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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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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.
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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.

Mentions: 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.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: 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.

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