Shotgun Proteomics at Sea: Coelomic Fluid of the Purple Sea Urchin

In a shotgun proteomics study of the coelomic fluid of the purple sea urchin (Strongylocentrotus purpuratus), Dheilly et al.¹ housed three sea urchins (S. purpuratus) for approximately six months and then returned the animals to full immune function with an injection of lipopolysaccharide prior to sample collection. Due to clotting factors, the researchers investigated whole coelomic fluid rather than evaluating coelomic fluid and coelomocytes in separate runs. After subjecting the samples to mass spectrometry (Thermo Scientific), 307 abundant proteins were identified. The data were evaluated for reproducibility by plotting the log (NSAF) values of the three sea urchins against each other, resulting in linear curves with high r² values and indicating proteomic similarity among the three individual animals.

The researchers grouped the identified proteins into 13 functional categories, including cell structure, shape, and mobility; cell adhesion; immune response; lysosomes, proteases, and peptidases; intracellular transport; exchangers and ATPases; cell signaling; stress response and detoxification; energy metabolism; nucleic acid and protein metabolism and processing; and cell proliferation, reproduction, and development. There were three proteins that were determined to be unclassifiable “others” and six “unknown” proteins. Of the 307 identified abundant proteins, a full 267 were predicted using the S. purpuratus genome. This is significant as it means that Dheilly et al.¹ were able to use shotgun proteomics to reliably confirm the presence of previously suspect proteins in coelomic fluid.

The researchers further assert that the majority of these identified proteins appear to be immunity related, although no meaningful relationship was observed between the number of identified proteins within each functional category and the relative abundance of the proteins in the whole coelomic fluid. Two categories, cell structure, shape, and mobility and cell signaling, were both highly represented in the samples, with 53 and 48 proteins, respectively. NSAF values indicate that the most abundant proteins were derived from the cell structure, shape, and mobility category and the immune response categories.

In the cell structure, shape, and mobility category, the most abundant protein was actin. Other highly abundant proteins important to cytoskeletal modification included profilin, fascin, cofilin, gelsolin, coronin, mysosin, tubulin, actinin, and collagen. The abundance of proteins from this category is not surprising since dynamic responses to the environment (such as phagocytosis and amoeboid movement) are highly represented in coelomocytes. Researchers noted 48 diverse proteins related to intracellular signaling, although none of these was abundant. Notable signaling proteins include calcistorin, a molecule related to clotting, and lipoproteins, reinforcing the potential relationship between lipid metabolism and coloelomocyte immune response. In the nucleic acid and protein metabolism and processing category, 37 low abundance proteins were identified, with histone H4 being the most abundant in the category. The researchers note that, while the antibacterial role of histones has not been studied in sea urchins, their abundance and conserved role in metazoans indicate a link between histones and sea urchin immune response. Thirty-three proteins were isolated in the immune response category, including the most abundant SpC3 and SpBf, which are central to the three identified complement activation pathways. Pathogen recognition molecules, Sp185/333 family proteins, and SCR proteins were also found in this category. Twenty-eight of the proteins were determined to fall into the cell adhesion category, including the highly abundant amassin variants, Von Willebrand factor, annexin, cadherin, selectin, talin, and galectin. Twenty-seven moderately abundant proteins were identified in the stress response and detoxification category, particularly heat shock proteins and chaperonin subunits as well as proteins involved in iron metabolism. For energy metabolism, 20 proteins were isolated, including transaldolase and transketolase. The 17 proteins from the lysosome, proteases, and peptidases category included a2-macroglobulin and thrombin. Echinonectin (several variants) and apextrin were both notable isolates in the 17 proteins from the cell proliferation, reproduction, and development category. In exchangers and ATPases, of the 13 identified proteins, a voltage-dependent anion channel, an H+ transporting ATPase, and a mitochondrial ATP synthase were most significant. Finally, six intracellular transport proteins were isolated, including adaptor protein 2, major vault protein, flotilin, and sorting nexin.

Dheilly et al.¹ assert that one noteworthy observation in this study is the relatively low number of peptides that matched to Sp185/333 sequences (73 percent). The researchers point to difficulties in identifying Sp185/333 proteins by mass spectrometry and suggest the possibility that only a small percentage of these transcripts is actually translated into proteins. It is also possible that the high variability within the species and individuals may account for differential expression of previously unsequenced Sp185/333 proteins. Other highly variable gene families (TLRs, NLRs, and fibrinogen domain-containing proteins) were absent from the experimental samples, suggesting that, under experimental conditions, the genes are either expressed at low levels in coelomic fluid or that the abundance may be below detectable levels for the method.

The researchers called for further shotgun proteomics studies to evaluate the integrated immune responses of coelomocytes, particularly those that are functionally specialized, and to shed light on the immune-related intracellular processes of the sea urchin, including lipid metabolism and clotting pathways.

References

1. Dheilly, N.M., et al. (2013) ‘Shotgun proteomics of coelomic fluid from the purple sea urchin, Strongylocentrotus purpuratus‘, Developmental and Comparative Immunology, published online January 23, 2013. http://dx.doi.org/10.1016/j.dci.2013.01.007