Abstract
The Down syndrome cell adhesion molecule (Dscam) gene is an extraordinary example of diversity that can produce thousands of isoforms and has so far been found only in insects and crustaceans. Cumulative evidence indicates that Dscam may contribute to the mechanistic foundations of specific immune responses in insects. However, the mechanism and functions of Dscam in relation to pathogens and immunity remain largely unknown. In this study, we identified the genome organization and alternative Dscam exons from Chinese mitten crab, Eriocheir sinensis. These variants, designated EsDscam, potentially produce 30,600 isoforms due to three alternatively spliced immunoglobulin (Ig) domains and a transmembrane domain. EsDscam was significantly upregulated after bacterial challenge at both mRNA and protein levels. Moreover, bacterial specific EsDscam isoforms were found to bind specifically with the original bacteria to facilitate efficient clearance. Furthermore, bacteria-specific binding of soluble EsDscam via the complete Ig1–Ig4 domain significantly enhanced elimination of the original bacteria via phagocytosis by hemocytes; this function was abolished by partial Ig1–Ig4 domain truncation. Further studies showed that knockdown of membrane-bound EsDscam inhibited the ability of EsDscam with the same extracellular region to promote bacterial phagocytosis. Immunocytochemistry indicated colocalization of the soluble and membrane-bound forms of EsDscam at the hemocyte surface. Far-Western and coimmunoprecipitation assays demonstrated homotypic interactions between EsDscam isoforms. This study provides insights into a mechanism by which soluble Dscam regulates hemocyte phagocytosis via bacteria-specific binding and specific interactions with membrane-bound Dscam as a phagocytic receptor.
Highlights
The specializations of the immune system that define adaptive immunity were believed to be unique to gnathostomes [1, 2], a possible alternative adaptive immune system has recently been identified in lamprey [3] and arthropods [4, 5]
These findings demonstrated that Eriocheir sinensis Dscam (EsDscam) forms the domain organization of the 9(Ig)-4(FNIII)-Ig-2(FNIII)-transmembrane domain (TM)-cytoplasmic tail, with the possible generation of 30,600 isoforms via alternative splicing of three Ig domain regions (Ig2, Ig3, and Ig7) and a TM
The results revealed marked upregulation of EsDscam post-stimulation with S. aureus, B. subtilis, A. hydrophila, and V. parahemolyticus (Figure 2H), indicating that soluble EsDscam may play a role in antimicrobial responses
Summary
The specializations of the immune system that define adaptive immunity were believed to be unique to gnathostomes (jawed vertebrates) [1, 2], a possible alternative adaptive immune system has recently been identified in lamprey [3] and arthropods [4, 5]. Arthropods do not produce antibodies, approximately 150 Ig domain protein-encoding genes have been identified in some insect genomes [12,13,14]. Most of these genes have been studied with regard to neuronal guidance, the implications for immunity have been investigated more recently [12,13,14]. The Down syndrome cell adhesion molecule gene, Dscam, which is a member of the arthropod Ig superfamily, can potentially produce 38,016 different forms through alternative splicing of exons in Drosophila melanogaster [14]. It is conceivable that a large protein isoform repertoire with the potential for recognizing diverse ligands and epitopes could be generated in crustaceans and insects
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