecular binding specificities to preselected protein regions. These peptides could represent either the antigenic sites of the protein or surface regions that are not immunogenic when the intact protein is the antigen. In either case the antibodies bind specifically to the intact protein, exclusively at the region used in immunization. In the present study we immunized BALb/c.ByJ and nude (BALb/c derived) strain mice with either Mb or synthetic antigenic site 5 of Mb. Radioimmune antibody binding studies showed that immunization of BALb/c.ByJ strain mice with either Mb or synthetic peptide resulted in the formation of antibodies that bound specifically to Mb, whereas nude mice did not produce such antibodies. These results indicate that the antibody response to both Mb and synthetic antigenic site is T-cell dependent.

Amino Acid Sequence↗
PubMed1985

Antigen presentation of lysozyme: T-cell recognition of peptide and intact protein after priming with synthetic overlapping peptides comprising the entire protein chain.

Recently, using synthetic overlapping peptides which encompass the entire protein chain of hen egg lysozyme, the full submolecular profile of continuous regions on the protein recognized by T cells (T sites) was localized. In the present report, we have examined in two mouse strains the proliferative response to peptides and to native protein of lymph node cells from mice primed with synthetic overlapping peptides, either individually or as a mixture. It was found that the pattern of T-cell recognition observed after priming with peptides differs from that obtained when the native protein is used as the immunogen. Some, but not all, of the T-site containing peptides were effective in priming for an anti-lysozyme T-cell response. Several peptides which were highly immunogenic as free synthetic peptides were not associated with any of the known protein T sites. Further, some peptides were effective in priming for T cells that respond in vitro to the priming peptide, but not to the whole protein. If antigen processing proceeds via fragmentation, then only those regions containing T sites would be expected to be effective in priming for a T-cell response to the intact protein. Since this was not found to be the case, it is unlikely that fragmentation of lysozyme is a prerequisite for antigen presentation. Rather, we suggest that the critical aspects in the presentation of a protein antigen predominantly involve recognition of an intact protein, the interaction of which with the cell membrane triggers cellular activating events.

Animals↗
PubMed1985

Discovery and implications of the immunogenicity of free small synthetic peptides: powerful tools for manipulating the immune system and for production of antibodies and T cells of preselected submolecular specificities.

Recent studies from this laboratory have found, contrary to a long-held belief, that synthetic peptides of a protein, as small as six residues, when immunized in their free form (i.e., without coupling to any carrier), elicit the formation of antibodies with submolecular binding specificities to preselected protein regions. These peptides could represent either the antigenic sites of the protein or surface regions that are not immunogenic when the intact protein is the antigen. In either case, the antibodies bind specifically to the intact protein, exclusively at the region used in immunization. Monoclonal antibodies with preselected specificities can also be produced by hybridoma technology from the spleens of the immunized animals. Furthermore, free synthetic peptides have been employed to generate T-cell lines and T-cell clones with specificities to preselected immunogenic locations and also to induce tolerance to such preselected locations. These fascinating breakthroughs indicate that free synthetic peptides may be used in the future as powerful tools in basic investigations and in therapeutic and diagnostic applications.

Adjuvants, Immunologic↗
PubMed1984

Localization and synthesis of the acetylcholine-binding site in the alpha-chain of the Torpedo californica acetylcholine receptor.

The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.

Acetylcholine↗
PubMed1984

Antigenic structures of proteins. Their determination has revealed important aspects of immune recognition and generated strategies for synthetic mimicking of protein binding sites.

Studies in this laboratory have resulted in the delineation and synthetic verification of several complete protein antigenic structures that are recognized by antibodies. More recently, for the first time, the full profiles of the sites that are recognized by T cells have been localized and confirmed by synthesis for two proteins, myoglobin and lysozyme. These have thus far constituted the only complete antigenic structures to be determined. The availability of these antigenic structures has enabled us to investigate in detail the molecular and cellular parameters responsible for immune recognition, responses to, and control and regulation of these responses to protein antigens at the molecular and submolecular levels. Moreover, these investigations have afforded general strategies for the synthetic mimicking of not only antigenic sites, but also protein binding sites involved in other biological activities.

Amino Acid Sequence↗
PubMed1984

Genetic control of the immune response to haemoglobin. V. Production and characterization of A beta restricted T cell lines and clones.

Monoclonal anti-Ia inhibition experiments were conducted to confirm and extend genetic mapping data of I-A gene control of immunity to human haemoglobin (Hb). It was found that the A beta gene is of critical importance in conferring immunity to the alpha-chain and beta-chain subunits of Hb. A possible involvement of I-E region genes in B10.D2 mice to beta-chain is discussed. Through the use of an alpha-chain specific T cell clone data, is obtained indicating that an intact Ia.8+ A beta chain is necessary for antigen presentation in vitro.

Animals↗
PubMed1984

T cell recognition of lysozyme. II. Shift in specificity during long-term culture determined by synthetic overlapping peptides comprising the entire protein chain.

Recently, by using a comprehensive synthetic strategy developed in this laboratory, we localized four sites (T sites) within the polypeptide chain of lysozyme recognized by T cells from two high responder mouse strains, DBA/1 and B10.BR. However, to detect minor specificities, the selective enrichment of lysozyme reactive cells, would be required. T cells from long-term cultures maintained by repeated stimulation with antigen are selectively enriched for that antigen. It is not known whether maintaining T cells for extended periods of time in vitro has any consequences on the profile of T cell recognition. In the present study, T cells from long-term cultures, derived from these two high responder lysozyme-primed mouse strains, were examined for their responsiveness to a series of synthetic overlapping peptides encompassing the entire polypeptide chain of the lysozyme molecule. We have found that the profile of T cell recognition of the long-term cultures may not reflect that of the lysozyme primed lymph node cells, but that it is subject to a shift in specificity towards submolecular features of the molecule. In addition, we have identified in B10.BR mice another region within the polypeptide chain of lysozyme (residues 72-84) which may potentially harbor a previously undetected (in lymph node cells) minor T site.

Animals↗
PubMed1984

A novel approach for localization of the continuous protein antigenic sites by comprehensive synthetic surface scanning: antibody and T-cell activity to several influenza hemagglutinin synthetic sites.

The determination in this laboratory of the complete antigenic structures of several proteins initially relied on a multi-approach complex chemical strategy and revealed that antigenic sites are surface locations which could be either 'continuous' or 'discontinuous' in architecture. More recently, we introduced a simplified comprehensive synthetic approach for the localization of the continuous antigenic sites of a protein. The approach depends on the synthesis of consecutive overlapping peptides, of uniform size and overlaps, and encompass the entire protein chain, from the beginning to the end. The latter approach is rather costly and labor-intensive, especially when applied to large protein molecules. All these studies showed, however, that protein antigenic sites occupy surface areas on a protein molecule. In order to render the determination of protein antigenic sites more feasible within a reasonable period of time, we considered that only the protein surface needs to be examined. Thus, for a protein of known three-dimensional structure, the protein surface can be readily screened for the continuous antigenic sites by the systematic synthesis and examination of immunochemical activity of all exposed segments of the protein. We have applied this approach here to influenza A virus hemagglutinin. Twelve peptides (11 reported for the first time here, and one reported previously), representing continuous surface segments of the molecule, have so far been synthesized, purified, characterized and their immunochemical activity studied. The peptides were found to bind anti-viral antibodies raised in outbred mice and antibodies in human sera from individuals that had suffered a recent influenza A infection. In one mouse strain (Balb/c; H-2d) so far examined, several of the peptides stimulated an in vitro proliferative response of T cells from virus (X-31)- primed mice. Finally, antisera to the peptides were raised in mice and, as expected, were found to bind to intact virus. In most cases, anti-peptide antibodies, did not bind disrupted virus. These studies indicate that protein 'continuous' antigenic sites can be localized by systematic synthetic scanning of the surface. It is emphasized that the approach is useful only for the localization of 'continuous' sites. The results also reveal that the antigenic structure of influenza virus hemagglutinin is more complex than has hitherto been suspected.

Amino Acid Sequence↗
PubMed1984

T-cell recognition of lysozyme. I. Localization of regions stimulating T-cell proliferative response by synthetic overlapping peptides encompassing the entire molecule.

The systematic localization of the full antigenic profile for T-cell recognition of a complex multivalent protein antigen has not been accomplished. Recently, this laboratory has developed a comprehensive strategy for the localization of all the continuous antigenic (as well as other binding) sites of a protein. The strategy depends on the synthesis of consecutive overlapping peptides that together systematically account for the entire polypeptide chain of the protein. This strategy was applied here for the localization of the 'continuous' T-cell recognition sites of hen egg lysozyme. Eight overlapping peptides encompassing the entire primary structure of lysozyme were synthesized and examined for their ability to stimulate in vitro proliferation of T cells from two high responder mouse strains (H-2k and H-2q) that had been primed with lysozyme. This approach enabled the identification of a full profile of in vitro active lysozyme peptides and the localization of four major T-cell recognition sites.

Amino Acid Sequence↗
PubMed1983

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites.

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes 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. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.

Animals↗
PubMed1983

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 Freund's 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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Localization, synthesis, and activity of an antigenic site on influenza virus hemagglutinin.

This paper reports the antigenicity of the fusion region of the influenza virus hemagglutinin (HA). Two peptides, comprising the fusion region (residues 1-11 of the HA2 part of HA) of strain A and strain B influenza virus, were synthesized and their abilities to bind rabbit, goat, and human anti-influenza antibodies were determined. In addition, 30 anti-HA monoclonal antibodies were examined for their ability to bind the synthetic peptides. In quantitative immunoadsorbent titrations, the two peptides bound considerable amounts of antibodies in rabbit and goat antisera against virus or HA of the A or B strain as well as in several human sera from patients recovering from influenza A. Of the 30 anti-HA monoclonal antibodies, 5 bound completely and 4 bound partially to the peptides. Antibodies were raised in rabbits against the peptides by immunizing with peptide-bovine serum albumin conjugates or with the free peptides. Anti-peptide antibodies were bound by HA and by the intact virus of the respective strain. However, these antisera failed to exhibit significant virus neutralizing activity. In contrast, the monoclonal antibodies that reacted with these peptides inhibited viral infectivity. The results clearly show that residues 1-11 of HA2 represent an important antigenic site on influenza virus.

Animals↗
PubMed1983

T-lymphocyte recognition of sperm-whale myoglobin. Recognition of synthetic peptides carrying antigenic site 5 by myoglobin-primed T-cells.

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm-whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T-cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145-153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time, up to 22 residues in length (peptide 132-153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr-151 and Ala-144. The T-cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb-primed mice afforded T-cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145-153 and 143-153) and more so to the longer peptides 135-153 and 132-153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity T-cells. No response was obtained also to intermediate-sized peptides. The latter result, due to an unfavourable mode of folding, suggested a conformational dependency in T-lymphocyte recognition.

Animals↗
PubMed1983

T-lymphocyte recognition of sperm-whale myoglobin. Specificity of T-cell recognition following neonatal tolerance with either myoglobin or synthetic peptides of an antigenic site.

Recently, in investigating the responses of T-cells from high responder mice that were primed with myoglobin (Mb) or with synthetic peptides containing antigenic site 5 and increasing in length stepwise by increments of two residues, we observed that T-cell recognition was highly dependent on conformation. In the present studies, tolerization experiments were carried out to further investigate this finding. Neonatal mice (BALB/cByJ) were either tolerized with Mb or with synthetic peptides of Mb containing antigenic site 5. Tolerization with Mb and subsequent immunization with Mb gave T-cells that did not proliferate in vitro to Mb or any of the peptides. T-cells from mice that were tolerized with a truncated peptide 139-153 (having deletions at Tyr-151 and Ala-144) and subsequently immunized with Mb proliferated in vitro to Mb and to peptides 132-153, 135-153 and 143-153. T-cells from mice that were tolerized with native Mb and subsequently immunized with peptides (which are unfolded in solution) did not proliferate in vitro to Mb, but responded well to the peptides. Conversely, tolerization with peptides had no effect on the recognition of, and the response to, native Mb by the T-cells, whereas the response to the peptides was completely removed. It was thus concluded that the recognition of protein antigens (or at least of Mb) by T-cells is (like the recognition by antibody) dependent on the conformation of the antigen.

Animals↗
PubMed1983

Genetic control of the immune response to myoglobin. XVI. Control of antibodies with preselected specificities following immunization with free synthetic peptides representing the antigenic sites or surface non-immunogenic locations in the protein.

Previous studies in this laboratory have resulted in the determination of the antigenic structure of myoglobin. The present work was carried out to investigate the genetic control of the murine antibody response to myoglobin following immunization with free (i.e., not coupled to a carrier) synthetic antigenic sites or other peptides corresponding to surface regions of myoglobin that are not immunogenic when the native molecule is the immunizing antigen. Synthetic peptides corresponding to antigenic 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 regions, peptide 1-6 and peptide 121-127, were injected in complete Freund's adjuvant in different strains of mice. Serum antibodies specific for myoglobin were subsequently obtained and were measured by means of a radioimmune plate binding assay in which Mb was used as the solid phase antigen. It was found that the genetic control of the antibody response to myoglobin following immunization with the free synthetic peptides was different from the genetic control obtained following immunization with native myoglobin. The significance of this finding is discussed.

Amino Acid Sequence↗
PubMed1983

Molecular localization of the full profile of the continuous regions recognized by myoglobin-primed T-cells using synthetic overlapping peptides encompassing the entire molecule.

The molecular localization of the full antigenic profile for T-cell recognition of a complex multi-determinant protein antigen has not to date been accomplished. Previously, this laboratory has introduced a comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein. This strategy depends on the synthesis of a series of overlapping peptides that together account for the entire structure of a protein. Such a strategy has been applied, in this report, to the delineation of the continuous sites of T-cell recognition of sperm whale myoglobin. Thirteen peptides, accounting for the entire protein chain, were synthesized and subsequently examined in vitro for their ability to stimulate lymph node cells from myoglobin primed DBA/2 (H-2d) mice, a known high responder. This strategy has enabled for the first time the localization of the full profile of the protein regions which contain the sites of T-cell recognition. Three regions gave a high response (one being immunodominant and coinciding with antigenic, i.e. antibody binding, site 4 of myoglobin). At least three regions appear to coincide with previously known antigenic (antibody binding) sites. Noteworthy is the finding of regions that are recognized by T-cells but to which no detectable antibody response is directed.

Animals↗