Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark
© Rumfelt et al; licensee BioMed Central Ltd. 2004
Received: 27 January 2004
Accepted: 06 May 2004
Published: 06 May 2004
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.
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.
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.
In all elasmobranchs examined, secretory IgW transcripts are expressed in two forms, a full-length, long form and a truncated, short form that is probably derived by alternative splicing [19, 20]. Recent molecular characterization of Ig H chains also identified both IgW forms in the African lungfish, Protopterus aethiopicus, a lobe-finned bony fish, which phylogenetic studies suggest are closely related to land vertebrates (tetrapods) (refer to Figure 1) [1, 21, 22]. Since molecular phylogenetic analyses determined that the cartilaginous fish (Chondrichthyes) separated prior to the divergence of bony fishes (Osteichthyes – lobe-finned and ray-finned) from the other jawed vertebrates  the discovery of IgW in the lungfish suggests that it was present in the common ancestor of bony and cartilaginous fishes 460 million years ago (MYA). As well this discovery provides an impetus for further work to determine if this Ig class is present in one or more groups of the ray-finned bony fish, i.e. chondrostean, and holostean, and teleost, and the cartilaginous fish group of holocephali [21, 24] since to date only IgM and IgD have been identified in the ray-finned bony fishes and only IgM in the holocephali .
To further our understanding of Ig class expression and regulation during ontogeny, we herein describe IgM and IgW VH family expression in the newborn nurse shark, Ginglymostoma cirratum.
Newborn IgM is expressed from multiple loci of one VH family
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 . 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+ . 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].
Deduced amino acid sequence percent similarity of canonical IgM (group V) VH regions to IgM1gj and newborn IgM groups I-IV, and VI.
Newborn IgM VH regions predominantly use only the DH1 gene. The aligned IgM cDNA clones from Figure 2 were analyzed for DH gene usage by comparison to the horn shark genomic DH genes (Accession #X13447 ). The DH gene portion that exactly matches the horn shark is underlined and italicized nucleotides indicate non-templated GC nucleotide additions. Clones that could not be conclusively identified as either DH gene are indicated by (?). CDR3 identical in sequence to both DH genes are indicated by (D1/D2). Clones 22E and 36E excluded due their non-productive rearrangement.
Horn shark X13447
CAACTACA GGACTGGG ATGATCCA
GACGGAGTGATA CTGGG A
AGGGGGGGGGA CTGGG GTAGGTTCGCT
AAGACCCACC TACAG TCTATA
GCCTC AGTGG AGCGGGAATTCGCT
GATGGTG GGGT AGGGGAATTCGCT (D1/D2)
GGGGG TGGG GCCGGT
ACAAGACGCATG AGTGGG TATGAATACT
ACAGGGGGGTAC TTGGGT (D1/D2)
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) . 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 ). Clone 36E VH has 56% and 48% identical AA residues, respectively, to the sandbar shark IgW (accession #U50606 ) and nurse shark IgW (accession #U51450 ), and only 36% identical AA to a representative sandbar shark IgM VH  (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 . 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
Deduced amino acid sequence percent similarity of IgW (group VI) VH regions to newborn IgW groups I-V.
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) . 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 IgM1gj where the Ig heavy chain protein does associate with Ig light chain protein , and modeling of other Cys more C-terminal in the CH1 domain has shown they likely are available for disulfide bonding to L chains .
IgW VH (#U51450 ) structure from IMGT  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
Newborn IgW and IgM CDR3 are shorter than in adults
IgM and IgW classes were present in the ancestors of all jawed vertebrates
We show that the newborn nurse shark expresses five IgM VH genes from one conventional VH family, as shown previously for the adult horn shark, sandbar shark, and ratfish . Additionally, newborn nurse sharks express at least two other VH families (36E and IgM1gj ). Newborn IgM VH CDR3 are diverse with N-nucleotide additions, which shows that TdT is active in embryonic life, as previously documented for newborn IgNAR CDR3 [5, 14]. The majority of IgM loci are expressed at birth. Among the five VH groups these loci differ mainly in the CDR1 and CDR2, implying that diversity-enhancing selection pressures are active in germline CDR as previously reported for other vertebrates . The IgM VH CDR3 length is significantly shorter in neonates, showing that a developmental program may control the CDR3 length. One explanation for this finding may be that there is expression of different VH clusters in adults that have longer DH and JH gene segments, as is seen in the mouse ; this possibility seems unlikely as germline diversity seems similar in neonates and adults (although the adult nurse shark repertoire must be studied in more detail to prove this point). Secondly, TdT expression in pro/pre B cells in adults may be higher than in young animals . Finally, TdT activity may be modified (e.g. decreased N-region addition or increased exonuclease activity – our data suggest that DH2 segments may be extensively trimmed in neonatal junctions) by expression of splice variants or developmental regulation of DNA-dependent protein kinase, a known modulator of TdT activity [58, 59].
Newborn IgW VH expression is from at least three rather divergent VH families . CDR3 lengths of IgW are also significantly shorter than that of adults, as seen for IgM, implying that a developmental program selects for increased CDR3 length as the animals mature.
Potential somatic hypermutation Ser AGY motifs are preferentially encoded in the FR1 and CDR1, whilst the untargeted TCN motif is present in FR3. No Ser residues at all are encoded in FR2. This tendency to target mutations predominantly in the CDR is expected, but is paradoxical in FR1 of IgM VH. There are generally fewer mutations in FR1 in Ig from all vertebrates so far examined. This suggests that despite the presence of hypermutation targeting motifs in this region, there is a higher order of control over the mutational mechanism that inhibits the targeting to FR1 AGY hotspots.
The IgM and IgW VH phylogenetic analysis suggest that both VH families were present in the cartilaginous and bony fish ancestral pool. A more thorough understanding of the evolution of these VH families will require identification of IgW in the cartilaginous fish class holocephali and in bony fish groups such as the chondrostean, holosteans and teleosts. Furthermore, isolation of all of the germline genes is required to examine relative expression levels of the various genes, and the level of diversity gained by somatic hypermutation after antigenic stimulation of B cells.
Most IgM loci expressed at birth in nurse shark are from one heterogeneous highly-related VH family which differs in the CDR1-2. This repertoire is increased in its diversity via N-region addition in CDR3. In spite of the N-region addition the newborn VDJ junction is significantly shorter than the adult, most likely due to extensive trimming of the DH2 segment during rearrangement. In contrast, nurse shark IgW VH genes have diverged to form at least three VH families expressed at birth.
Nurse shark pups were delivered by Caesarian section from a gestating female shark near term as described .
cDNA library construction and screening
Tissues were dissected from nurse shark pups and total RNA was isolated as described . The newborn pup spleen and epigonal cDNA libraries were constructed as described previously . 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  and aligned in ClustalW v1.8  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 . 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 ; G. cirratum IgW U51450; G. cirratum IgM1gj 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.
Adult and newborn CDR3 length comparisons
Adult nurse shark IgM VH cDNA Genbank accession numbers AY608337-AY608404 (clones Mary M2-Mary M34, Jesus M3-M47, and Joseph M1-M27). Newborn nurse shark IgM cDNA clones1E, 2E, 2S, 6E, 14E, 15S, 17S, 21S, 22E, 24S, 26E, 27S, 29S, 30E, 35S, 36E, 46S, 47S, 49S, 50S, 54S, 55E, 57S, 72S, 73S (Genbank accession numbers AY609247-AY609274). Newborn nurse shark IgW cDNA clones 1E, 5E, 8S, 9E, 14S, 20E, 25E, 27S, 37E, 40S, 76S, 90S, 92S, 99S, 105S, 114S, 130S (Genbank accession numbers AY609225-AY609246, AY531553-AY531554, AY524297). Sandbar shark and horn shark IgM accession numbers listed in the phylogenetic tree analysis were used for determination of CDR3 length. Human (Hs) preterm neonate and adult IgM VH cDNA sequences were obtained from Zemlin et al  and Xenopus laevis (Xl) 5–48 day larvae and adult IgM VH cDNA sequences published in Schwager et al  and Du Pasquier et al .
This study was supported by NIH grant RR06603 (MFF).
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