The Dscam PRR

Unlike vertebrates, invertebrates lack the adaptive capacity and memory that allow the vertebrate immune surveillance system to distinguish a broad spectrum of micro-organisms by making use of a large and diverse collection of recognition receptors that are generated by somatic recombination of antibody immunoglobulin domains and clonal selection. The number of putative PRRs in the mosquito is limited, with only about 150 predicted PRR genes being identified in the A. gambiae genome and the total number of PRRs and effector genes numbering less than 1000 in most invertebrates (reviewed by Schulenburg et al., 2007).

One member of the immunoglobulin gene superfamily in D. melanogaster, the Down syndrome cell-adhesion molecule (Dscam) gene can potentially generate 38 016 different alternative splice isoforms through alternative splicing of 101 exons (Graveley, 2005). The Drosophila Dscam was named by analogy to the human protein DSCAM, which is a candidate disease gene for the mental retardation associated with Down's syndrome and which has been positionally cloned on chromosome 21 (Yamakawa et al, 1998). The remarkable molecular diversity of this Drosophila molecule is indicated by its gene organization. The molecule contains 10 immunoglobulin domains and six fibronectin type III domains as well as four arrays of alternative exon cassettes (the exon 4, 6, 9, and 17 cassettes) that have 12, 48, 33, and two variable exons, respectively. The analogue gene of Dscam in A. gambiae (AgDscam) has a similar number of alternative exons; it also includes exon cassettes of exons 4, 6, and 10, consisting of 14, 30, or 38 alternatively spliced immunoglobulin domain exons, respectively (see Figure 5.4 for Dscam domain organization). In theory, these opportunities for alternative splicing can result in 31 920 alternatively spliced forms in A. gambiae. Because of the existence of a mutually exclusive splicing mechanism in Dscam for generating sequence variability in its three immunoglobulin ecto-domains, D2, D3, and D7, the resulting protein isoforms all have the same domain architecture (Schmucker and Flanagan, 2004; Graveley, 2005). A regulator of Dscam's mutually exclusive splicing, the heterogeneous nuclear ribonucleoprotein hrp36, has been shown to act specifically within the exon 6 cassette to prevent the inclusion of multiple exons or multiple exon 6 variants (Olson et al., 2007).

The majority of the constant exons are highly conserved between Drosophila Dscam and AgDscam

Exon4(14) Exon6(30) Exon10(38)

Transcript m M M

Figure 5.4 Gene and protein domain organization of AgDscam. AgDscam gene has three major arrays of alternative exons, with only one single exon from each array being incorporated into each mRNA molecule. The number above the transcript indicates the corresponding exon numbers. AgDscam protein has ten Ig-like domains, six fibronectin type III domains (FN_III), and a transmembrane domain (TM). Splicing variants are generated in the Ig2, Ig3, and Ig7 domains which are shaded correspondingly to splicing exons. aa, amino acids.

(70-95% sequence similarity at the amino acid level), while the alternative-splicing exons are more variable, with only 30-70% homology between these two arthropods. This exon-sequence divergence pattern suggests that constant and alternative exons are under different functional constraints, with the constitutive exons perhaps being involved in conserved functions and the hypervariable immunoglobulin domains reflecting the profound differences in the lifestyles and environmental exposure of the two insects.

Both the nervous system and the immune system require the extraordinary diversity and specificity characteristic of recognition receptors. A landmark study by Wojtowicz et al. (2004) has clearly demonstrated that the isoform diversity of Dscam involves a binding specificity that is mainly dependent on homophilic rather than heterophilic binding; this mechanism resembles that of many other immunoglobulin cell-adhesion molecules, which bind homophilically (Agarwala et al., 2001; Wojtowicz et al, 2004) and is consistent with a separate study showing that all three of the variable immunoglobulin domains (Ig2 (D2), Ig3 (D3), and Ig7 (D7)) contribute to the binding. In a more recent study, these researchers have provided evidence that more than 18 000 isoforms exhibit striking isoform-specific homophilic binding, and that through the binding of the same variable domain, self-binding domains can assemble in different combinations to generate an enormous repertoire of homophilic binding proteins (Wojtowicz et al., 2007). Drosophila is likely using this vast repertoire to generate a unique identity and homotrophic binding specificity for each neuron as a means of helping neuronal processes to discriminate self and non-self more efficiently and specifically. The X-ray structure of the N-terminal four immuno-globulin domains (D1-D4) of two distinct Dscam isoforms (expressed using a baculovirus system) has revealed a horseshoe configuration in which the variable domains of D2 and D3 make up two independent surface epitopes (epitopes I and II) on either side of the receptor. Epitope I contributes to the homophilic binding specificity of full-length Dscam hypervariable receptors, as has been confirmed by mutagenesis studies and swapping of peptide segments (Meijers et al., 2007).

Dscam has a dual role in both neural development and the immune system in insects. It contributes to axon guidance and neuron wiring in the nervous system (Schmucker and Flanagan, 2004; Chen et al, 2006; Hattori et al, 2007), while recent studies have established that Dscam also plays a role as a hypervariable PRR in the innate immune system of insects (Watson et al., 2005; Dong et al., 2006b). Transcriptional analysis based on micro-array hybridization has indicated that Dscam repertoires are differentially expressed in haemocytes and in the nervous system, with the choice of splice variants being regulated both spatially and temporally (Neves et al., 2004; Watson et al., 2005). More interestingly, single-cell real-time PCR has demonstrated that individual cells belonging to the same cell type express diverse repertoires of Dscam isoforms, suggesting a mechanism for generating unique cell identity in both the nervous system and other tissues (Neves et al, 2004). Tissue-specific expression profiling of brain, fat body, and haemo-cytes (based on a 50-mer oligo-microarray hybridization) has indicated that 59 of 60 alternative exon 4 and exon 6 sequences are expressed in all three different cell types, with only a subset of 14 being expressed in a tissue-specific manner, in either the fat body or haemocytes (Watson et al, 2005).

Immunocompetent cells in Drosophila have the potential to express more than 18 000 isoforms of Dscam in the form of membrane-binding proteins that can serve as recognition receptors; these cells also express isoforms of Dscam that are secreted into the haemolymph. RNAi-mediated gene silencing of Dscam in larval haemocytes significantly decreases their efficiency in phagocytosing both Gram-negative and Gram-positive bacteria, suggesting that Dscam acts as either a recognition or signalling receptor during phagocytosis. Preliminary data obtained by expressing all of the extracellular domains, including all three variable immunoglobulin domains, has shown a strong binding of Dscam to bacteria, whereas binding by the isoform containing only the first two immuno-globulin domains was barely detectable, suggesting that specific homophilic binding and binding to bacteria utilize different immunoglobulin domains (Watson et al, 2005; Meijers et al, 2007). However, the molecular mechanisms by which Dscam binds to bacteria remain unknown, and their elucidation requires detailed studies in the future.

Using quantitative real-time PCR and selecting exon cassette 4 as a proof of principle, Dong and colleagues have been able to show that alternative splicing of AgDscam is important for immune responsiveness to bacteria, malaria parasites, fungi, and the bacterial surface molecules LPS and peptidoglycan (Dong et al, 2006b). Depletion of AgDscam by RNAi-mediated gene silencing of its constant domain caused a decrease in the phago-cytic efficiency of the mosquito's immunocompe-tent cells with regard to both Gram-negative and Gram-positive bacteria. RNAi-mediated gene silencing and in vitro bacterial binding assays revealed that AgDscam was binding directly to the bacterial surfaces.

Interestingly, AgDscam was shown by exon-specific gene silencing to produce pathogen challenge-specific splice form repertoires that were enriched in receptor molecules with an increased affinity and defence-related specificity for the eliciting pathogen. In vivo gene silencing of AgDscam in A. gambiae increased the mosquitoes' susceptibility to infection with both bacteria and the malaria parasites. Depletion of certain isoforms that had been enriched by the bacterial challenge led to a decrease in the mosquitoes' survival rate when infected with the same micro-organism, suggesting that AgDscam responds to bacteria in a splice-form-specific manner. At the cellular level, AgDscam has been shown to colocalize with both Gram-positive and Gram-negative bacteria, as well as with rodent and human malaria parasites. Small interfering RNA-mediated gene silencing specifically targeting candidate alternative isoforms has shown a significant increase in parasite number in the mosquito's midgut epithelium, suggesting that AgDscam is also defending the insect against malaria parasites in a splice-form-specific manner (Y. Dong and G. Dimopoulos, unpublished results). The regulators of mosquito innate immune-signalling transduction pathways are also involved in regulating the alternative splicing of AgDscam (Y. Dong and G. Dimopoulos, unpublished results). Further analyses are currently addressing the mechanisms by which AgDscam binds to both bacteria and parasites and, more importantly, are attempting to determine whether the differential association of specific splice forms of AgDscam is the source of the binding specificity for the different pathogens' surface molecules.

The vertebrate Dscam molecule has been linked to Down's syndrome, but neither of the two human Dscam paralogues that have been studied has displayed a significant degree of alternative splicing (Agarwala et al., 2001). The Dscam of zebrafish has been shown to be essential for cell migration (Yimlamai et al, 2005). Dscam-like sequences have been identified in the Diptera and Hymenoptera; in four beetles, including Tribolium castaneum (Coleoptera); and in the silk moth B. mori (Lepidoptera); orthologous genes have been identified in all these species through comparative gen-omic analysis (Graveley, 2005; Watson et al., 2005).

The Dscam gene with its alternatively spliced exons has evolved within several insect orders over 250 million years. However, homology in terms of the origin of the alternative spliced exons has not been found outside the Insecta (Crayton et al., 2006). In a more recent study, comparative structural, expression, and evolutionary analyses of a Dscam homologue in two species of the crustacean Daphnia (Cladocera) has suggested that the diversification of Dscam is functioning outside the insect world and that more than 13 000 different transcripts can be produced through the alternative splicing of variable exons in Daphnia Dscam (Brites et al., 2008). Daphnia Dscam is thought to function in both the nervous and immune systems, on the basis of the alternative expression of variable exons observed in brain cells and haemocytes.

It is possible that during evolution, different routes have been taken that have achieved functionally similar ends: Invertebrate immunity shows astounding analogies to the vertebrate adaptive immune system at the molecular level, with the alternative splicing of Dscam generating a massive quantity of recognition receptors (Kurtz and Armitage, 2006; Schulenburg et al., 2007). However, many questions remain to be addressed regarding both recognition specificity and clonal selection (Du Pasquier, 2005). Considering that recognition diversity has been achieved through alternative splicing but not through the somatic DNA rearrangements seen during the development of antibody diversity, selection likely occurs through the regulation of splicing, or it occurs after Dscam is expressed on the cell surface. How stable is the isoform repertoire after selection? How is the expression of Dscam regulated in a cell population as the cells are renewed? It has not yet been firmly established whether the different isoform repertoires mediate the specificity of the pattern recognition, or whether this recognition specificity has memory. Moreover, whether the Dscam diversification mechanisms that generate the massive expansion of receptors are active outside of the arthropod class is also as yet unknown (see reviews by Kurtz and Armitage, 2006; Schulenburg et al, 2007).

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