Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

Abstract

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium. Keywords: 185/333 proteins; sea urchin; invertebrate immunity; coelomocytes; intestine; transmission electron microscopy The immune system of sea urchins lacks antibody-based adaptive immune responses. 1,2 However, there are strong similarities between other components of sea urchin and vertebrate immune systems. The sea urchin genome revealed broad homologies between the immune response genes of these deuterostome invertebrates and those of vertebrates. 3 Many of the sea urchin gene families that are putatively involved in pathogen recognition are highly diversified compared with their vertebrate homologues. 4 It has been suggested that gut associated pathogenesis and regulation of normal gut flora might have driven the expansion of these immune response gene families in sea urchins. 4 The 185/333 gene family is unique to the sea urchins and lacks homology to all other gene families identified thus far. Nair et al. 5 first revealed the importance of 185/333 molecules in sea urchin immune responses. They showed that 185/333 mRNA expression increased by up to 75-fold in immunologically activated coelomocytes, so that over 60% of sequences in a subtracted cDNA library of immune response expressed sequence tags were 185/333 mRNAs. These results suggest a fundamental role for 185/333 proteins in the immune responses of sea urchins. Further studies revealed high levels of variability within this new gene family 5,6 that are caused by the insertion or deletion of short sequence stretches called elements, single nucleotide substitutions and sequence repeats. Proteomic analyses indicate that each sea urchin is capable of expressing a diverse repertoire of 185/333 proteins and that the suite of 185/333 proteins expressed differs when sea urchins are chal...

TheSp185/333immune response genes and proteins are expressed in cells dispersed within all major organs of the adult purple sea urchin

“…16 This is the same location as reported here for Sp185/333 + cells in S. purpuratus, which are also associated with the coelomic epithelium that covers the outer surface of the intestine. However, Sp185/333 + cells present in the columnar epithelium of the intestine in S. purpuratus were not reported in H. erythrogramma.…”

“…However, Sp185/333 + cells present in the columnar epithelium of the intestine in S. purpuratus were not reported in H. erythrogramma. 16 The intestinal localization of the Sp185/333 + cells within the columnar epithelium is strikingly similar to the variable region-containing chitin-binding proteins (VCBPs) expressed in intestine-associated cells in amphioxus, Branchiostoma floridae, and in the solitary tunicate, Ciona intestinalis. 29,30 It is noteworthy that both the Sp185/333 and the VCBP gene families have been described as highly diverse, 12,13,[29][30][31][32][33][34][35] that the VCBPs bind to bacteria and augment phagocytosis by hemocytes in C. intestinalis cells in the columnar epithelium of the intestine may be intestine cells, while the cells associated with the coelomic epithelium surrounding the intestine in contact with the coelomic fluid may be coelomocytes.…”

“…9 The Sp185/333 proteins are localized to two subtypes of coelomocytes-polygonal phagocytes and small phagocytes-where they associate with membranes and are localized to perinuclear vesicles, and are present on the cell surface of small phagocytes. 15,16 Following immune challenge in vivo, the proportion of Sp185/333-positive (Sp185/333 + ) cells in the coelomic cavity significantly increases relative to pre-challenge. 15 However, it is not known whether Sp185/333 proteins are present in various organs and tissues throughout the body.…”

Single Sea Urchin Phagocytes Express Messages of a Single Sequence from the DiverseSp185/333Gene Family in Response to Bacterial Challenge

“…Of the expressed sequence tags from those screens, 73% matched to two previously uncharacterized sequences, DD185 (11) and EST333 (13). In S. purpuratus the gene family is called Sp185/333 because it has been reported in another echinoid species (14).…”

Variable immune molecules in invertebrates

“…The functional significance of this is unclear at present, although it has been suggested that they may form bridges between immune cells (Brockton et al, 2008). They do not appear to bind bacteria in the coelomic fluid but an immunostaining study has suggested that gut amoebocytes phagocytose 185/333-covered bacteria (Dheilly et al, 2011). Conclusive evidence for the exact role of these proteins in sea urchin immunity is eagerly awaited.…”