Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

Erwin De Genst,Karen Silence,Mehdi Arbabi Ghahroudi,Klaas Decanniere,Remy Loris,Jörg Kinne,Lode Wyns,Serge Muyldermans

doi:10.1074/jbc.m413011200

Abstract

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.

Highlights

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites
Ontogeny of D2-L19 and cAb-Lys2—Two variable domain of these heavy-chain antibodies (VHH), cAb-Lys2 and D2-L19, were isolated by phage display [14, 15]. These VHHs apparently employed the same D gene that was rearranged to different V and J segments
A search in the VHH germ line data base [10] revealed that the V region of cAb-Lys2 most probably originates from the cvhhp08 germ line gene (89% sequence identity, AJ245114.1), onto which 17 nucleotide transitions and 13 transversions accumulated, whereas the V region of D2-L19 most probably originates from the cvhhp11 germ line gene (88.5% sequence identity, AJ245117.1)

Summary

Introduction

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Because the number of possible V(D)J recombinations exceeds by far the number of crystal structures that are available, it is as yet impossible to investigate whether the D-encoded part of the loop adopts different or similar backbone architectures when present in a different V-J surrounding Likewise, it remains an open question whether the same epitope on a large antigen will be recognized by antibodies originating from different B cell lineages that employed the same D gene in their V(D)J recombination. This would mean that the structural repertoire of the H3 loop is less diverse than predicted by the immense sequence diversity

Methods

Results

Conclusion