Trends in Antibody Sequence Changes during the Somatic Hypermutation Process Louis A. Clark, Skanth Ganesan, Sarah Papp and Herman W. T. van Vlijmen This information is current as of September 27, 2021. J Immunol 2006; 177:333-340; ; doi: 10.4049/jimmunol.177.1.333 http://www.jimmunol.org/content/177/1/333 Downloaded from References This article cites 32 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/177/1/333.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Trends in Antibody Sequence Changes during the Somatic Hypermutation Process Louis A. Clark,1 Skanth Ganesan, Sarah Papp, and Herman W. T. van Vlijmen1 Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing Downloaded from changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are ϳ4% for core residues, ϳ5% for surface residues, and ϳ12% for residues in common Ag-contacting positions, excepting the those coded by the D gene. The Journal of Immunology, 2006, 177: 333–340. http://www.jimmunol.org/ he versatility of the mammalian adaptive immune system single-base transitions over transversions at an ϳ3:1 ratio (7). In- rests on its ability to generate high-affinity Abs to foreign sertions and deletions also occur but are considerably less common T Ags. The set of naive B cells expresses a repertoire of (8, 9). Certain four-base DNA sequence motifs, called hotspots, surface-bound Ab precursors called B cell receptors or BCRs, a are correlated with the mutation locations. The two most com- subset of which can bind most new Ags with low affinity. To make monly cited four-base motifs are RGYW (10) and its inverse re- this possible, the V(D)J recombination process has evolved to mix peat, WRCY (11), where R denotes a purine base, Y a pyrimidine V, D, and J genes during cell development to provide a large base, and W an A or a T base. sequence diversity in the germline L and H chains. Considering It is not always clear how the mutations selected during the by guest on September 27, 2021 only the number of human V, D, and J genes and neglecting a gain affinity maturation process contribute to improving binding affinity in diversity from the joining mechanisms, one gets an estimate of or other properties such as selectivity or stability. Only a few stud- 320 L chain variable domain and 11,000 H chain variants. The ies exist that look at the effect of single somatic hypermutations assumption that any H chain can pair with any L chain yields 3.5 ϫ 6 (12). One expects the residues in contact with the Ag to be the 10 theoretical specificities (1). Additional adaptability comes most critical, but often large affinity improvement come from non- from the somatic hypermutation process, where the recombined surface mutations. Daugherty et al. (13) used phage display on Ab genes undergo error-prone replication during an in vivo selec- Escherichia coli to improve the affinity of a single-chain Fv mol- tion process. Those B cells that produce Abs with higher affinities ecule to cardiac glycoside digoxigenin and found that all of the for their Ags selectively proliferate, leading to a further enormous affinity enhancements occurred at non-Ag-contacting residues. increase in diversity and a fine tuning of the affinity. Similarly, Zahnd et al. (14) used a ribosome display to improve Much of somatic hypermutation research to date has focused on single-chain Fv binding to a peptide Ag and found that the most defining the molecular mechanism. This aspect has been reviewed recently (2–5) and, although much remains to be learned, certain effective mutation was not at the interface with the Ag. From our sequence-related aspects are clear. The enzyme AID (activation- own work redesigning Ab-Ag interfaces (34), we find that even induced cytidine deaminase) deamidates cytidine residues, con- Ag-contacting mutation effects are not always rationalizable or verting them to uracil and initiating the somatic hypermutation predictable from three-dimensional structure-based energetic cal- process (6). Further processing of the local DNA may involve culations. Thus, there is great potential to learn from the natural excision of the uracil base and repair by certain error-prone poly- affinity maturation process. merases (see reviews in Refs. 3–5). The overall process favors The work presented here seeks to better define the results of the somatic hypermutation process and to investigate sequence-related aspects of protein-protein recognition. Given a mature sequence, a Biogen Idec Inc., Cambridge, MA 02142 probable germline sequence resulting from the recombination of Received for publication November 4, 2005. Accepted for publication April 19, 2006. species-specific V, D, and J genes (15, 16) can be derived using The costs of publication of this article were defrayed in part by the payment of page sequence matching and consideration of the known mechanistic charges. This article must therefore be hereby marked advertisement in accordance rules of V(D)J recombination (see Materials and Methods). Once with 18 U.S.C. Section 1734 solely to indicate this fact. the germline chain sequence is known, simple comparison with the 1 Address correspondence and reprint requests to Dr. Louis A. Clark and Dr. Herman W. T. van Vlijmen, Biogen Idec Inc., 14 Cambridge Center, Cambridge, MA 02142. aligned mature sequence yields the position and type of mutation E-mail addresses: [email protected] and [email protected] that occurred during the affinity maturation process. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 334 TRENDS IN SOMATIC HYPERMUTATIONS Tomlinson et al. (17) have previously used a similar type of the best gene are always assumed to be identical with those in the mature analysis to analyze the diversity of amino acids at specific posi- sequence, and no mutations are recorded. tions in the germline and mature Ab sequences. They found that Algorithm testing was performed in cases where the V(D)J fitting pro- cedure was performed manually. Additionally, D gene fitting was tested on the frequency of somatic hypermutation and the diversity of the cases where the DNA sequence was known. In the small number of cases germline sequences is highest in the CDRs. Rather than focus on examined, translating from the protein sequence to the most probable DNA the mutation frequencies, we examine the type of mutation and its sequence was sufficient to distinguish between available D gene sequences. functional implications deduced from the location in the structure. Typically, one or two related D genes had match scores clearly separated from all other lower-scoring D gene matches The results indicate that residue type changes during the somatic In some cases the algorithm clearly returns nonsensical matches, as hypermutation process are significant and have underlying func- indicated by long stretches of poor base matches. The failures can be traced tional rationales. back to humanized Abs, to Abs from species outside the human and mouse libraries used, or to insertions/deletions. Germline sequence determinations requiring Ͼ20-amino acid mutations relative to the mature sequence are Materials and Methods discarded on the assumption that the problem is inappropriate to the se- Germline V(D)J fitting procedure quence or that the algorithm has failed. Sequences with more than five consecutive mutated residues are discarded for similar reasons. Some se- The V(D)J germline fitting algorithm is intended to provide reasonable quences have so little information in the D and J gene regions that fitting solutions for the germline Ab sequence based on the mature protein se- is impractical.