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

Rumfelt LL, Lohr RL, Dooley H, Flajnik MF - BMC Immunol. (2004)

Bottom Line: Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101, USA. Lynn.Rumfelt@sw.ca

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.

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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.
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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.

Mentions: 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.


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

Rumfelt LL, Lohr RL, Dooley H, Flajnik MF - BMC Immunol. (2004)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC420240&req=5

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.
Mentions: 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.

Bottom Line: Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101, USA. Lynn.Rumfelt@sw.ca

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.

Show MeSH
Related in: MedlinePlus