Newborn IgM is expressed from multiple loci of one VH family

Developmental regulation of IgM expression was examined by screening two cDNA libraries constructed from newborn nurse shark epigonal organ (primary lymphoid tissue) and spleen (secondary lymphoid tissue). Libraries were probed with a canonical IgM VH and partial CH1 probe under high and low stringency hybridization conditions [5]. Deduced amino acid (AA) sequences of the leader, VH, and partial CH1 for 25 positive clones were compared to the predicted AA sequence of the single previously published adult nurse shark IgM (Genbank accession #M92851 [26]) and aligned in Clustal W (Figure 2) [27]. These clones formed six groups (I-VI) that differ in VH leader, FR and CDR. Clones in Groups I-V are all clustered within one VH family, and as has been reported for other elasmobranchs; nevertheless CDR1 and CDR2 are diverse among the different groups. Group VI, which consists only of clone 36E, is distinct from the other groups and is discussed separately below.

Excluding the positions of VDJ rearrangement (CDR3), VH members within a group are quite similar in FR and CDR1-2, suggesting that they are expressed from one locus or several highly related loci. Indeed, microheterogeneity of 5'UT sequences of the 24 clones identified expression of 15 highly related loci from the five groups (data not shown). We estimated from previous Southern blotting analysis that there were ~15–25 IgM loci in the nurse shark [5]. Thus, our library screening results would suggest that most IgM loci are expressed at birth, which is consistent with our previous immunohistochemistry study demonstrating that the great majority of splenic B cells in newborn nurse sharks are IgM⁺ [12]. If this estimate of gene number is confirmed in future studies of nurse shark VH genes, then this elasmobranch species has far fewer IgM genes as compared to the published estimates of 100 IgM loci for the horn shark and 50 loci for the skate [1, 28].

The IgM clones were analyzed for the conserved residues necessary to maintain the Ig structural fold using the ImMunoGeneTics (IMGT) system [29–31]. Representatives of the most divergent groups I and VI were compared to the originally isolated nurse shark IgM M92851 VH (Figure 3) [32]. Residues essential for the Ig fold are the hydrophobic GlyLeuGluTrp in FR2 positions 49–52, Trp41, and Cys in strands B and F at positions 23 and 104 required for the canonical disulfide bridge [31]. These residues and their positions are conserved in members of the five IgM groups, indicating preservation of the Ig fold structure. The FR and CDR for the clones were analyzed for their similarity to the original VH (Table 1). All groups were least similar to the original VH in the FR1 (69–83% identity) and CDR1 (33%), and most similar in FR2 and FR3 (78–93%). CDR2 was more conserved than CDR1, with groups II-IV most similar to the original sequence. FR conservation and variation in the CDR are consistent with findings in the horn shark, sandbar shark, and higher vertebrates [33]. Membership within a specific VH family has been defined as greater than or equal to 75% identical AA (greater than or equal to 70% identical nucleotides); thus VH in nurse shark groups I-V are within a single family (refer to Table 1) [31, 35]. Additionally an ATG codon (Methionine) in CDR1 is present in all IgM clones, consistent with a previous study showing this codon at this position is preserved throughout vertebrate phylogeny (refer to Fig 2) [35]. Any nucleotide substitution in this codon leads to an AA replacement, suggesting a role for its evolutionary conservation. Generally, CDR1 and CDR2 contain codons that either target the hypermutation machinery (see below) or are minimally degenerate (e.g. ATG) to maximize the potential for AA modifications in these regions of the V domain [36].

cDNA clone 36E in group VI is an unrearranged VH containing intronic sequence. The deduced sequence of 36E has only 38–50% AA identity with the conventional VH, and thus forms a separate VH family (Table 1) [31]. This VH family contains the residues important for the Ig fold, with the exception of an Arg replacing the typical Gly at position 49 (Figure 3). Clone 36E VH has more charged residues, especially in CDR1 and FR2, than the canonical VH, and the CDR are not enriched in Ser, unlike the other IgM VH. National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) [38–40] identified this VH family is most similar to a monotypic horn shark IgM VH (79%) (accession #Z11776 [41]). Clone 36E VH has 56% and 48% identical AA residues, respectively, to the sandbar shark IgW (accession #U50606 [42]) and nurse shark IgW (accession #U51450 [43]), and only 36% identical AA to a representative sandbar shark IgM VH [44] (data not shown). This novel VH family, therefore, is most related to the unusual horn shark VH and it was present in their common ancestral gene pool ~120 MYA [24]. In addition, this VH has similarity to both IgM and IgW VH genes, suggesting a more ancient origin (see below). Identification of a productively rearranged transcript containing the constant gene exons for this novel IgM VH locus is needed to permit its placement in the appropriate Ig class and enable further study.

IgW VH s are diverse and consist of multiple families expressed at birth

The ontogeny of IgW expression in neonate sharks was investigated by screening nurse shark pup spleen and epigonal organ cDNA libraries with an IgW VH and partial CH1 probe [5]. Clones were negatively selected for IgM VH⁺ cross-hybridizing clones and positively selected for strong, intermediate, and weak hybridization intensity signals. Their deduced AA sequences were aligned to the published nurse shark IgW VH (accession #U51450 [43]) in Clustal W and shown to form six groups (Figure 4). The percent similarity of the FR and CDR AA sequences to the original VH region is summarized in Table 3. Groups I and II are members of the original VH family, with 73–78% identity. Groups III-V form three new IgW VH families, with 46–56% AA identity to the original VH [31]. Interestingly, similar to our observations for the novel IgM VH family clone 36E, CDR1 in groups II and V contain more charged residues than are found in the other groups. CDR3s of each IgW clone in groups I and V are diverse and contain unique AA residues, again suggesting that TdT is active on rearranging IgW loci in neonates. A genomic clone for nurse shark IgW has not been isolated and therefore identification of DH gene usage in the junctions is not possible. 5'UT microheterogeneity shows that IgW VH groups are expressed from 11 loci, suggesting that several highly related loci are represented by each group (data not shown). High stringency Southern blotting analysis of the conventional IgW VH family indicated that 4–6 loci exist in the nurse shark, and low stringency conditions revealed more VH⁺ bands, which may have been divergent IgW (or IgM) VH families [43]. Multiple IgW VH families were previously indicated by in situ hybridization results in the skate, Raja eglanteria [45]. Thus these data together suggest that the nurse shark and skate have an equivalent number of IgW VH loci.

In the first constant domain (CH1) of conventional Ig H chains a Cys in the A strand is typically present that forms a disulfide bond with light (L) chains; this position is Gly in group II clones and Arg in group I clone 8S (Figure 4) [31]. A Cys located in a more C-terminal position may be available for L chain association (data not shown). Mutation of the canonical Cys used for L chain association also is seen in IgM_1gj where the Ig heavy chain protein does associate with Ig light chain protein [5], and modeling of other Cys more C-terminal in the CH1 domain has shown they likely are available for disulfide bonding to L chains [5].

IgW VH (#U51450 [43]) structure from IMGT [46] was compared to representative members of the divergent groups III and V (refer to Figure 3). Both of these VH families maintain the conserved residues and positions required for the Ig fold. In addition, they contain more Pro residues in strands A and C than conventional IgW VH, which are also present in these same strands in cartilaginous fish IgM VH (ratfish, nurse and horn sharks, Figure 3) [26, 33, 47]. In summary, our results, along with previous data, demonstrate the existence and expression of several IgW VH families in newborns. These VH gene families conserve residues necessary for maintenance of the Ig fold, and they undergo rearrangements containing N-region additions in neonates.

The potential somatic hypermutation motif AGY is biased to FR1 and CDR1

The V region of secretory IgNAR in the adult nurse shark has a high frequency of mutation in adult sharks, yet this region is seldom mutated in young pups suggesting that the newborn immune system requires further maturation to enable an effective immune responsive environment [14, 48]. Previously the Ser codon motif AGY (Y=C/T) had been identified as a heavily targeted site of IgNAR somatic hypermutation [48]. Therefore, we analyzed the newborn IgM and IgW VH FR and CDR for the AGY potential hypermutation motif and Ser codon TCN (N=C/G/T/A), which tends not to be somatically mutated (Figure 5). The AGY motif was utilized significantly more frequently in IgM VH FR1 (p < 0.01) and CDR1 (p < 0.01) than the TCN codons and IgW also preferred the AGY codon motif for CDR1 (p < 0.01). Both IgM and IgW VH genes encode the TCN sequence more frequently than AGY in FR3 (p < 0.01), but encode neither AGY nor TCN in FR2 implying a resistance to hypermutation in these regions, as seen in previous studies of tetrapod Ig genes [34]. Thus, the nurse shark VH genes have potential hypermutation sites focused in FR1 and CDR1, while FR2 and FR3 lack motifs conducive to targeting somatic hypermutation.

Newborn IgW and IgM CDR3 are shorter than in adults

Newborn mouse, human, and frog VH CDR3s are less diverse and shorter in length due to non-random VH gene usage and lack of TdT activity [16, 17, 49–51]. In newborn elasmobranchs TdT is expressed and functional early in shark ontogeny, as shown in previous studies of TdT expression and implied by the diversity of the newborn CDR3 repertoire in the IgM and IgW classes (Figures 2, 4, Table 2) and IgNAR types 1 and 2 [5, 14, 48]. The frequency of IgW and IgM VH CDR3 lengths in newborn nurse sharks were compared to adult nurse, horn, and sandbar sharks (Figure 6A). Newborn IgM VH utilize significantly shorter CDR3 than the adult with the newborn mean length 7.8 AA (p < 0.01, range 3–17 AA, n = 23) and the adult mean length 11.6 AA (p < 0.01, range 4–18 AA, n = 64). This is consistent with our findings that expressed newborn IgM VH tend to have only one recognizable DH gene in CDR3 (Table 2). As well, newborn IgW VH CDR3s are significantly shorter than adult CDR3s with the newborn mean 7.6 AA (p < 0.05, range 2–13, n = 16) and the adult mean 10.6 AA (p < 0.05, range 7–15, n = 12). Previously published newborn and adult IgNAR were also included in the VH CDR3 length analysis, although IgNAR CDR3s are longer because this VH gene undergoes four rearrangement events (V-D1-D2-D3-JH). Neonatal IgNAR VH Type 1 CDR3 mean length 14.1 AA (p < 0.01, range 8–20 AA, n = 18, [14]) was also significantly shorter than those of the adult CDR3 mean 20 AA (p < 0.01, range 15–26, n = 20, [14]). As stated previously, the IgNAR Type 3 VH locus has two germline-joined DH genes and its length is always 16 AA (n = 32) for all clones analyzed, suggesting that this VH is positively selected during ontogeny on a self-ligand [14]. As Type 3 VH expression wanes during postnatal maturation it was not possible to compare this gene's expression in adult life [14]. Together these results show that newborn IgW, IgM, and IgNAR VH CDR3 are significantly shorter than those of adults, indicating that the postnatal Ig VH repertoire is not entirely mature. In addition, the results suggest that the rearrangement of Ig genes and the development of B cells from immature precursors persist in primary lymphoid tissues after birth in cartilaginous fish. To confirm these findings the shark Ig VH CDR3 lengths were compared to previously published newborn and adult frog and human IgM VH CDR3s (Figure 6B). Despite the fact that all newborn sharks VH CDR3 contain N-region additions, indicative of functional TdT gene activity not apparent in frog and human newborn sequences, the contrast of CDR3 sizes in young and old sharks is similar to that of tetrapod CDR3 during ontogeny. Thus, a developmental program of postnatal maturation, defined by longer CDR3, is demonstrated in sharks, frogs, and humans, which spans 460 million years of evolution.

IgM and IgW classes were present in the ancestors of all jawed vertebrates

¹The identification of IgW in the lungfish, close relatives of land vertebrates, substantially changed the phylogenetic distribution and evolution of this Ig class, since these lobe-finned bony fish shared a common ancestor with cartilaginous fish more than 460 MYA (refer to Figure 1) [21]. ²Accordingly, IgW VH family is more ancient than previously believed and may be the primordial Ig rather than IgM, as initially hypothesized [43]. ³The lungfish discovery allowed a more thorough phylogenetic analysis of the IgM and IgW VH families (Figure 7). ⁴The neighbor-joining phylogram was constructed using VH FR1-3, excluding the CDR to reduce effects caused by positive selection, and was rooted using the outgroup mouse (M. musculus) L chain V kappa (accession #29725591) [52–55]. ⁵Representative members from each newborn IgM and IgW VH group were included in the tree, in addition to representative IgM and IgW VH sequences from the lungfish (P. aethiopicus), ratfish (H. colliei-holocephali), skate (R. erinacea-elasmobranch), guitarfish (R. productus-ray, elasmobranch), sandbar shark (C. plumbeus-elasmobranch), horn shark (H. francisci-elasmobranch), and nurse shark (G. cirratum) (refer to Figures 1,2 and 4). Notably this analysis reveals that IgM and IgW cluster as separate VH families among the cartilaginous and bony fish lineages, demonstrating that the IgW VH family is ancient. The monotypic horn shark IgM (accession #Z11776) and its ortholog, the nurse shark divergent IgM group VI clone 36E, segregate with the IgW VH group indicating that they have features of both the IgM and IgW VH families. This phylogenetic analysis confirms the findings of the lungfish IgW discovery, namely that IgM and IgW VH are ancient gene families that were present in the common ancestor of cartilaginous and bony fish, which most likely was a placoderm, an extinct heavily-armored fish group considered to be the earliest vertebrates with jaws [24]. Finally, most of the shark and skate IgM sequences, excluding clone 36E, cluster within a species, which suggests that the various VH groups emerged after the divergence of each species.

Animals

Nurse shark pups were delivered by Caesarian section from a gestating female shark near term as described [60].

cDNA library construction and screening

Tissues were dissected from nurse shark pups and total RNA was isolated as described [5]. The newborn pup spleen and epigonal cDNA libraries were constructed as described previously [5]. Libraries were plated and screened with canonical nurse shark IgM VH probe under both high and low stringency conditions as described [5, 61]. Canonical nurse shark IgW VH and partial CH1 probe was amplified by PCR from plasmid DNA containing cloned cDNA insert using specific primers and labeled as described [5, 43]. Clones were selected and isolated based on hybridization signal intensity of strong, intermediate and weak with more than 60 clones analyzed for each Ig class.

Alignment and phylogenetic analysis

IgW and IgM cDNA clones were translated into amino acid sequences using the EXPASY translate tool [62] and aligned in ClustalW v1.8 [63] for alignment analysis or ClustalX v 1.8 for phylogenetic tree analysis [27, 55]. Phylogenetic tree analysis was performed using the VH regions from FR1-FR3 excluding CDR1-2. Amino acid sequences were aligned in ClustalX v1.8 using the multiple alignment parameter which does pairwise alignments in the Gonnet series protein weight matrix under default conditions of 10.00 gap opening, 0.20 gap extension, and 30% delay divergent sequences. A neighbor-joining (NJ) tree in PHYLIP output was drawn using a dendrogram as the guide and the reliability of branching order was determined by 1000 replications (bootstrap analysis) [52, 55]. The NJ phylogram tree was drawn in Treeview v1.6.6 and rooted using mouse VL kappa as outgroup [54]. The NJ tree was labeled in Canvas v9.0 (ACD Deneba Software, Miami, FL, USA). Genbank and Swiss-Prot and TrEMBL accession numbers used for phylogenetic analysis are as follows: M. musculus kappa LC 29725591; P. aethiopicus IgW AF437727 clone 28; P. aethiopicus IgM AF437734 clone 76; P. aethiopicus IgM AF437724 clone 27; R. erinacea IgM S10387; R. erinacea IgM S12838; R. erinacea IgW S12839; R. erinacea IgW REU08009; C. plumbeus IgW 1117935; C. plumbeus IgW1255130; C. plumbeus IgW 1255132; H. colliei IgM AAC12920; H. colliei IgM 2653745; H. colliei IgM 2653755; H. colliei IgM 2653743; H. francisci IgM 64003; H. francisci IgM 64005; H. francisci IgM Z11776; H. francisci IgW C6-26m13f (clone W26) P83907; H. francisci IgM clone 14-1 AY612427; C. plumbeus IgM clones 12, 19, 26, 27, 35 [44]; G. cirratum IgW U51450; G. cirratum IgM_1gj AF327520; G. cirratum IgM M92851; G. cirratum IgM 57S AY609270; G. cirratum IgM 30E AY609260; G. cirratum IgM 36E AY609263; G. cirratum IgM 72S AY609272; G. cirratum IgM 24S AY609256; G. cirratum IgW14S AY609229-AY609230; G. cirratum IgW 20E AY609231-AY609232; G. cirratum IgW 25E AY531553-AY531554; G. cirratum IgW 99S AY609242-AY609243; G. cirratum IgW 130S AY609246; R. productus IgM clone 23A AY612424-AY612425; R. productus IgM 3-1 AY612426.