Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + V{lambda} pairings

Surinder S. Saini1, William Farrugia2, Paul A. Ramsland2 and Azad K. Kaushik1

1 Departments of Pathobiology and Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada 2 Structural Immunology Laboratory, The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, Australia

Correspondence to: A. K. Kaushik; E-mail: akaushik{at}uoguelph.ca
Transmitting editor: S. Izui


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgM{lambda} antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody’s combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the {lambda}-light chains that pair with µ-heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V{lambda}1 genes (V{lambda}1x and two new V{lambda}1d and V{lambda}1e genes) in IgM with unusually long CDR3H. The V{lambda}1d and V{lambda}1e genes are similar to each other, but diverge from the other V{lambda}1 genes into two closely related subfamilies. The available bovine V{lambda} genes are classified into three V{lambda} gene families: V{lambda}1, V{lambda}2 and V{lambda}3 based on nucleotide similarity >=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V{lambda}1 subfamilies. Patterns of Ser residues differ for V{lambda} domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These ‘Ser signatures’ can be used to track divergent evolution in {lambda}-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V{lambda} domains that pair with VH regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V{lambda} domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V{lambda} domains also occupied smaller volumes if paired to VH domains with extremely long CDR3H (>=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were <=22 residues. Thus, the role of the {lambda}-light chains in these unusual cattle antibodies is probably to act as a relatively featureless supporting platform for the extremely long CDR3H regions, which undoubtedly are dominantly involved in binding to an antigen.

Keywords: bovine V{lambda} gene, V{lambda}1 gene family, VH + V{lambda} pairing, VDJ rearrangement, VJ rearrangement


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In mice and humans, enormous antibody diversity is generated via rearrangements of large numbers of divergent Ig gene segments to produce the VJ and V(D)J genes encoding the V regions of light and heavy chains respectively. This combinatorial Ig gene shuffling occurs in the bone marrow throughout life (15). By contrast, species with germline Ig gene pools of limited diversity, such as chicken, rabbit, sheep and cattle, employ alternative post-rearrangement mechanisms for antibody diversification. For example, the pre-immune antibody repertoire in chickens is diversified from single functional VH and V{lambda} genes, via gene conversion in the bursa of Fabricius (6,7). Both gene conversion events and hypermutation in the appendix contribute to antibody diversification in rabbits where abundant germline Ig genes are not stochastically rearranged (8,9). Yet another mechanism is used by sheep to generate antibody diversity by non-antigen-dependent somatic hypermutations (10). While antibody diversity in cattle (Bos taurus) is generated by similar mechanisms to those outlined above, the bovine IgM heavy chains appear to be unique in the generation of exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) regions (11). These uncommonly long CDR3H regions can exceed 60 residues in length and frequently contain multiple Cys residues. Although multiple Cys residues have been observed in antibody CDR3H of other vertebrates like sharks, camels, dolphins, monotremes (platypus) and occasionally in humans, these Cys-rich CDR3H certainly do not attain the massive sizes observed in cattle antibodies [reviewed in (12)]. The generation of exceptionally long CDR3H regions in cattle IgM antibodies seems to compensate for reduced germline VH gene diversity (consisting of only a single polymorphic gene family), which is further diversified by somatic hypermutation (1315).

Species-related differences also exist with regard to the relative abundance of {kappa}- and {lambda}-light chain antibody isotypes [reviewed in (16)]. For example, human and swine express {kappa}- and {lambda}-light chains at 60 and 40% frequencies respectively. The {kappa}-light chains contribute almost entirely to the antibody repertoires of mouse (95%) and rabbit (90%), whereas {lambda}-light chains are predominantly expressed (>90%) in cattle, sheep and horses. Chicken antibodies exclusively employ {lambda}-light chains. By contrast, some camel (17) and nurse shark (18) antibodies are secreted as homodimers of the heavy chains and are devoid of light chains altogether.

The bovine immune system develops in the absence of possible influences from maternal antibodies since transplacental passage of Ig is prevented by a syndesmochorial or cotyledonary type of placenta (16). Unlike mice and humans, bovine B cells do not express surface IgD and probably belong to a unique lineage with characteristics common to both B1 and B2 lymphocytes (19). A single polymorphic VH gene family, BovVH1, is expressed in the antibody repertoire of cattle (14). Two V{lambda} gene families, V{lambda}1 and V{lambda}2, have been identified in cattle (20,21). The members of the bovine V{lambda}1 gene family are further grouped into V{lambda}1a, V{lambda}1b, V{lambda}1c and V{lambda}1x subfamilies. The V{lambda}1a and V{lambda}1b genes are, however, predominantly expressed in bovine antibodies. Although V{lambda}1x genes constitute ~50% of the bovine V{lambda} germline genes, these were observed to be not expressed (20).

Earlier, we observed that VDJ rearrangements encoding bovine IgM antibodies with CDR3H lengths ranging between 56 and 61 amino acids paired with V{lambda}1x genes (11). To gain insight into such restricted VH + V{lambda} pairings, we analyzed the primary sequences and structural characteristics of VJ rearrangements that pair with VDJ rearrangements encoding IgM antibodies with exceptionally long CDR3H regions. These experiments led to the identification of two novel V{lambda}1 genes, designated as V{lambda}1d and V{lambda}1e subfamilies. In contrast to predominantly expressed V{lambda}1a and V{lambda}1b gene subfamilies, amino acid residues Ser90L and Ala91L are strictly conserved in the V{lambda}1x, V{lambda}1d and V{lambda}1e gene-encoded complementarity-determining region 3 of the light chain (CDR3L) regions. We propose that the conserved Ser90L has a structural role in fitting the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment, FR1L and CDR1L. In general, CDR3L of V{lambda} domains have smaller volumes if paired to VH domains with extremely long CDR3H (>=48 residues), while CDR3L are more variable in their conformation and occupy larger volumes if CDR3Hs are <=22 residues. These data support our previous proposal that the cattle {lambda}-light chains have only a minimal (if any) role in antigen binding and instead function as a support platform for the bulky CDR3H regions, which by themselves may bind antigen (12).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hybridomas
Murine x bovine heterohybridomas were developed from B cells, stimulated in vitro with 25 µg/ml of LPS from Escherichia coli (serotype 0127:B8) and 300 ng/ml of phorbol 12-myristate 13-acetate (Sigma, St Louis, MO) for 48 h (14). The clone BF1H1 originated from splenic B cells of a 125-day-old bovine fetus, while other hybridomas (BLV8C11, BLV1H12, BLV4H9, BLV5B8, BLV5D3, BLV5H4, BLV2C7, BLV7B7 and BLV7G1) were constructed from peripheral blood B cells of an adult Holstein cow. The supernates from all the clones were tested for IgM secretion in an ELISA (14) using unlabeled murine monoclonal anti-bovine IgM isotype (Sigma) as a capture antibody and biotin-conjugated murine monoclonal anti-bovine IgM (Sigma) and avidin-conjugated alkaline phosphatase (Pierce, Rockford, IL) as the secondary reagent. With the exception of clone BF1H1, all test hybridomas secreted bovine IgM.

PCR, cloning and sequencing of VJ rearrangements
Total RNA was extracted from the hybridoma cells using TRIzol reagent (Gibco/BRL, Gaithersburg, MD) and cDNA was synthesized from 7 µg of total RNA using a poly(dT) oligonucleotide (Pharmacia LKB Biotechnology, Uppsala, Sweden). The possible V{lambda}–J{lambda} rearrangements were PCR amplified using a 5' primer (5'-AGGTCGACTCTGCACAGGATCCTG-3') from the bovine V{lambda} leader sequence and a 3' primer (5'-AGTCTAGAACAGGGTGACCGAG-3') from the bovine C{lambda} gene (22) with built-in restriction sites for SalI and XbaI endonucleases respectively. The PCR conditions included denaturation at 95°C for 1 min, annealing at 68°C for 1 min and extension at 72°C for 1 min up to a total of 30 cycles. The PCR products from clones BLV8C11 and BF1H1 were cloned in the Bluescript KS+/– vector (Stratagene, La Jolla, CA) as described (14). Recombinant plasmids extracted from at least two colonies were purified using a mini-affinity column (Qiagen, Stanford, CA), and sequenced in both directions with T7 and T3 primers by automated DNA sequencing (MOBIX; McMaster University, Hamilton, Ontario, Canada). The PCR products from other clones were sequenced directly using 3' C{lambda} primer.

Sequence analysis and homology modeling of bovine V{lambda} domains
Codons and translated amino acid sequences of the bovine V{lambda} domains were numbered according to Kabat et al. (2), and analyzed using the GenBank BLAST program (23). Phylogenetic relationships between nucleotide sequence of cattle, sheep and human V{lambda} genes were determined using the MEGALIGN program (DNA Star, Madison, WI). Evolutionary relationships between the sequences were further examined by the numbers and locations within the sequences of Ser residues because the bovine V{lambda} domain sequences were richly populated by this amino acid.

Homology models of the bovine V{lambda} domains were constructed using the Homology module of the Insight II program suite, version 98.0 (MSI, San Diego, CA) and following procedures similar to those previously used to model human Fv molecules (24). Templates for the initial models were selected from the Protein Data Bank (PDB) on the basis of sharing the highest amino acid similarities to the bovine sequences and where possible sharing CDRL loops of the same canonical structures (25). Coordinates from two high-resolution crystal structures of human V{lambda} domains used as templates were the 1.94-Å resolution structure of the Sea {lambda} Bence–Jones dimer (PDB code 1JVK) (26) and the 1.84-Å structure of the B7-15A2 IgG1({lambda}) Fab (PDB code 1AQK) (27). Template V{lambda} sequences were aligned with the bovine sequences and assigned weighting values (for derivation of spatial restraints) using the Align2D fitting algorithm of the Modeller program, which was then used to construct the atomic models by the satisfaction of spatial restraints method (28). Homology models were subjected to conjugate-gradient energy minimization until convergence (0.001 kcal/mol) using the Crystallography and NMR Suite (29). Harmonic restraints of 10.0 kcal/(mol Å2) were imposed on all main-chain heavy atoms throughout the energy minimizations. Stereochemistry and geometry of the final models were checked using the PROCHECK program (30). Structural comparisons of the bovine V{lambda} domain models and figures were prepared with the MOLMOL program, version 2K.1 (31).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bovine V{lambda} genes are classified into three families
Nucleotide sequences were determined for VJ {lambda}-light chain rearrangements (Fig. 1) from bovine heterohybridoma cell lines (BLV8C11, BF1H1, BLV4H9, BLV5H4, BLV2C7, BLV7B7 and BLV7G1). The nucleotide sequences for the corresponding VDJ heavy chain rearrangements had previously been determined (11,14,32). Based upon sharing >=80% nucleotide similarity (Fig. 2), the V{lambda} discussed here all fall into the known single V{lambda}1 gene family. Furthermore, nucleotide sequence comparisons of the available 81 bovine V{lambda} genes (11,20,21) defines three bovine V{lambda} gene families, designated as V{lambda}1, V{lambda}2 and V{lambda}3, and representative members are shown in Fig. 2. The majority of the bovine V{lambda} genes are grouped into the V{lambda}1 gene family, while V{lambda}2 and V{lambda}3 gene families comprise one and two members respectively.



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Fig. 1. Comparison of nucleotide sequences of bovine V{lambda}1 gene subfamilies. The codons are numbered according to Kabat et al. (2) and the CDRs are shown in bold letters. The sequences, BLV1H12 (11) and clone 15 (20) are included for comparison. Dashes indicate nucleotide identities and dots represent a lack of a nucleotide at particular positions.

 


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Fig. 2. Percent nucleotide identity matrix for bovine V{lambda} genes. Sequences are compared for the VJ rearrangements isolated from the bovine heterohybridomas and for representative members of the three bovine V{lambda} gene families.

 
The bovine V{lambda}1 and V{lambda}2 gene families share highest nucleotide homology with the corresponding sheep V{lambda}I (92.6%) and V{lambda}II (88.9%), and human V{lambda}I (74.8%) and V{lambda}II (79.3%) gene families respectively. The bovine V{lambda}3 gene family, however, shows highest nucleotide homology to an unclassified sheep V{lambda}6b gene (75.0%) and also with the human LBR1104 V{lambda} gene (73.8%), a member of the human V{lambda}III gene family. Phylogenetic analysis revealed that representative members of cattle, sheep and human V{lambda} gene families cluster together into three major branches with the exception of sheep clone 3.1, a member of the sheep V{lambda}III gene family, which falls within group V{lambda}1 (Fig. 3). The unclassified sheep V{lambda}6b gene clusters with group V{lambda}3 genes together with the bovine clone C4 V{lambda} gene.



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Fig. 3. Phylogenetic relationship between members of bovine (B) V{lambda}1, V{lambda}2 and V{lambda}3 gene families, and the corresponding V{lambda} genes of sheep (S) and human (H). Clones 10 and 13 (20); clone C4 (21); clones 5.3, 6.1 and 3.1 (33); clone V{lambda} 6b, (GenBank accession no. AF038146); clone DPL 8 (34); clone HSLV 2066 (35); and clone LBR 1104 (36).

 
Classification of two novel bovine V{lambda}1 genes into V{lambda}1d and V{lambda}1e subfamilies
For the VJ sequences reported here, two clones (BLV4H9 and BLV5H4) are encoded by V{lambda}1a subfamily genes and three others (BLV2C7, BLV7B7 and BLV7G1) are encoded by V{lambda}1b subfamily genes (Fig. 2). However, the V{lambda}1 gene encoding VJ rearrangements in two clones (BLV8C11 and BF1H1) could not be grouped into existing bovine V{lambda}1 subfamilies as these do not share at least 91% nucleotide homology (Fig. 2) and also lack the characteristic structural features associated with the members of V{lambda}1a, V{lambda}1b, V{lambda}1c or V{lambda}1x gene subfamilies (20). For example, codon TCC/TCA at position 8 is replaced with CCC, codon TCC at position 20 is replaced with ACC, codon GGT/CGT at position 50 is replaced with AAT, codon ACC/AGC at position 52 is replaced with AAC, codon TCC at position 65 is replaced with ACC and codon AGC at position 76 is replaced with codon GCT (Fig. 1). Although high nucleotide similarity (88.8%) exists between V{lambda} gene-encoded region of the BLV8C11 and BF1H1 VJ rearrangements, in the CDR1L region of BF1H1 the rearranged V{lambda} gene is missing one codon (Fig. 1). Therefore, the novel BLV8C11 and BF1H1 V{lambda}1 genes are classified into new subfamilies, designated as V{lambda}1d and V{lambda}1e respectively. Similar to other V{lambda}1 genes, the consensus hot-spot triplet AGC/AGT occurs at a high frequency in the CDR1L in comparison to the CDR2L region of BLV8C11 and BF1H1 V{lambda} genes.

The V{lambda}1c and V{lambda}1x genes are members of a closely related V{lambda}1 subgroup
Although the representative member of V{lambda}1x subfamily (BLV1H12) shares nucleotide homology >=91% with members of both the V{lambda}1a (BLV4H9 and BLV5H4) and V{lambda}1c (clone 15) subfamilies (Fig. 2), V{lambda}1a genes encode a CDR1L length of 14 codons, while V{lambda}1c and V{lambda}1x genes encode a CDR1L comprising of 13 codons (Fig. 1). In addition, V{lambda}1c and V{lambda}1x genes express identical codons at positions 27b (AAT), 30 (GGA), 34 (AGC) and 51 (GAC), and appear sufficiently different from the other V{lambda}1 subfamilies where codons AAC, GGC/AGA, AAC/GGC and GCG/AGT occupy these positions respectively (Fig. 1). It seems that V{lambda}1c and V{lambda}1x genes constitute a closely related V{lambda}1 subgroup.

Variable Ser content and distinct Ser distribution patterns in bovine V{lambda} domains
While searching for conserved sequence motifs in the amino acid sequences of the bovine V{lambda} domains (Fig. 4), we observed interesting patterns of Ser residues (‘Ser signatures’), which allow classification of the sequences into three V{lambda}1 groups (Fig. 5). The newly described V{lambda}1d and V{lambda}1e gene-expressing VJ rearrangements separate into group 1 (with lower total Ser content of 14.7% and divergent Ser distributions, compared to other groups; 18.4–20.2% total Ser in groups 2 and 3), V{lambda}1a into group 2 and V{lambda}1b into group 3. The Ser signature of the V{lambda}1x containing VJ sequence (BLV1H12) shows that its sequence is highly related to groups 2 and 3, but retains a few key Ser residues from group 1 sequences (e.g. Ser90L in CDR3L). The division of the V{lambda} domains on the basis of Ser content alone closely matches the phylogenetic tree constructed from the nucleotide sequences (Fig. 3), where V{lambda}1 family corresponds to a unique branch separating V{lambda}1d and V{lambda}1e sequences from the remaining V{lambda}1 sequences.



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Fig. 4. Comparative sequence alignment for the deduced amino acids of the expressed bovine V{lambda} domains. The sequences are available in GenBank (accession nos AF015796, AF015797, AF015802, AF015803, AF015805, AF023842 and AF097214). The sequence BLV1H12 is included for comparison (11). Note the conserved Ser90L and Ala 91L residues in the CDR3L of V{lambda}1x, V{lambda}1d and V{lambda}1e subfamilies in contrast to V{lambda}1a and V{lambda}1b subfamilies.

 


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Fig. 5. Ser signatures of the bovine V{lambda} domains. Locations of Ser residues conserved within and across bovine V{lambda}1 gene-containing domains are indicated with crosses. Ser signatures are listed for all groups and the overall Ser pattern observed in the bovine V{lambda}1 rearranged light chains is presented.

 
Restricted VH + V{lambda} gene pairings occur in mitogen-stimulated B lymphocytes of cattle
The V{lambda}1a- and V{lambda}1b-encoded VJ rearrangements pair with VDJ rearrangements encoding CDR3H of lengths ranging from 15 to 22 codons (Table 1) consistent with our previous observations (11). Interestingly, the novel V{lambda}1d (BLV8C11) and V{lambda}1e (BF1H1) genes only pair with the VDJ rearrangements that encode exceptionally long CDR3H (58 and >48 codons; Table 1). Similarly, VJ rearrangements encoded by V{lambda}1x genes, also exclusively pair with VDJ rearrangements encoding an exceptionally long CDR3H (56–61 codons; Table 1). Such restricted VH + V{lambda} gene pairings are surprising in polyclonally activated B cells that are expected to represent a non-antigen-selected population of B cells. Therefore, we have assumed that a specific limited set of VJ rearrangements will only pair with certain bovine VDJ rearrangements because of the structural constraints that are imposed by the extremely long and bulky CDR3H regions they encode.


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Table 1. VH + V{lambda} pairings in the context of CDR3H lengths in non-antigen activated B lymphocytes of cattle
 
Although it would have been ideal to model VH and Fv for the bovine antibodies, we previously assessed that homology modeling techniques could not be used to produce accurate models of the extremely long CDR3H structures (12). Therefore, we prepared homology models of the bovine V{lambda} domains to examine the structural features that may facilitate interactions with VH domains containing exceptionally long CDR3H regions. As illustrated (Fig. 6), the most obvious differences are predicted to be within CDR3L. The BLV8C11 CDR3L is compact and probably does not protrude beyond the globular portion of the V{lambda} domain (Fig. 6). A similar compact CDR3L structure was predicted for clone BF1H1 and to a lesser extent for BLV1H12. Thus, in general for bovine antibodies with extremely long CDR3H regions, a compact and relatively featureless surface is presented by the partner V{lambda} domain. Predicted structures of CDR3L were more variable and always protruded from the globular V{lambda} domain for antibodies with CDR3H of <=22 residues as shown for BLV4H9 (Fig. 6).



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Fig. 6. Homology models of bovine V{lambda} domains. BLV8C11 is a representative V{lambda} domain found in antibodies with exceptionally long CDR3H regions (48–61 residues). BLV4H9 represents a V{lambda} from a bovine antibody with a shorter CDR3H (<22 residues). Layers of anti-parallel ß-pleated sheet that interact with the partner VH domain are depicted as black directional arrows. Note the compact versus protruding CDR3L structures in BLV8C11 versus BLV4H9 V{lambda} domains. Location of the Ser100L, characteristic of {lambda}-light chains from domestic ruminants, is indicated (Gly–Ser–Gly). Lower panels show close-up views of structurally important residues at position 90L as CPK spheres.

 
Similar to bovine V{lambda}1x genes that pair with the VDJ rearrangements (BLV1H12, BLV5B8 and BLV5D3) encoding exceptionally long CDR3H (Table 1), Ser90L and Ala91L are strictly conserved in the CDR3L of V{lambda}1d (BLV8C11)- and V{lambda}1e (BF1H1)-encoded VJ rearrangements (Fig. 4). The characteristic Ser90L and Ala91L amino acids in the CDR3L encoded by V{lambda}1d, V{lambda}1e and V{lambda}1x genes is in contrast to VJ rearrangements encoded by the predominantly expressed V{lambda}1a and V{lambda}1b genes where several amino acid substitutions are evident (Fig. 4). In models of the bovine V{lambda} domains (Fig. 6), the side-chain of the residue at position 90L is buried in the globular core of the protein in the space between the N-terminal segment (residues 1–4), the end of FR1L and CDR1L. The relatively small Ser90L side-chain (encoded by V{lambda}1d, V{lambda}1e and V{lambda}1x genes) fits snugly in this pocket and probably functions to hold in place the ascending polypeptide of CDR3L. The small Ser90L side-chain contains a polar hydroxyl group that makes this residue compatible with hydrogen bonding to several polar groups, which line the respective pocket. Only alanine and glycine residues are smaller than Ser, but these residues would not perform the same function as Ser90L because they have non-polar side-chains (Ala) or are too flexible (Gly) to play a supportive role in CDR3L. Larger residues at position 90L, such as tyrosine (BLV4H9 and BLV5H4) with a bulky phenolic side-chain, will not easily fit into the same pocket as Ser (Fig. 6). The regions surrounding Tyr90L in the protein appear to mutually adjust with resulting differences in the relative orientation of the CDR3L ascending polypeptide. The side-chain of the residue at position 91L points toward the center of the combining site and could interact with antigen or residues from the heavy chain. In cattle antibodies with exceptionally long CDR3H regions, the conservation of Ala91L (a residue not typically involved in antigen binding) signifies that it probably does not participate in binding antigen, but instead forms part of an extensive interface between CDR3H and light chain.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We previously observed that 9% of VDJ rearrangements in polyclonally activated B lymphocytes of adult cattle encode functional IgM antibodies with a CDR3H length ranging from 56 to 61 amino acids (11). Interestingly, these VDJ rearrangements paired exclusively with V{lambda}1x genes. Such restricted VH + V{lambda} pairings in mitogen-activated B cells raised the question whether these originated from unique configurational requirements associated with the generation of an antigen combining site with an exceptionally long CDR3H. While H and L re-association would confirm these pairings, such independent observations in both fetal and adult B cells without antigen stimulation suggested that these depended upon required structural support. Since VDJ rearrangements encoding exceptionally long CDR3H are evident early during B cell ontogeny (32), we analyzed the VJ rearrangements associated with such VDJ rearrangements and examined their structural properties. These experiments led to the isolation of two new V{lambda}1 genes, BLV8C11 and BF1H1, that have been classified into closely related V{lambda}1d and V{lambda}1e subfamilies respectively, based on the criteria developed earlier (20).

The newly classified bovine V{lambda}1, V{lambda}2 and V{lambda}3 genes cluster together with the human V{lambda}I, V{lambda}II and V{lambda}III gene families and with sheep V{lambda}I and V{lambda}II families and the unclassified clone 6b gene respectively. Since clone 3.1, classified as a member of sheep V{lambda}III gene family, clusters with the bovine V{lambda}1, sheep V{lambda}I and human V{lambda}I genes, it would be appropriate to reclassify it as V{lambda}1 gene.

At the protein level, we identified interesting patterns of Ser residues that we term ‘Ser signatures’, which can readily be used to classify the bovine V{lambda}1 genes into their separate groups. These Ser signatures can also be used to track divergent evolution in {lambda}-light chains. For example, in domestic ruminants (cattle, sheep and goats), a Ser at position 100L is distinctive, since this residue is not found in almost all the {lambda} chain sequences from other vertebrate species (2). The residue at position 100L is within the characteristic ß-bulge (Gly–X–Gly) of the J segment of variable domains, which is a key structural feature for the formation of the VL–VH domain interface (see Fig. 6). In {lambda}-light chains of primates and pigs, the same sequence is almost invariantly Gly–Gly–Gly and in chickens an Ala residue is found at 100L (2). Although we are not certain of the relevance of Ser signatures to antibody function, it is well-known that Ser and Thr residues in variable domains are potential O-linked glycosylation sites and are also reactive with complement through the formation of an ester linkage of the carbonyl group, of activated components (C3a or C4a), and the available hydroxylated side-chain [reviewed in (37)]. Thus, the variations between species noted in the Ser signatures should relate to carbohydrate content (solubility) and complement-mediated effector functions of the antibodies with {lambda}-light chains.

In the bovine IgM antibodies, all the VDJ rearrangements with exceptionally long CDR3H paired with VJ rearrangements were encoded either by V{lambda}1x (11), V{lambda}1d or V{lambda}1e genes. It was surprising that none of the predominantly expressed V{lambda}1a and V{lambda}1b gene-encoded VJ rearrangements paired with VDJ rearrangements with unusually long CDR3H. The earlier observations that suggested lack of expression of V{lambda}1x genes (20) are, in part, explained by the fact that these associate with ~9% of VDJ rearrangements that encode exceptionally long CDR3H. Further, such restricted VH + V{lambda} pairings in IgM antibodies are distributed among V{lambda}1x, V{lambda}1d or V{lambda}1e genes and, therefore, their individual expression is expected to be at low frequencies. While an elaborate analysis in future is likely to extend these observations, the conservation of Ser90L (see below) in VJ rearrangements encoded by V{lambda}1x, V{lambda}1d or V{lambda}1e genes seems to be the structural requirement of {lambda}-light chain in IgM antibodies with unusually long CDR3H. Since a significant proportion of the cattle antibody repertoire consists of functional IgM antibodies with exceptionally long CDR3H (11), it is obvious that many circulating B lymphocytes express such restricted VH + V{lambda} pairings in cattle. Similar highly restricted VH + VL pairings have not been observed in non-antigen-selected B lymphocytes in other species such as mice where these are mostly stochastic (38) with some exceptions (3941).

Inspection of the translated amino acids of V{lambda}1x-, V{lambda}1d- and V{lambda}1e-encoded VJ rearrangements showed absolute conservation of Ser90L and Ala91L within the CDR3L region. This was in contrast to varying, and often bulky, amino acid substitutions noted at positions 90L and 91L in the VJ rearrangements encoded by the V{lambda}1a and V{lambda}1b genes. These residues are located in the ascending polypeptide of the CDR3L, and so will exert significant influence over its conformation and relative position within the antigen-binding site of the antibody (see Fig. 6). The side-chain of Ser90L is predicted to be buried in the globular core of the V{lambda} domain and may act to pin the ascending CDR3L polypeptide in place by interacting with neighboring residues from the N-terminal segment, FR1L and CDR1L. The VJ rearrangements containing V{lambda}1d and V{lambda}1e genes contained CDR3L regions that were shorter by 1 or 2 residues and were predicted by homology modeling to form compact non-protruding loops when compared to V{lambda}1a, V{lambda}1b gene-containing light chains. Thus, the {lambda}-light chains that pair with µ-heavy chains with exceptionally long CDR3H are, in general, compact globular domains with relatively featureless surfaces, which may function as a supportive platform for the bulky and obtrusive CDR3H regions as we previously proposed (12). The extremely long CDR3H regions, because they contain multiple Cys residues, are likely to fold into stabilized mini-domains capable of binding antigen without the direct participation of residues from the light chain.


    Acknowledgements
 
We thank Dr Brian Allore (Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada) for his help in DNA sequencing. S. S. S. is a recipient of a studentship from the Canadian Commonwealth Scholarship and Fellowship Program. This work was supported by NSERC and OMAFRA grants to A. K.


    Abbreviations
 
CDR3H—complementarity-determining region 3 of the heavy chain

CDR3L —complementarity-determining region 3 of the light chain

PDB—Protein Data Bank


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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