The Unusual Genetics and Biochemistry of Bovine Immunoglobulins

Abstract

Antibodies are the key circulating molecules that have evolved to fight infection by the adaptive immune system of vertebrates. Typical antibodies of most species contain six complementarity-determining regions (CDRs), where the third CDR of the heavy chain (CDR H3) has the greatest diversity and often makes the most significant contact with antigen. Generally, the process of V(D)J recombination produces a vast repertoire of antibodies; multiple V, D, and J gene segments recombine with additional junctional diversity at the V-D and D-J joints, and additional combinatorial possibilities occur through heavy- and light-chain pairing. Despite these processes, the overall structure of the resulting antibody is largely conserved, and binding to antigen occurs predominantly through the CDR loops of the immunoglobulin V domains. Bovines have deviated from this general paradigm by having few VH regions and thus little germline combinatorial diversity, but their antibodies contain long CDR H3 regions, with substantial diversity generated through somatic hypermutation. A subset of the repertoire comprises antibodies with ultralong CDR H3s, which can reach over 70 amino acids in length. Structurally, these unusual antibodies form a β-ribbon "stalk" and disulfide-bonded "knob" that protrude far from the antibody surface. These long CDR H3s allow cows to mount a particularly robust immune response when immunized with viral antigens, particularly to broadly neutralizing epitopes on a stabilized HIV gp140 trimer, which has been a challenge for other species. The unusual genetics and structural biology of cows provide for a unique paradigm for creation of immune diversity and could enable generation of antibodies against especially challenging targets and epitopes.

Keywords: Antibody; Antibody diversity; Antibody repertoire; Bovine immunoglobulin; Cow antibody; Knob; Long complementarity-determining region; Ruminant antibody; Stalk; Ultralong CDR3.

Figure 1. Comparison of ‘normal’ and ‘ultralong’ CDR H3s

Structure of an antibody with a ‘normal’ CDR H3 length, Ofatumumab (left, PDB: 3giz) and an ultralong CDR H3 cow antibody, BLV1H12 (right, PDB: 4k3d). The CDR H3s are colored red with the heavy chain in light blue and light chain in grey. The β-ribbon ‘stalk’ and disulfide bonded ‘knob’ motifs are labelled.

Figure 2. Bovine IGH locus genomic organization

Schematic of the orientation of coding elements of the cattle immunoglobulin heavy chain locus. Key at bottom left shows the purple variable (V) segments, green diversity (D), orange joining (J) and blue constant (C) genes. Pseudo V, J and C genes are paler tones. Elements used preferentially in the ultralong CDR H3 antibodies are marked by red triangles.

Figure 3. Genetic basis for encoding ultralong CDR H3s

(A) A germline insertion in germline IGHV1-7. The nucleotide and encoded amino acid sequence of the 3′ end of a typical vertebrate IGVH region is shown above and IGHV1-7 shown below. The coding region nucleotides are in uppercase letters. The recombination signal sequence (RSS) is shown in small letters, with the heptamer and nonamer in brown. IGHV1-7, which is used in ultralong CDR H3 antibodies, has an 8 base-pair duplication in the germline (boxed). This duplication shifts the reading frame and extends the amino acid sequence, allowing the initial portion of the ‘stalk’ β-strand to be encoded. (B) Nucleotide and amino acid sequence of the germline IGHD8-2 gene. Nucleotides which can be mutated to a residue that encodes a cysteine are shown in red, as are the corresponding amino acids. RGYW/WRCY mutational hotspots for AID are boxed. Note that a large fraction of codons of IGHD8-2 are mutational hotspots that can be mutated to cysteine. (C) Sequences of the five Fabs with ultralong CDR H3s whose structures have been determined. The germline regions of the IGHV1-7, IGHD8-2, and IGHJ2-4 are shown above, with the CDR H3 lengths and number of cysteines indicated to the right. The cysteines are highlighted in yellow, and the disulfide connectivities of the knob region are indicated. The cysteine and tryptophan, which define the boundaries of CDR H3, are highlighted in yellow and cyan, respectively. The amino acid numbers, using the system of Stanfield et al., are shown below (Stanfield, Wilson and Smider, 2016).

Figure 4. Genetic components mapped onto the Fab structure of BLV5B8

The rearranged genes (left) and their corresponding structural components (right) are color coded (blue, IGHV1-7; green, IGHD8-2; orange, IGHJ2-4, Pink, both IGLV30 and IGLJ3). In yellow is the area of V-D junction (labeled N) that encodes the ascending β-strand. This region is conserved and it is unclear whether it is derived from typical TdT catalyzed N-region insertions or other mechanisms like nucleotide capture. The C_H1 and C_λ regions are indicated on the right.

Figure 5. Structural diversity in the bovine ultralong CDR H3 repertoire

These include diverse disulfide patterns, driven by cysteine mutations, different loop lengths and amino acid content formed through AID point mutations as well as diversity in deletions, knob orientation, size, and stalk length.

Figure 6. The stalk and knob domains of the five currently known bovine Fab ultralong CDR H3 regions

The three strands in the core knob domain are colored yellow, green and blue and the disulfide bonds are shown as sticks. The orange disulfide is always formed from the first Cys in the CPDG motif to a Cys in the second core β-strand.

Figure 7. The bovine stalk and surrounding CDR residues

The Fab heavy and light chains are shown in light blue and pink, respectively The stalk regions all have very similar take-off angles from the body of the Fabs and appear to be structurally stable. Ladders of alternating aromatic residues may add to the stabilization of the stalks. The non-H3 CDR loops are well conserved and may also aid in support for the long stalk.

Figure 8. Genetic and structural model for ultralong CDR H3 antibody repertoire formation

One or more germline V-D-J recombination events using IGHV1-7 (dark purple), IGHD8-2 (dark green), and IGHJ2-4 (dark orange) is shown in the upper left. Junctional diversity, including insertions and deletions, can potentially alter the length of the stalk, as the V-D and D-J joints occur in regions encoding the ascending and descending strands, respectively. Presumably, two disulfide bonds can form in the knob of the naïve/germline antibody (shown in red). The primary repertoire is further diversified in the gut associated lymphoid tissue by AID-mediated processes (upper right). Nucleotide changes that alter amino acid content, particularly to cysteines, can change disulfide patterns and loops (red lines). Larger deletions (black rectangles) in the D_H region can also potentially change loop lengths and disulfide patterns. The β-strands of the knob core and stalk domain are represented by black arrows.