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Three-finger proteins (TFPs), or Ly6/uPAR proteins, are characterized by the beta-structural LU domain containing three protruding "fingers" and stabilized by four conserved disulfide bonds. TFPs were initially characterized as snake alpha-neurotoxins, but later many studies showed their regulatory roles in different organisms. Despite a known expression of TFPs in vertebrates, they are poorly studied in other taxa. The presence of TFPs in starfish was previously shown, but their targets and functional role still remain unknown. Here, we analyzed expression, target, and possible function of the Lystar5 protein from the Asterias rubens starfish using bioinformatics, qPCR, and immunoassay. First, the presence of Lystar5 homologues in all classes of echinoderms was demonstrated. qPCR revealed that mRNA of Lystar5 and LyAr2 are expressed mainly in coelomocytes and coelomic epithelium of Asterias, while mRNA of other TFPs, LyAr3, LyAr4, and LyAr5, were also found in a starfish body wall. Using anti-Lystar5 serum from mice immunized by a recombinant Lystar5, we confirmed that this protein is expressed on the surface of coelomocytes and coelomic epithelium cells. According to ELISA, a recombinant analogue of Lystar5 bound to the membrane fraction of coelomocytes and coelomic epithelium but not to the body wall or starfish arm tip. Analysis by LC-MALDI MS/MS suggested integrin α-8-like protein expressed in the coelomocytes and coelomic epithelium as a target of Lystar5. Thus, our insights propose the important role of TFPs in regulation of starfish physiology and show prospects for their further research. [ABSTRACT FROM AUTHOR]

Three-finger proteins (TFPs), or Ly6/uPAR proteins, are characterized by the beta-structural LU domain containing three protruding “fingers” and stabilized by four conserved disulfide bonds. TFPs were initially characterized as snake alpha-neurotoxins, but later many studies showed their regulatory roles in different organisms. Despite a known expression of TFPs in vertebrates, they are poorly studied in other taxa. The presence of TFPs in starfish was previously shown, but their targets and functional role still remain unknown. Here, we analyzed expression, target, and possible function of the Lystar5 protein from the Asterias rubens starfish using bioinformatics, qPCR, and immunoassay. First, the presence of Lystar5 homologues in all classes of echinoderms was demonstrated. qPCR revealed that mRNA of Lystar5 and LyAr2 are expressed mainly in coelomocytes and coelomic epithelium of Asterias, while mRNA of other TFPs, LyAr3, LyAr4, and LyAr5, were also found in a starfish body wall. Using anti-Lystar5 serum from mice immunized by a recombinant Lystar5, we confirmed that this protein is expressed on the surface of coelomocytes and coelomic epithelium cells. According to ELISA, a recombinant analogue of Lystar5 bound to the membrane fraction of coelomocytes and coelomic epithelium but not to the body wall or starfish arm tip. Analysis by LC-MALDI MS/MS suggested integrin α-8-like protein expressed in the coelomocytes and coelomic epithelium as a target of Lystar5. Thus, our insights propose the important role of TFPs in regulation of starfish physiology and show prospects for their further research.