May 28, 2023

Summary

Researchers are investigating why antibodies seem to recognize certain parts of antigens more readily than others. By conducting meticulous experiments, they have discovered that germline-encoded motifs known as GRAB interact with specific amino acid sequences during antibody immunity. These findings lay the groundwork for deeper insights into humoral immunity.

Background

Antibody production is a crucial aspect of humoral immunity in the immune response. Through DNA recombination, many species have the ability to create antibodies that can recognize all kinds of proteins. This suggests the presence of a large antibody library in organisms.

Interestingly, when different individuals are exposed to the threat of the same pathogen, the different antibodies they produce often only target specific epitopes. These epitopes are called "public epitopes,” their existence has yet to be clearly explained.

Experiment and Result

"Public Epitopes" Widely Present

To find viral sequences that can be widely recognized, Ellen et al. analyzed 569 human serum samples and identified 363 virus peptides. Among them, 199 can be recognized by at least 30% of individuals. These 363 peptides are derived from 62 viruses involving multiple classes. The antibody response against the same peptides does not depend on individuals’ age or geographic location, proving that these "public recognition peptides" are universally present in humans.

To reveal whether single or multiple epitopes within these peptides cause this public recognition, they further used truncation or serine/threonine mutagenesis screening. They found that different individuals often produce antibody responses to the same epitopes.

A Light Chain Preference for "Public Epitopes.”

The light chains of IgG are divided into κ and λ light chains, and there is no known functional difference between the two light chains. One antibody only has one type of light chain. The authors hypothesize that if different antibodies can recognize the same epitope, they should have structural similarities.

Further experimental results do indeed show this preference. For example, most antibodies produced against a "public recognition peptide" consisting of 56 amino acids in EBV are λ light chain type. In a "public recognition peptide" of a B-type rhinovirus, there are two "public epitopes,” each corresponding to an antibody with either κ or λ light chain type. These data indicate a light chain preference for the same "public epitope.”

Identification of "Public Epitopes" in Germline-encoded Sequences

After confirming the characteristics of the "public epitope,” researchers selected the "common recognition peptide segment" of three different viruses and isolated the corresponding B cell receptors (BCRs). The results showed that BCRs often have the same V gene segment, and the corresponding heavy chain CDR3 segment is not so conservative. This indicates that antibodies recognize the "public epitope" through the germline-encoded sequences in the V gene segment.

Further experiments also proved that these antibodies' recognition of the "public epitope" is particular, rather than multiple reactive antibodies that "happen" to recognize these epitopes.

Earlier, researchers found that edge lysine is essential in the "public epitope" of the λ light chain type. Therefore, the research team further screened nearly a thousand antibody-antigen complexes and found that the 51st amino acid of the λ light chain could directly react with lysine in the antigen. In this reaction, the 51st amino acid is often aspartic acid.

To further verify this, researchers selected two antibodies that recognize EBV, one of which can recognize edge lysine, and the other cannot. After mutating their 51st amino acids, the former could no longer recognize edge lysine, while the latter's binding was not affected. This proved that aspartic acid located at position 51 of the light chain plays a crucial role in recognizing edge lysine.

By screening for similar light chain 51st-lysine interactions, researchers found six different V gene segments and named them germline-encoded amino acid binding motifs (GRAB). In 32 antibody-antigen interactions related to these six GRABs, three-quarters were lysine-GRAB interactions, and five of these six GRABs could similarly bind edge lysine.

GRAB Exists in Other V Gene Segments

After confirming the existence of GRAB in the V gene segment of the λ light chain, researchers turned their attention to other parts of the V gene segment. Through further searches of protein databases, researchers found other GRABs in the heavy chain segment, κ light chain segment, and other λ light chain segments, which can recognize "public epitopes" containing different amino acids such as aspartic acid, glutamic acid, and proline, proving the universality of GRAB in "public epitope" recognition.

Finally, researchers mutated GRAB and found that these mutations significantly reduced the binding of the corresponding antibodies to the influenza virus "public epitope.” Mutations in sites outside GRAB did not considerably impact high-resolution antibody footprinting.

By comparing the antibody responses of humans, non-human primates, and mice to the same virus, researchers found that different species often recognize different "public epitopes." Further research on GRAB found that the GRAB of other species only showed partial similarities. This may be why "public epitopes" differ between different species.

Conclusion

In summary, this study addresses an important issue in immunology, which is why antibodies more easily recognize certain regions of antigens than others. Through careful experimental design, researchers identified the existence of GRAB and revealed how they interact with specific amino acid sequences in antibody immunity. This discovery provides a solid foundation for further understanding humoral immunity.

This study involved many peptide segments for experiments, some of which were synthesized by GenScript.

Reference

[1] Shrock et al., Science 380, eadc9498 (2023). DOI: 10.1126/science.adc9498