Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire

Abstract

The antigen-binding site of the camel heavy-chain antibodies devoid of light chain consists of a single variable domain (V(H)H) that obviously lacks the V(H)-V(L) combinatorial diversity. To evaluate the extent of the V(H)H antigen-binding repertoire, a germline database was constructed from PCR-amplified V(H)H/V(H) segments of a single specimen of Camelus dromedarius. A total of 33 V(H)H and 39 V()H unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the V(H)Hs evolved within the V(H) subgroup III. Nevertheless, the V(H)H germline segments are highly diverse, leading to a broad structural repertoire of the antigen-binding loops. Seven V(H)H subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged V(H)Hs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the V(H)H germline genes at the regions affecting the structure of the antigen-binding loops.

Fig. 1. Southern blot analysis of dromedary liver genomic DNA hybridized with a camel V_HH probe. Dromedary DNA was digested with BamHI, EcoRI and BamHI–EcoRI, as indicated on top of the lanes. Phage λ PstI restriction fragments are used as size marker.

Fig. 2. The deduced amino acid sequences of the dromedary germline V_H (upper) and V_HH segments (lower). Braces group the V_HH sequences in subfamilies as defined by their CDR2 length and the position of the additional Cys at indicated positions. Boxes indicate V genes that differ by at least 5 nt, but encode an identical amino acid sequence. The amino acid length is given in the column following the primary structure. The types of the predicted H1 and H2 canonical structures are in the last columns. Asterisks denote where the predicted loop may deviate from loop type defined by Chothia et al. (1992) due to novel residue at the key-site. X, unpredictable; PS, potentiality to switch (see text).

Fig. 3. Neighbour-joining phylogenetic tree of the dromedary germline V_H and V_HH segments. The tree was constructed by using clustalW, phylip packages with 1000 replicates for Bootstrap of nucleotide sequences encoding the V_H/V_HH portions. Filled and open circles denote a V_H and V_HH member, respectively. The two filled rectangles indicate pseudogenes. V_HH subfamilies are indicated.

Fig. 4. The common usage of the D element in V_H and V_HH: a genomic D element (camD4) is flanked at both sides with the RSS containing a nonamer (N) separated from a heptamer (H) by a 12-nt spacer. The sequences derived from the germline camD4 (upper line), V_H-cDNA clone i5h50 (middle line) and V_HH-cDNA clone med83 (lower line) are boxed. The CDR3 region between the FR3 and FR4 is indicated.

Fig. 5. Amino acid sequence variability plots for the V_H and V_HH germline (upper panels) and cDNA sequences (lower panels). The variability index (bars) was calculated from FR1 to FR3, as described in the results. The grey and open bars are for amino acid positions of the CDR and FR regions, respectively, and the black bars are for the extra hypervariable region in the V_HH. The CDR1 and CDR2, as defined by Kabat et al. (1991), are shaded and placed between dotted lines.

Fig. 6. Distribution and frequency of AGY (filled bars) and TAY (open bars) triplets in the germline V_H (upper) and V_HH (lower) segments. The occurrence of AGY and TAY triplets were scored in all reading frames. The numbers in between refer to the position of V_H codons (Kabat numbering). The shaded background originated from the cDNA variability plots shown in Figure 5. The dotted regions denote the CDR1 and CDR2, as defined by Kabat et al. (1991).

Fig. 7. (A) Nucleotide alignment of cDNA clone l3a11 (lower line) and its putative germline gene (cvhhp11, upper line), revealing a deletion of 6 nt (dotted). The palindromic sequence is in bold and underlined. Numbers indicate nucleotide positions of the germline V_HH element. (B) Alignment of the cDNA (lower lines) and the closest germline sequences (upper lines) indicates insertions, which are non-templated (pair 1) or are duplicated (boxed in pair 2). (C) Frequency and distribution of the RSS-like elements in the camel germline V_Hs and V_HHs. Numbers indicate the incidence of RSS found in 50 V_Hs (upper) and 42 V_HHs (lower). (D) Improper joining adjacent to the RSS signal resulting in clones with a deletion: the heptameric sequences are boxed and the expected cleavage site is indicated by an arrow. The three camel V_HH cDNA sequences (l3a08, med64 and hhat20) show a codon deletion when aligned with their putative corresponding genes (cvhhp28 and cvhhp11).