Diversification of Ig Superfamily Genes in an Invertebrate

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Science  09 Jul 2004:
Vol. 305, Issue 5681, pp. 251-254
DOI: 10.1126/science.1088069


The freshwater snail Biomphalaria glabrata possesses a diverse family of fibrinogen-related proteins (FREPs), hemolymph polypeptides that consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyl-terminal fibrinogen domain. Here, we show that the IgSF1 domain of the FREP3 subfamily is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants are derived from a small set of nine source sequences by point mutation and recombinatorial processes. Diverse FREP3 transcripts are also produced. We hypothesize a mechanism present in snails that is capable of diversifying molecules involved in internal defense.

Invertebrates do not possess the lymphocyte-based adaptive immune system that plays a conspicuous role in vertebrate immunity, yet invertebrates nonetheless are capable of effective immune responses (1). Although much has been learned in recent years from the Drosophila model system (24), we have not yet achieved a comprehensive view of innate immunity across the broad spectrum of invertebrate phyla (5). We studied the innate immune system in a prominent invertebrate phylum, the Mollusca, with the use of the pulmonate gastropod Biomphalaria glabrata, an intermediate host for the human parasite Schistosoma mansoni, as a model organism. Our previous investigations have revealed that B. glabrata possesses a diverse family of hemolymph proteins termed fibrinogen-related proteins or FREPs (69) (Fig. 1A). FREPs are an unusual juxta-position of one or two N-terminal immunoglobulin superfamily (IgSF) domains with a C-terminal fibrinogen (FBG) domain (Fig. 1B) (68). FREPs may have a role in immune defense (6): They are produced in increased abundance by the circulating defense cells (hemocytes) of snails after infection with trematode parasites, and they have lectin-like properties allowing them to precipitate soluble antigens derived from trematodes (6). At present, 13 different FREP subfamilies have been identified (69). Full-length sequences have been obtained for four FREP genes (7, 8).

Fig. 1.

Recovery of diverse sequences of the FREP3 gene subfamily of B. glabrata. (A) Southern blot representing genomic DNA from one snail digested by different restriction endonucleases. The probe contained an FBG-encoding sequence that is relatively conserved among FREP gene subfamilies. Numerous cross-reactive bands (over 23 in the EcoR I digest) demonstrate an extensive FREP gene family in the genome of B. glabrata (79). Size indicated in kilobases (same in Fig. 2A). (B) Top, domain organization of FREP3 at the polypeptide level. Bottom, the intron-exon structure of FREP3, indicating the location of a 330-nt region of IgSF1 targeted for PCR amplification with the use of primers F3GSP23a and F3GSP16a. All primers used in this study are indicated. SP, signal peptide; IgSF, immunoglobulin superfamily domain; SCR, small connecting region; ICR, interceding region; and FBG, fibrinogen domain. Colors identify corresponding coding and protein regions. (C) The number of cloned sequences analyzed for two individual snails (A and B) and numbers of sequences of IgSF1 present within and shared between these snails [at nt and amino acid (aa) levels].

We previously noted that FREP genomic sequences were surprisingly variable, both within IgSF and FBG domain-encoding regions (7, 8). In this study, we chose to investigate a gene region that is particularly variable, namely a 330-nucleotide (nt) fragment of the N-terminal IgSF1 domain of FREP3 (referred to hereafter as IgSF1) that spans most of exon 2 of FREP3 and includes two cysteine codons that are conserved among IgSF domains. This gene region was amplified by polymerase chain reaction (PCR) from whole-body genomic DNA of individual snails, cloned, and sequenced (10) (Fig. 1B).

Analysis of 183 clones from one individual (snail A) yielded 45 different sequences and, from a second individual (snail B), 37 different sequences were obtained from 173 clones. Only one sequence was common to both snails (Fig. 1C). A total of 314 different sequences (recovered from 1493 clones) was eventually derived from 22 snails (including snails A and B and different strains and field isolates) and embryos derived from one parent (11). All of these sequences belong to the FREP3 subfamily (nt identity ≥ 86%) and cluster together in a gene tree, separate from other FREP subfamilies (fig. S1).

The results of several control experiments ruled out experimental artifacts such as template switching (12) and polymerase error (13) as major contributors to the large sequence diversity that was observed [for details, see Supporting Online Material (SOM) Text]. The nonrandom nature of sequence diversity was evident from the absence of insertions or deletions, as well as a significant excess of synonymous nucleotide substitutions and a deficit in lethal mutations (yielding stop codons) among the inferred point mutations. The codons for the cysteine residues that are conserved among IgSF sequences were only rarely affected. The distribution of nucleotide differences among IgSF1 sequences did not indicate obvious hot spots for point mutations. The diversity of IgSF1 sequences was three- to fourfold higher (P = 7 × 10–4) compared to control genes, myoglobin (14) and heat shock protein 70 (hsp70) (15), obtained with the use of strictly comparable experimental methods (10). Notably, an analysis of FREP3, myoglobin, and actin sequences contained within an EST (expressed sequence tag) data set collected by an independent research group, with the use of another strain of B. glabrata and different methods, also provided support for increased sequence diversity of FREP3 relative to these other genes (SOM Text).

Next, investigation of the number of FREP3 loci by Southern hybridization showed that only three to five restriction fragments were present in DNA of individual snails (Fig. 2A). The resulting estimate for the number of FREP3 loci thus was between two and five (2 to 10 alleles present in a single individual), which is too low to account for the extensive sequence diversity observed. Computational analysis performed to address this apparent inconsistency led us to conclude that a minimal set of nine source sequences is sufficient to generate all other FREP3 sequence variants through modification by (i) nucleotide point mutations, (ii) concatenation of segments from pairs of source sequences (recombinatorial diversification), or (iii) a combination of these two processes. In fact, all nine inferred source sequences concorded with particular FREP variants that were recovered more frequently than other sequences, and from multiple snails (Fig. 2B). The source sequences may represent germline FREP3 genes: Between two and five source sequences were present in individual snails, a number consistent with the estimated number of FREP3 loci. Also all FREP3 diversity within a single snail derives from its source sequences (Fig. 2C and fig. S2).

Fig. 2.

Diversity among IgSF1 sequences of FREP3 subfamily. (A) Southern analysis showing that genomic DNA from one snail, digested by four different restriction endonucleases, contains three to five bands that strongly hybridized with a FREP3-specific probe. (B) The nine computationally inferred source sequences. The considerable similarity of these source sequences is demonstrated by a hypothetical generation of these sequences from a pair of ancestral prototype sequences via recombinatorial diversification (gray and light gray) and point mutation (black). Only the variable nucleotide positions along the 330-nt length of the IgSF1 sequences are depicted. The color (key) of the source sequences is also used in (C) and figs. S2 to S4. (C) Diversity of all IgSF1 sequences recovered from snail A can be derived from five source sequences by point mutations, recombinatorial diversification, or both. The source sequences are identified by different colors. These colors show the contribution of source sequences to variant sequences, with color changes indicating recombinatorial diversification. White backgrounds denote point mutations. Only the variable nucleotide positions along the 330-nt length of the IgSF1 sequences are depicted.

To verify that B. glabrata produces diversified FREP3 transcripts, we recorded the diversity of cDNA of three juvenile snails. The diversity among 48 different FREP3 sequences recovered was elevated three- to fourfold over that of control genes. As with the genomic sequences described above, point mutations and recombinatorial diversifications best explained the variant FREP3 cDNA sequences (16). The variability of cDNA sequences is comparable to that of genomic FREP3 clones (P = 0.47) (SOM Text).

To determine whether IgSF1 sequences from a parent are similar to those found in offspring produced by self-fertilization (B. glabrata is hermaphroditic), we sampled genomic DNA from circulating hemocytes of a snail and from ∼30 pooled embryos [gastrula or early trochophore stage (17)] derived from this snail. Diversification of both parental and offspring IgSF1 sequences by both mutational and recombinatorial processes was again evident (Fig. 3A). The frequency of parental point mutations was higher than that of the offspring (P = 0.02), but the rate of recombinatorial diversification was not different. All three source sequences recovered from the parent were also observed in the offspring. The offspring yielded one additional source sequence, suggesting that our sampling may not have recovered the full complement of source sequences from the parent (fig. S3). Three of the four source sequences recovered from a parent and its progeny were held in common, in contrast to only one of nine source sequences from snails A and B. One interpretation of the intergenerational diversity of FREP3 genes observed here is that this enables a hermaphrodite such as B. glabrata to prevent tracking by pathogens across host generations (18).

Fig. 3.

Diversity of FREP3 variants between parent and offspring and between different tissues. (A) Distribution of distinct FREP sequences from one parent (P) snail and its offspring (O) produced by selfing. The number of clones analyzed is indicated in parentheses. Left diagram, the sequences are regarded as distinct if they differ by at least one nucleotide. Right diagram, sequences are considered equivalent if they differ by inferred point mutation only. This is a more stringent approach to describing sequence diversity because it is insensitive to nucleotide misincorporations during PCR. Note that previously distinct sequences may be rendered equivalent by ignoring point mutations. (B) The diversity of FREP3 sequences obtained from different cells types of the snail, shown in the same fashion as in (A). Stomach wall (S; mostly muscle cells), cells of nervous (N) systems from cerebral ganglia, and hemocytes (H).

Efforts to localize the diversification process to a particular tissue or cell-type resulted in recovery of similar rates of diversity of FREP3 sequences from DNA isolated from hemocytes, central nervous system tissue, and muscle from the stomach wall, all derived from the same juvenile snail (Fig. 3B). In addition to four shared source sequences, many variant sequences were observed. Although we are not able to rule out hemocyte contamination of the tissue samples and may thus overestimate the diversity in nonhemocyte tissues, these results suggest that IgSF1 diversification occurred in all three tissues (fig. S4).

The overall diversity of B. glabrata FREPs is likely considerably greater than that suggested by the IgSF1 region of FREP3 alone. FREP proteins exist in their native configuration as complex multimers (19). Moreover, previous observations indicated that other regions of FREP genes were also potentially variable (79). We confirmed this experimentally by using PCR to amplify a 539-nt region of the C-terminal FBG sequence of FREP3 from the DNA of a single snail. Analysis of 21 clones revealed 8 distinct FBG sequences (92 to 99% nt identity) that differed from each other by point mutations and, in one instance, recombinatorial diversification (fig. S5). Similar diversification was indicated among independently obtained EST data (SOM Text).

FREP3 sequences are extraordinarily diverse. The low estimated number of FREP3 loci and the frequent recovery of a small number of source sequences stands in sharp contrast to the low frequency of recovery of the vast majority of remaining variant FREP3 sequences. One hypothesis to account for this pattern is that germline DNA contains FREP3 source sequences that are diversified in all somatic cells at a low frequency. This diversification selectively targets FREP3 genes because control genes do not incur elevated mutations. Somatic recombinatorial diversification occurs in vertebrates, yeast, and plants (2022) but has not been reported from jawless vertebrates or invertebrates. Considering also that recombination and mutation occur spontaneously during meiosis, such modification of germline FREP3 source sequences may provide individual snails with different starting repertoires of IgSF1 sequences.

We emphasize that we are not implying that the mechanisms for diversification of IgSF1 of FREP3 are related to those of vertebrate immunoglobulin diversification. However, the concept of diversified IgSF molecules functioning in immune defense may have to be extended not only to invertebrates, but specifically to the protostome lineage, which is distantly related to vertebrates (deuterostomes). Further study is needed to characterize the underlying processes in the gastropod B. glabrata.

Our findings help to understand how molluscs, without the benefit of adaptive immunity, have nonetheless thrived despite challenge by varied and omnipresent pathogens. These results, along with other studies, such as of the Toll pathway (24) and of Amphioxus VCBP proteins (23), further blur the distinction between the internal defenses of invertebrates and vertebrates. It will be interesting to learn if diversification of nonself recognition molecules also occurs in other invertebrate phyla long separated from one another by independent evolutionary histories.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S5


References and Notes

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