Generation of clade- and symbiont-specific antibodies to characterize marker molecules during Cnidaria-Symbiodinium endosymbiosis.

The endosymbiosis between cnidarians and dinoflagellates is responsible for the formation of coral reefs. Changes in molecules have been identified during the process of cnidaria-Symbiodinium endosymbiosis. However, the complexity of the molecular interaction has prevented the establishment of a mechanistic explanation of cellular regulation in this mutualistic symbiosis. To date, no marker molecules have been identified to specifically represent the symbiotic status. Because the endosymbiotic association occurs in the symbiotic gastrodermal cells (SGCs), whole cells of isolated SGCs were used as an antigen to generate monoclonal antibodies (mAb) to screen possible molecular candidates of symbiotic markers. The results showed that one of the generated monoclonal antibodies, 2-6F, specifically recognized clade C symbiotic Symbiodinium but not its free-living counterpart or other Symbiodinium clades. The expression levels of 2-6F mAb-recognized proteins are highly correlated with the symbiotic status, and these proteins were characterized as N-linked glycoproteins via treatment with peptide N-glycosidase F. Furthermore, their glycan moieties were markedly different from those of free-living Symbiodinium, potentially suggesting host regulation of post-translational modification. Consequently, the 2-6F mAb can be used to detect the symbiotic state of corals and investigate the complex molecular interactions in cnidaria-Symbiodinium endosymbiosis.

Heterogeneous nuclear ribonucleoprotein hrp36 acts as an alternative splicing repressor in Litopenaeus vannamei Dscam.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are highly conserved from nematode to mammal because they play an important role in several aspects of pre-mRNA maturation, including RNA packaging and alternative splicing. In Drosophila, the hnRNP A1 homolog hrp36 regulates alternative splicing in several genes, including the Down syndrome cell adhesion molecule (Dscam), which produces tens of thousands of isoforms from one locus. In this study, the first hrp36 gene was identified and characterized from Litopenaeus vannamei (Lvhrp36). Its open reading frame (ORF) contains 1101 bp encoding 366 amino acids. The deduced Lvhrp36 protein includes two copies of the RNA recognition motif (RRM), a C-terminal glycine-rich domain (GRD), the highly degenerate RNP consensus sequences RNP-1 and RNP-2, and two RGG boxes. Tissue tropism analysis indicated that Lvhrp36 is expressed ubiquitously and at high levels in most tissues. dsRNA silencing of shrimp Lvhrp36 in vivo induced abnormal exon inclusions in LvDscam, especially in the Ig3 variable region. In the Ig3 region, a total of 14 different combinations were arranged in three different types of abnormal inclusion pattern. This compares to a single combination (one abnormal pattern) in Ig2 and three different combinations (one abnormal pattern) in Ig7. This is the first evidence to suggest that hrp36 may be involved in the regulation of the Ig7 variable region, and it is noteworthy because, at least in Drosophila, there are no hrp36 binding sequences in the Ig7 exon cluster. The above aberrant events were not observed in all of the Lvhrp36-silenced shrimp, and even when they occurred, the normal patterns of inclusion were far more common. We hypothesize that this continued prevalence of normal inclusions was probably due to other unsilenced proteins that were able to rescue Lvhrp36's functionality. Taken together, our results suggest that Lvhrp36 acts as a splicing repressor that regulates alternative splicing events in the Ig2, Ig3 and Ig7 variable regions of shrimp L. vannamei Dscam.

Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported.

Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.

The putative invertebrate adaptive immune protein Litopenaeus vannamei Dscam (LvDscam) is the first reported Dscam to lack a transmembrane domain and cytoplasmic tail.

It has recently been suggested that Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in the alternative adaptive immune system of invertebrates. Here, we isolated and characterized the first shrimp Dscam from Litopenaeus vannamei. The LvDscam protein had an extracellular domain but lacked the expected transmembrane domain and cytoplasmic tail, both of which are found in all other members of the Dscam family (and may also be found in other L. vannamei Dscams that have not yet been isolated). In nervous tissue, expression levels of LvDscam were unexpectedly low. Phylogenetic analysis suggests that LvDscam is far from the Dscams found in other invertebrates. Nevertheless, the domain architecture of the extracellular region of LvDscam is similar to other invertebrate Dscams, and it exhibits the typical configuration of 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD). Cloning and characterization of a total of 62 cDNAs from hemocytes collected from WSSV-free, WSSV-persistent and WSSV-acute-infected shrimp revealed 23 alternative amino acid sequences in the N-terminal of Ig2, 30 in the N-terminal of Ig3 and 13 in the Ig7 domain. This implies that LvDscam can potentially encode at least 8970 unique isoforms. Further analysis suggested that the LvDscam Ig2 and Ig3 regions are more functionally important than Ig7 in the shrimp's specific immune response against WSSV. We discuss how this tail-less, soluble Dscam can still play an active role in alternative adaptive immune response even while its axonal guidance functionality may be impaired.