Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark

Abstract

Background: Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families.

Results: IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species.

Conclusion: As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions.

Figure 1

Phylogenetic tree. Cartilaginous fish, which includes the Holocephali ratfish and Elasmobranch sharks, skates and rays, shared a common ancestry with the ray-finned and lobe-finned bony fishes 460 million years ago (MYA). Cartilaginous fish have been identified as the earliest extant vertebrate group with major components of the adaptive immune system, i.e., Ig, TCR and MHC.

Figure 2

Alignment of VH and partial CH1 of IgM cDNA clones. The deduced amino acid sequences of 25 IgM cDNA clones (Genbank accession # AY609247-AY609274) were aligned with the conventional adult nurse shark IgM (Genbank accession #M92851 [32]) in ClustalW. Dashes (-) indicate identity with the first sequence and gaps introduced to correct alignment are indicated by slashes (/). Identification of framework and CDR are based on previous work [26]. Differences in the framework and CDR separated the clones into six groups (I-VI). The CDR3 varies in length and sequence thus most clones are unique, demonstrating diversity is present at birth in the nurse shark.

Figure 3

Newborn IgM and IgW VH families conserve residues important for the Ig fold motif. The VH structures are formatted per the IMGT numbering system [29]. (A) IgM Ig fold is maintained in IgM VH family members. The nurse shark canonical IgM Genbank accession # M92851 [32] was compared to the more divergent members from group I clone 30E and group VI clone 36E. Hydrophobic amino acids (hydropathy index with positive value: I, V, L, F, C, M, A) and tryptophan (W) at positions at which more than 50 % of analysed IG and TR sequences were positive are highlighted in blue [32]. Prolines (P) are marked in yellow and conserved Ig fold residues Cys at positions 23 and 104, and Tryp (W) at position 41 are highlighted in yellow lettering. This modeling demonstrates that Ig fold motifs strand B and F Cys and hydrophobic core residues are conserved in these VH domains. Hatched circles represent residues of the canonical VH IMGT structure that are not utilized in the shark VH. (B) The IMGT numbering system was used to compare the VH domains of the conventional IgW U51450 [46] to representatives of the IgW VH divergent groups III and V. These divergent VH families maintain the Ig fold motif and utilize Pro in the FR similar to IgM (refer to part A).

Figure 4

IgW VH form multiple families. The deduced amino acid sequences of the IgW cDNA clones (Genbank accession #AY609225-AY609246, AY531553-AY531554, AY524297) were compared to the predicted amino acid sequence of conventional IgW U51450 [43] and aligned in ClustalW with differences in FR and CDR separating the clones into six groups. Dashes (-) indicate identity with the first sequence and gaps introduced to correct alignment are indicated by slashes (/). The CDR3 are unique for most clones demonstrating the neonatal repertoire is diverse in the IgW isotype. The Cys (*) typically important for light chain association is mutated in several members of groups I and II.

Figure 5

Potential somatic hypermutation motifs are restricted to the FR1 and CDR1 for IgM and IgW VH genes. The FR and CDR nucleotide sequences for all IgM and IgW cDNA clones were analyzed for the Ser codon potential somatic hypermutation motif AGC/T (AGY red square) and the non-hypermutated Ser codon nucleotide sequence TCC/T/A/G (TCN yellow square). IgM significantly uses the AGY sequence in the FR1 and CDR1 (p < 0.01, one-way factorial analysis of variance (ANOVA), n = 23) while IgW uses this motif in the CDR1 (p < 0.01, ANOVA, n = 16). The non-hypermutated motif was significantly enriched in the FR3 region for both Ig VH families (p < 0.01, ANOVA) and neither motif was present in all FR2 analyzed.

Figure 6

Newborn IgW and IgM CDR3 are significantly shorter than the adult correlate. A). Newborn and adult nurse sharks (Gc) frequency of specific CDR3 region lengths for IgM, IgW and IgNAR were compared. Newborn VH CDR3 is shorter in length more frequently than the adult for each Ig isotype except IgNAR type 3 (which is not expressed in detectable amounts in the adult). Sample size for each Ig isotype in newborns and adults are listed in part B. B). The mean CDR3 lengths for Xenopus laevis (Xl) [49,51] and human (Hs) [64] newborn and adult IgM VH sequences were compared to the newborn and adult shark IgM, IgW and IgNAR VH CDR3 mean lengths. Significantly the newborn shark CDR3 lengths for each Ig class follow the same pattern of preference for shorter CDR3 lengths as seen in the newborn frog (larvae) and human. Significance determined by ANOVA test with ** = p value <0.01 and * = p value <0.05.

Figure 7

IgW and IgM VH are ancient gene families. A neighbor-joining tree was drawn in PHYLIP using cartilaginous fish and lobe-finned bony fish IgM and IgW VH sequences FR1-FR3 omitting the CDR1-2 from published sources (accession numbers noted next to sample) and this report (identified by clone numbers from Figures 2 and 4). The tree shows the various fish species cluster together by VH family rather than by their evolutionary relatedness indicating the IgW VH gene family is as ancient as the IgM VH gene family.