In recent years, transcriptomic databases have become one of the main sources for protein discovery. In our studies of nervous system and digestive tract regeneration in echinoderms, we have identified several transcripts that have attracted our attention. One of these molecules corresponds to a previously unidentified transcript (Orpin) from the sea cucumber Holothuria glaberrima that appeared to be upregulated during intestinal regeneration. We have now identified a second highly similar sequence and analyzed the predicted proteins using bioinformatics tools. Both sequences have EF-hand motifs characteristic of calcium-binding proteins (CaBPs) and N-terminal signal peptides. Sequence comparison analyses such as multiple sequence alignments and phylogenetic analyses only showed significant similarity to sequences from other echinoderms or from hemichordates. Semi-quantitative RT-PCR analyses revealed that transcripts from these sequences are expressed in various tissues including muscle, haemal system, gonads, and mesentery. However, contrary to previous reports, there was no significant differential expression in regenerating tissues. Nonetheless, the identification of unique features in the predicted proteins suggests that these might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade.
In recent years, transcriptomic databases have become one of the main sources for protein discovery. In our studies of nervous system and digestive tract regeneration in echinoderms, we have identified several transcripts that have attracted our attention. One of these molecules corresponds to a previously unidentified transcript (Orpin) from the sea cucumber Holothuria glaberrima that appeared to be upregulated during intestinal regeneration. We have now identified a second highly similar sequence and analyzed the predicted proteins using bioinformatics tools. Both sequences have EF-hand motifs characteristic of calcium-binding proteins (CaBPs) and N-terminal signal peptides.Sequence comparison analyses such as multiple sequence alignments and phylogenetic analyses only showed significant similarity to sequences from other echinoderms or from hemichordates. Semi-quantitative RT-PCR analyses revealed that transcripts from these sequences are expressed in various tissues including muscle, haemal system, gonads, and mesentery. However, contrary to previous reports, there was no significant differential expression in regenerating tissues. Nonetheless, the identification of unique features in the predicted proteins suggests that these might comprise a novel subfamily of EF-hand containing proteins specific to the Ambulacraria clade..
Holothuria glaberrima exhibits the capacity to regenerate body parts after injury or loss. This ability is mediated by an initial blastemal formation which requires proliferation and reorganization of precursor cells. Accordingly, various molecules play key roles in different aspects of the regeneration of a body part. The use of microarrays and high‐throughput sequencing lead our laboratory to identify a series of new molecules associated to different processes of regeneration. Such is the case for the protein Orpin. Based on an EST database, we found that Orpin gene is over expressed at early stages of intestinal regeneration. Its expression occurs concomitantly with remodeling of the extracellular matrix. Also, another isoform was identified and both sequences were present in libraries from regenerating nerve and intestinal tissues. The characterization between those sequences indicates that both encode two EF‐hand motifs. Unlike other known calcium binding proteins, they exhibit a signal peptide. The use of Orpin antibodies showed the labeling of the luminal epithelium as well as the radial nerve. Moreover, these antibodies are being used to determine the tissue and temporal specificity of Orpin and its isoform. In order to evaluate the effect of Orpin during regeneration, two His‐tagged fusion proteins are expressed to be injected into regenerating animals. Thus, determining the role of Orpin and its isoform during the regeneration process would help to elucidate the molecular mechanisms involved in different aspects of nerve and intestinal regeneration. Grant Funding Source: NIH (1SC1GM084770)
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