The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus

Jingshan Ren,Joanne E Nettleship,Gemma Harris,William Mwangi,Nahid Rhaman,Clare Grant,Abhay Kotecha,Elizabeth Fry,Bryan Charleston,David I Stuart,John Hammond,Raymond J Owens

doi:10.1016/j.molimm.2019.04.026

Abstract

Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.

Highlights

In comparison to human and mice, germ-line immunoglobulin variable region gene diversity in cattle is highly limited
Synthetic genes encoding the variable regions were inserted into plasmids containing resident signal sequences and light and heavy chain constant regions to produce B4 and B13 Cλ light chain (LC) and IgG1Fd HC expression vectors respectively
Transfection of HEK cells with either B4 and B13 HC alone did not produce any heavy chain only antibody fragments confirming that assembly with the LC was essential for production of secreted protein

Summary

Introduction

In comparison to human and mice, germ-line immunoglobulin variable region gene diversity in cattle is highly limited. The limited sequence diversity in the expressed Vλ repertoire in cattle has led to the suggestion that it contributes relatively little to antigen recognition and that most of the immune response resides in the VH (Saini et al, 2003). A unique feature of the antibody response in cattle is the generation of a subset (up to 10%) of heavy chains that have a highly extended H3 of over sixty residues. This compares to an average of 20 residues for most bovine heavy chains, which in itself is longer than in other species such as human and mouse, typically 8–16 residues. The ultra-long H3s contain a large number of cysteine residues that cross-link to stabilise the structure (Saini et al, 1999; Wang et al, 2013)

Methods

Results

Conclusion