Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

Abstract

A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems.

Formation of ultralong DH regions through genomic rearrangement

“…However, local sequence alignment reveals that the 5’ end of IGHD6-3 is 91.2% identical to IGHD8-2 (89.4% for IGHD6-2) over the first 85 nucleotides, whereas IGHD3-3 (and IGHD3-4) is only 80% over the first 62 residues (Supplemental Figure 7). Of note, IGHD6 family sequences share a cysteine in the same position as the conserved cysteine in IGHD8-2, which is highly conserved in deep sequenced ultralong CDR H3 antibodies, and forms a conserved disulfide bond at the base of the ultralong CDR H3 stalk [23, 26]. Thus, donation of an IGHD6 to the 5’ end of an IGHD7 through a recombinational or gene conversion process is a likely mechanism to produce IGHD8-2.…”

“…The homologous DH regions are cysteine rich, and diversity can be generated through both germline and somatically generated cysteines, which can form a diverse array of potential disulfide bonded loops[23, 35, 36]. In the knob region of ultralong CDR H3s, a diversity of disulfide bond patterns has been observed in several crystal structures [23, 25, 26], and mutations to and from cysteine have been confirmed through deep sequence analysis. Thus, novel cysteines encoded in the DH regions contributes to structural diversity in bovine antibodies.…”

“…Bovines are remarkable in having very long CDR H3 regions[14–24], with an average length of 26 amino acids [16] but with an exceptionally long subset of the repertoire (the “ultralong” CDR H3 antibodies) that can have CDR H3 lengths of up to seventy amino acids. These CDR H3 regions form their own independently folding mini domains comprised of a β-ribbon “stalk” that protrudes far from the typicalparatope surface upon which sits a disulfide-bonded “knob”[21, 23, 25, 26]. Cows are the only species thus far investigated that can produce a broadly neutralizing antibody response against HIV, which is characterized by ultralong CDR H3 regions that penetrate the glycan shield of the spike protein to bind a conserved broadly neutralizing epitope in the CD4 binding region [27].…”

Broadly Neutralizing Bovine Antibodies: Highly Effective New Tools against Evasive Pathogens?

“…Seven recent structures reveal that small changes (<7 • ) in the angle of the stalk can lead to large changes in the orientation of the putative antigen binding site [46]. Notably, a threonine is nearly always found at the second position of this junctional insertion and is positioned directly opposite alternating tyrosines (YxYxY) encoded in the germline and present in the descending side of the stem in the 3D structure [18,47]. How changes specific to this V-D junctional sequence affect the binding or stability of the structure has not yet been explored but could feasibly be examined via the alanine-scanning mutagenesis of an Ab with a known antigen target of the ultralong CDR H3.…”

The structure of a LAIR1-containing human antibody reveals a novel mechanism of antigen recognition

“…These domains adopt a conserved β-sheet structure that displays variable loops and are each presented on an elongated, but rigid β-hairpin (Stanfield et al, 2016; Wang et al, 2013). While it is clear that the additional domains play an important role in ligand binding, the remaining five CDR loops are also exposed and further studies are needed to see the contribution that they make (Wang et al, 2013).…”