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Mar Drugs
2024 Oct 30;2211:. doi: 10.3390/md22110488.
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In Search of the Role of Three-Finger Starfish Proteins.
Lyukmanova EN
,
Bychkov ML
,
Chernikov AM
,
Kukushkin ID
,
Kulbatskii DS
,
Shabelnikov SV
,
Shulepko MA
,
Zhao R
,
Guo W
,
Kirpichnikov MP
,
Shenkarev ZO
,
Paramonov AS
.
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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.
Figure 1. Comparison of human and A. rubens TFPs. (a) Heatmap of pairwise sequence similarity of the LU domain sequences of the human and A. rubens TFPs. Only starfish proteins having high similarity (>53%) with human proteins are shown. (b) Superimposition of spatial structures of the LU domains of the human and A.sterias rubens TFPs with the highest sequence similarity in ribbon representation. Protein names and sequence similarity (%) are shown on the panels. Experimental NMR structures of Lypd6 and Lynx2 (PDB: 6IB6 and 6ZSS, respectively) are shown. For other cases, sequence-based structures predicted by ESMFold2 [36] are shown.
Figure 2. Maximum likehood guide tree of the LU domains of the human and A. rubens TFPs. Different types of proteins are signed by colors.
Figure 3. Maximum likehood dendrogram and phylogenetic relationships of A. rubens Lystar5 homologues among Echinodermata species. Class and species are shown. Numbers denote similarity distance.
Figure 4. Expression of the TFPs genes and integrin α-8-like gene in different tissues of A. rubens: (a) Data represented as mRNA level of starfish TFPs normalized to the level of mRNAs encoding 40s ribosomal protein S13 and glyceraldehyde-3-phosphate dehydrogenase ± SEM (n = 5). (* p < 0.05) indicates significant difference between the data groups according to one-way ANOVA followed by Tukey’s test. (# p < 0.05) indicates a significant difference between the data groups according to the Kruskal–Wallis test followed by Dunn’s test. (b) Heatmap representing the mean normalized level of mRNAs encoded different TFPs (Lystar5, LyAr2, LyAr3, LyAr4, and LyAr5).
Figure 5. Analysis of Lystar5 expression and localization in different A. amurensis tissues: coelomocytes, coelomic epithelium cells, cells of the body wall, and arm tip. (a) Representative flow cytometry histograms of cells’ distribution by fluorescence intensity of fluorescent dye (TRITC) conjugated with secondary antibodies. (b) Quantification of the Lystar5 expression in different tissues revealed from flow cytometry data. Data presented as normalized MFI ± SEM (n = 4–5). (**** p < 0.0001) indicate a significant difference between the data groups according to the one-way ANOVA followed by Tukey’s test. The shift of the median of the cells’ distribution histogram to the right in comparison to control serum indicates the specific binding of anti-Lystar5 serum to analyzed cells. (c) Lystar5 localization in the cells of different tissues assayed by confocal microscopy (n = 5). Scale bar = 10 µm; nuclei were stained by Hoechst 33342.
Figure 6. Interaction of immobilized recombinant Lystar5 with lysates of tissues of A. amurensis by BLI. (a) Traces represent average of three double-referenced (after subtraction of non-specific binding) BLI sensograms for each tissue ± SEM (n = 3). (b) Bar graphs represent the mean values for maximal response (at 870 seconds) for BLI biosensors with immobilized Lystar5 and without Lystar5 (control) ± SEM (n = 3). * p < 0.05 indicates a significant difference between the responses measured by using the Lystar5-coupled and control biosensors according to the unpaired parametric t-test.
Figure 7. ELISA analysis of the Lystar5 interaction with immobilized membrane fraction of different tissues of A. rubens. Parameters describing the curve fit for coelomocytes: A1 = 1.97 ± 0.09, EC50 = 54.5 ± 15.4 nM, nH = 0.71 ± 0.1; for coelomic epithelium: A1 = 2.85 ± 0.12, EC50 = 13.4 ± 6.6 nM, nH = 0.75 ± 0.10. Curves were not fitted for the body wall and arm tip. Data present as the background-subtracted data, O.D. ± SEM (n=5).
