Ohta Y and Flajnik M (2006), IgD, like IgM, is a primordial immunoglobulin c...

XB-ART-162

Proc Natl Acad Sci U S A 2006 Jul 11;10328:10723-8. doi: 10.1073/pnas.0601407103.

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IgD, like IgM, is a primordial immunoglobulin class perpetuated in most jawed vertebrates.

Ohta Y , Flajnik M .

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IgD has remained a mysterious Ig class and a bane to immunology students since its discovery >40 years ago. Its spotty occurrence in mammals and birds and the discovery of an isotype with similarities to IgD in bony fish are perplexing. We have identified IgD heavy (H) chain (delta) from the amphibian Xenopus tropicalis during examination of the IgH locus. The Xenopus delta gene is in the same position, immediately 3' of the IgM gene, as in mammals, and it is expressed only in the spleen at low levels, primarily as a transmembrane receptor by surface IgM(+) cells. Our data suggest that frog IgD is expressed on mature B cells, like in mouse/human. Unexpectedly, Xenopus IgD is orthologous to IgW, an Ig isotype found only in cartilaginous fish and lungfish, demonstrating that IgD/W, like IgM, was present in the ancestor of all living jawed vertebrates. In striking contrast to IgM, IgD/W is evolutionarily labile, showing many duplications/deletions of domains, the presence of multiple splice forms, existence as predominantly a secretory or transmembrane form, or loss of the entire gene in a species-specific manner. Our study suggests that IgD/W has played varied roles in different vertebrate taxa since the inception of the adaptive immune system, and it may have been preserved as a flexible locus over evolutionary time to complement steadfast IgM.

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Species referenced: Xenopus tropicalis
Genes referenced: igh ighx nos1 snrpb XB5798883

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	Fig. 1. The X. tropicalis δ gene is immediately 3′ of the μ gene. (A) X. tropicalis genome scaffold (version 3.0) 840 contains the IgH locus. C, secretory, and TM regions are indicated as gray, black, and open boxes, respectively. The approximate size of the Ig genes and intergenic regions is shown. The χC1 domain is only a prediction, because it has not been assembled into the scaffold. The C domains are located at the edge of the scaffold spanning an ≈90-kb region. (B) AGCT motifs are rich in 5′ upstream region of μ, χ, υ, but not in that of δ. The distance and positions of AGCT motifs are shown as bars. The numbers of AGCT motifs found in these regions are also shown under each Ig isotype. All diagrams are not to scale.
	Fig. 2. Alignment of Xenopus IgD H chain to IgD and IgW H chains from other species. Amino acid sequences were deduced from the δ cDNA sequence (GenBank accession no. DQ387453) isolated from X. tropicalis spleen. Alignments were made by using clustal x with minor manual adjustments. Domain designation was based on exon/intron boundaries. Bars and dots indicate identical amino acids and gaps, respectively. Potential IgSF strands are indicated as A–G with lines over the sequences. Noncanonical cys are marked with an open oval, and canonical cys and trp found in most IgSF domains are shaded. Glycosylation sites are underlined.
	Fig. 3. Cartoon of Xenopus IgD H-chain structure. The V and C domain are shown as large ovals, and sugars and noncanonical cys residues are indicated as small open ovals (on the left) and ticks (on the right). GenBank accession nos. are shown in the legend Fig. 6.
	Fig. 4. Xenopus IgD is expressed predominantly in the spleen. Twenty micrograms of total RNA from various tissues of an X. tropicalis frog was loaded on the gel, and the Northern blot was hybridized with X. tropicalis δC1 and TM probes separately. An X. tropicalis μ C1-TM probe was also used to compare the level of expression and tissue distribution. Two bands detected with the μ probe account for the different 3′-UTR lengths between secretory and TM forms or unknown splice variants specific to this species. Ubiquitously expressed LMPX was used as the loading control. Although the loading amounts are not equal on this blot, IgD is found only in the spleen, despite the fact that less spleen RNA was loaded. RNA size markers are shown on the right.
	Fig. 5. Relative expression level of four Ig isotypes in IgM+ vs. IgM− splenocytes. qPCR analysis was done by using SYBR green, and each run was performed in triplicate. The relative expression level was calculated by using 2−δδCt method as 10A9+ vs. 10A9− cells and plotted on a log scale in sigma plot. Primer sets are shown underneath. The figure represents the mean value of Experiment 1 (three separate runs) with standard deviations shown in bars. Mean values of two separate experiments are also shown underneath. Each threshold cycle (Ct) was normalized to the Ct of LMPX. The raw threshold cycle (Ct) values are presented in Table 1.
	Fig. 6. Phylogenetic relationship of Xenopus IgD to IgD and IgW from other species. Neighbor-joining bootstrapping trees (1,000 runs) of C1 (A) and TM (B) domains were made. Gaps were included, and multiple substitutions were not taken into account. The scales show the genetic distances. The scale shown as a bar represents the genetic distance (i.e., number of amino acid changes in the given scale). GenBank accession nos. for each sequence are as follows: Human-IgD (AAH21276), Mouse-IgD (AAB59654), Rat-IgD (AAO19643), Horse-IgD (AAU09793; ref. 44), Lungfish-IgW (AAO52811), Sandbar shark-IgW (AAB03680), Nurse shark-IgW (AAB08972), Skate-IgX (AAA49546), Catfish-IgD (AAC60133), Halibut-IgD (BAB41204), Atlantic Cod-IgD (AAF72566), Rainbow trout-IgD (AAW66976), Human-IgM (AAH11857), Mouse-IgM (AAH18315), Rat-IgM (AAH92582), Chicken-IgM (P01875), X. laevis-IgM (AAA49774), X. tropicalis-IgM (AAH89679), Lungfish-IgM (AAO52808), Nurse shark-IgM (AAA50817), Horn shark-IgM (P23085), and Skate-IgM (AAA49547).
	Fig. 7. Plasticity of IgD/W in different vertebrate species. TM and secretory forms are obviously displayed. TM forms are the major type in frog, mouse, and human. The TM and secretory forms are shown for IgW to emphasize the alternative splicing. No TM forms have been described in lungfish, and the TM/secretory forms in bony fish are the same size. L-chain association is predicted only in bony fish, Xenopus, and lungfish. Only one form of ray-finned bony fish IgD (the original catfish model) among several other forms is shown here, and the light-red domain is the C1 domain donated from the μC1 domain. The hinge regions are not shown for mouse and human δ chains.

References [+] :

Anderson, Complete genomic sequence and patterns of transcription of a member of an unusual family of closely related, chromosomally dispersed Ig gene clusters in Raja. 1994, Pubmed