Structurally inherent antigenic sites. Localization of the antigenic sites of the α-chain of human haemoglobin in three host species by a comprehensive synthetic approach

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major ‘continuous’ antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of ‘structurally inherent antigenic sites’, namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

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Antigenic structure of human haemoglobin. Localization of the antigenic sites of the β-chain in three host species by synthetic overlapping peptides representing the entire chain

Biochemical Journal ◽

10.1042/bj2340441 ◽

1986 ◽

Vol 234 (2) ◽

pp. 441-447 ◽

Cited By ~ 34

Author(s):

N Yoshioka ◽

M Z Atassi

Keyword(s):

Host Species ◽

Structural Characteristics ◽

Three Dimensional ◽

Antigenic Structure ◽

Synthetic Approach ◽

Immune Recognition ◽

Beta Chain ◽

Human Haemoglobin ◽

Antigenic Sites ◽

Full Profile

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

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A novel and comprehensive synthetic approach for the elucidation of protein antigenic structures. Determination of the full antigenic profile of the α-chain of human haemoglobin

Biochemical Journal ◽

10.1042/bj1910261 ◽

1980 ◽

Vol 191 (1) ◽

pp. 261-264 ◽

Cited By ~ 81

Author(s):

A L Kazim ◽

M Z Atassi

Keyword(s):

Primary Structure ◽

Synthetic Approach ◽

Human Haemoglobin ◽

Alpha Chain ◽

Antigenic Sites ◽

Full Profile ◽

Α Chain ◽

First Time ◽

Antigenic Profile

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.

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The antigenic structure of poliovirus

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1989.0024 ◽

1989 ◽

Vol 323 (1217) ◽

pp. 467-478 ◽

Cited By ~ 27

Keyword(s):

Monoclonal Antibodies ◽

Virus Particle ◽

Structural Changes ◽

Three Dimensional ◽

Antigenic Structure ◽

X Ray ◽

Antigenic Sites ◽

Type 3 ◽

Insight Into

We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus by X -ray crystallographic methods. By providing a three-dimensional framework for the interpretation of a wealth of experimental data, the structures have yielded insight into the architecture and assembly of the virus particle, have provided information regarding the entry of virus into susceptible cells, and defined the sites on the virus particle that are recognized by neutralizing monoclonal antibodies. Thus locating mutations in variants selected for resistance to neutralizing monoclonal antibodies has defined three antigenic sites of the surface of the virion, and provided clues as to the mechanisms by which viruses escape neutralization. Finally, comparison of the structures of the two strains, together with analysis of sequences of many poliovirus strains, have begun to define the structural changes associated with serotypic differences between polioviruses.

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Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the α-chain of human haemoglobin

Biochemical Journal ◽

10.1042/bj1970507 ◽

1981 ◽

Vol 197 (2) ◽

pp. 507-510 ◽

Cited By ~ 18

Author(s):

A L Kazim ◽

M Z Atassi

Keyword(s):

Binding Site ◽

Synthetic Approach ◽

Protein Chain ◽

Human Haemoglobin ◽

Alpha Chain ◽

Α Chain

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

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Prediction and conformation by synthesis of two antigenic sites in human haemoglobin by extrapolation from the known antigenic structure of sperm-whale myoglobin

Biochemical Journal ◽

10.1042/bj1670275 ◽

1977 ◽

Vol 167 (1) ◽

pp. 275-278 ◽

Cited By ~ 42

Author(s):

A L Kazim ◽

M Z Atassi

Keyword(s):

Sperm Whale ◽

Antigenic Structure ◽

Homologous Proteins ◽

Human Haemoglobin ◽

Antigenic Sites ◽

Inductive Effects ◽

Structural Analogy ◽

Starting Model ◽

Sperm Whale Myoglobin

The complete antigenic structure of sperm-whale myoglobin was previously determined in our laboratory. By structural analogy with myoglobin, two regions in human haemoglobin were predicted to comprise antigenic sites. One region was on the alpha-chain [alpha-(15-23)] and the other on the beta-chain [beta-(16-23)]. These two regions were synthesized, purified and characterized, and their immunochemistry was studied. Each peptide was able specifically to bind considerable amounts of haemoglobin antibodies. In a set of homologous proteins, barring any drastic conformational or electrostatic inductive effects exerted by the substitutions, and allowing for obstruction due to subunit interaction, the determination of the antigenic structure of one protein may serve as a useful starting model for the others.

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The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species

Biochemical Journal ◽

10.1042/bj1910681 ◽

1980 ◽

Vol 191 (3) ◽

pp. 681-697 ◽

Cited By ~ 53

Author(s):

S S Twining ◽

H Lehmann ◽

M Z Atassi

Keyword(s):

Amino Acid ◽

Antibody Response ◽

Three Dimensional ◽

Sperm Whale ◽

Cross Reactivity ◽

Antigenic Structure ◽

Amino Acid Substitutions ◽

Antigenic Sites ◽

The Cross ◽

Sperm Whale Myoglobin

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.

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Subunit interacting surfaces of human haemoglobin. Localization of the α-subunit-β-subunit interacting surfaces on the β-chain by a comprehensive synthetic strategy

Biochemical Journal ◽

10.1042/bj2340457 ◽

1986 ◽

Vol 234 (2) ◽

pp. 457-461 ◽

Cited By ~ 6

Author(s):

N Yoshioka ◽

M Z Atassi

Keyword(s):

Binding Capacity ◽

Binding Activity ◽

The Other ◽

Synthetic Approach ◽

Beta Chain ◽

Human Haemoglobin ◽

Α Subunit ◽

Alpha Chain ◽

Synthetic Strategy ◽

Interacting Surfaces

A synthetic approach is introduced for localization of subunit interacting surfaces in oligomeric proteins. It consists of studying the binding activity of consecutive uniform overlapping peptides encompassing an entire subunit to the other, radiolabelled, subunit. This permits the establishment of the full profile of peptides that bind the other intact subunit. This approach has been demonstrated with haemoglobin, and its application here with the beta-chain peptides has enabled the localization on the beta-chain of the submolecular regions responsible for its binding to alpha-chain in solution. There was good agreement between the binding surfaces found here in solution and those expected from the crystal structure. There were also, however, some significant differences in the levels of binding found in solution and those expected from the crystal. Peptide 21-35 possessed much higher binding activity than would be expected from its contribution to subunit association in the crystal. Conversely, other regions expected to possess considerable binding capacity for alpha-chain either showed low (peptides 111-125 and 121-135) or almost no binding (peptides 91-105 and 101-115) capacity. On the other hand, two interacting surfaces (within peptides 11-25 and 71-85) that make a contribution in solution do not appear to play a role in the crystal. It is concluded that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should serve as an effective method for localization of subunit interacting surfaces of unknown proteins, even those that can be isolated only in traces.

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Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the β-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain

Biochemical Journal ◽

10.1042/bj2340453 ◽

1986 ◽

Vol 234 (2) ◽

pp. 453-456 ◽

Cited By ~ 21

Author(s):

N Yoshioka ◽

M Z Atassi

Keyword(s):

Binding Sites ◽

Solid Phase ◽

Three Dimensional ◽

Binding Activity ◽

Dimensional Structure ◽

Synthetic Approach ◽

Protein Chain ◽

Beta Chain ◽

Human Haemoglobin ◽

Β Chain

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

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