Crow RS et al. (2024), Recombinant SpTransformer proteins bind to spec...

ECB-ART-53559

Front Immunol 2024 Jan 01;15:1496832. doi: 10.3389/fimmu.2024.1496832.

Show Gene links Show Anatomy links

Recombinant SpTransformer proteins bind to specific sites on sea urchin phagocytes and modulate SpTransformer gene expression and immune responsiveness.

Crow RS , Grayfer L , Smith LC .

???displayArticle.abstract???
INTRODUCTION: The California purple sea urchin, Strongylocentrotus purpuratus, relies exclusively on an innate immune system to survive in its pathogen rich marine environment. Central to this defense is the SpTransformer (SpTrf) gene family that is unique to the euechinoid group of echinoderms. These genes were initially identified based on their striking upregulation in response to immune challenge. The SpTrf gene family encodes structurally similar proteins with a wide range of sequence diversity within and among individual sea urchins. A recombinant (r)SpTrf protein interacts specifically with a variety of non-self targets. Other rSpTrf proteins cross-linked to inert beads show distinct functions for cell binding and augmenting phagocytosis . However, whether the rSpTrf proteins bind to sea urchin phagocytes, and the cellular consequences of binding are largely unexplored. METHODS: rSpTrf protein binding to, and responses by phagocytes was investigated by cytology, flow cytometry, binding competitions using In-cell ELISA, and gene expression analyses. RESULTS: Soluble rSpTrf proteins bind specifically and exclusively to both live and fixed polygonal and small phagocytes. The different rSpTrf proteins appear to bind shared receptor(s) or other form of cell surface binding site. The phagocyte response to bound rSpTrf proteins culminates in modulated expression of the SpTrf gene family as well as other immune-related genes. CONCLUSIONS: These findings underscore the multifaceted and dynamic functions of SpTrf proteins within the innate immune system of the purple sea urchin. Their varied functions enable a robust immune response while also providing a unique modulatory mechanism by which response levels are controlled and adjusted to the level of the foreign threat.

???displayArticle.pubmedLink??? 39936151
???displayArticle.pmcLink??? PMC11810900
???displayArticle.link??? Front Immunol

???attribute.lit??? ???displayArticles.show???

	Figure 1. A cartoon alignment shows element patterns in exon 2 of the SpTrf genes. (A) Gene names, shown to the left, are based on the sequence of element 10, indicated in lower case letters, which is associated with distinct patterns of the surrounding elements. The maximum number of elements (colored boxes) from all known SpTrf genes and cDNAs from S. purpuratus (28, 37, 38) are shown at the top. Tandem and interspersed repeats in the second exon are shown at the bottom. The intron (int) for most genes is about 400 nucleotides (nt) and is not shown to scale. The intron labels are based on a phylogenetic analysis of introns to establish clades of similar sequence (28). This figure is modified from (39). (B) Many SpTrf-E2 messages are edited to SpTrf-E2.1. Most of the SpTrf messages are edited that alters the sequence, which expands the range of proteins encoded by individual genes (39). Many of the SpTrf-E2 messages are edited at a specific glycine codon to create a stop in element 8 that encodes a truncated SpTrf-E2.1 protein (38). The message is not degraded in sea urchin cells because the SpTrf-E2.1 protein is present in the coelomic fluid (43). The white striped elements in the second exon of the SpTrf-E2.1 message are 3′ of the edited stop codon and are not transcribed.
	Figure 2. rSpTrf proteins bind to live phagocytes. rSpTrf-A6 binds to a subset of live polygonal phagocytes in variable, punctate, perinuclear patterns. (A–C) Phagocytes incubated with rSpTrf-A6 are labeled with RαV5-549 followed by GαRIg-555. Most phagocytes bound rSpTrf-A6, while some showed no binding [red arrow in (A)] or very little binding [red arrow in (B)]. The arrangement of bound rSpTrf-A6 show patterns that (A) surround the nucleus, (B) are distributed partially over the nuclear area, or (C) are an asymmetrical accumulation on one side of the nucleus. The rSpTrf proteins are generally not associated with the edges of the cells. (D) Phagocytes incubated without rSpTrf-A6 are negative for background labeling with RαV5-549 and GαRIg-555. The phagocyte type is identified based on actin cytoskeletal structure, using MαActin followed by GαMIg-488 antibodies. Most cells are polygonal phagocytes, but some small phagocytes with very little actin cytoskeleton associated with the nucleus are present (A, B, D). The insets in each panel show the nuclear areas (DNA labeled with DAPI) of the cells with the most rSpTrf-A6 and without the actin label. Imaging was done on an LSM 800 confocal microscope (Zeiss) and false color editing was done with the Zeiss image processing program associated with the microscope. Scale bars indicate 10 µm.
	Figure 3. rSpTrf-A6 binds to live phagocytes with variable expression of natSpTrf proteins. Both polygonal and small phagocytes show variable binding of rSpTrf-A6 and variable expression of natSpTrf proteins. (A–F) Phagocytes incubated in aCF with rSpTrf-A6 are labeled with ChαV5 followed by GαChIg-405. Cells are also labeled with rabbit-anti-natSpTrf antibodies (α-66 and α-68) followed by GαRIg-555. α-66 and α-68 bind well to natSpTrf proteins but poorly to 36.6 nM rSpTrf-A6. (G, H) Phagocytes are incubated as in (A–F) but without rSpTrf-A6. All cells are also incubated with MαActin followed by GαMIg-488 and panels in the right column show merges of phagocytes plus actin to identify differences in cytoskeletal structure of different types phagocytes. Nuclear DNA labeling with DAPI is omitted because the emission spectrum overlaps with GαChIg-405. Outlines of cells in the left column of panels are indicated by dotted lines and correlate with the actin staining of the cells in the right column of panels. (A, B) Cell 1 binds rSpTrf-A6 (yellow) and shows a single vesicle with natSpTrf proteins. Cell 2 binds rSpTrf-A6 and expresses natSpTrf proteins (red) in a perinuclear pattern. Both cells are polygonal phagocytes. (C, D) Cells 3, 5 and 6 are polygonal phagocytes that bind rSpTrf-A6 but do not express natSpTrf proteins. Cells 4 and 7 are small phagocytes and cell 4 binds rSpTrf-A6 and expresses natSpTrf proteins, whereas cell 7 is negative for both. (E, F) Cell 8 is a small phagocyte that expresses natSpTrf proteins but does not bind rSpTrf-A6. The inset in (F) shows cell 8 with ChαV5 and MαActin labeling without the anti-natSpTrf labeling to confirm the absence of rSpTrf-A6 binding. Cells 9 and 10 are polygonal phagocytes that do not expresses natSpTrf proteins even though cell 10 binds rSpTrf-A6 and cell 9 does not. (G, H) Phagocytes incubated without rSpTrf-A6 do not show background labeling for ChαV5 followed by GαChIg-405. However, cells 11 and 13, which are small phagocytes, express natSpTrf proteins. Labeling with α-66 and α-68 followed by GαRIg-555 does not show binding artifacts in the 405 channel. Imaging was done on an LSM 800 confocal microscope (Zeiss) and false color editing for all panels was done with the Zeiss image processing program associated with the microscope. Scale bars indicate 10 µm.
	Figure 4. rSpTrf-E2-4 binds to fixed phagocytes. Fixed, impermeable phagocytes were incubated with rSpTrf-E2-4, permeabilized, and labeled with ChαV5 and MαActin followed by GαChIg-405 and GαMIg-488. (A–C) Phagocytes bind rSpTrf-E2-4 in a pattern that is distributed over the cell surface with most of the binding near the periphery of the cells. (D–F) Fixed phagocytes incubated as in (A–C) but without rSpTrf-E2-4 do not show background labeling with ChαV5 and GαChIg-405. MαActin and GαMIg-488 label the cytoskeletal structure to identify cell type. Imaging was carried out on an LSM 800 confocal microscope (Zeiss) with false color editing using associated imaging program. Scale bars indicate 10 µm.
	Figure 5. rSpTrf-A6 binds to phagocytes but not red spherule cells. Coelomocytes from two sea urchins (SU-A, SU-B) were incubated with or without rSpTrf-A6 followed by labeling with MαV5-488. Washed cells were evaluated by flow cytometry with a BD Celesta Cell Analyzer based on gates optimized for coelomocytes as reported previously (30, 63, 68). Cells were evaluated for fluorescence with the 488 nm laser to detect MαV5-488 (boxed in green), and the 647 nm laser to detect the autofluorescence of the red spherule cells (boxed in red). Detection of red spherule cells in Quadrats 1 and 4 (Q1 and Q4) in each of the four scatter plots was established based on gating profiles from Yakovenko et al. (30). Q1 and Q2 were defined based on the fluorescence levels of negative control cells incubated without rSpTrf-A6 (two lower scatter plots) as detected by the 488 nm laser. The percentage of cells in each quadrat is indicated. For gating details, see Supplementary Figure S4 .
	Figure 6. The rSpTrf proteins show distinct levels of binding to phagocytes. The rSpTrf proteins and rCSF-1 (negative control) were incubated with cells collected from three different sea urchins. Binding was evaluated with MαV5-HRP using In-cell ELISA. (A) The rSpTrf proteins bind to phagocytes. Each rSpTrf protein shows increased binding with increasing concentration except for rSpTrf-C1, which shows a binding plateau. Cells from the three sea urchins show binding variability that is also dependent on the rSpTrf protein. No binding is evident for rCSF-1. (B) There are significant differences in cell binding among the rSpTrf proteins. The binding curves in panel (A) were fitted with a linear regression model (y = mx + b) to estimate the marginal effect of protein concentration on the change in OD450. The marginal effects for each protein were compared to each other, and statistically significant differences were established using a Tukey test with significance set at p < 0.05. Letters indicate significant differences, and shared letters indicate no differences. rSpTrf-E2-4, -A6, and -01 show the steepest slopes and thus exhibit significantly more binding. In contrast rSpTrf D1, -E2-3, and -C1 have shallower slopes, indicating significantly less binding. Binding by all rSpTrf proteins is higher than rCSF-1, which does not bind to the cells. (C) Elevated levels of natSpTrf proteins on cell surfaces correlate negatively with rSpTrf binding to cells. Coelomocytes in separate wells were evaluated for natSpTrf proteins with α-71 and GαRIg-HRP and for rSpTrf proteins with MαV5-HRP in separate wells using In-cell ELISA. The level of natSpTrf proteins on the surface of coelomocytes is negatively correlated with binding by the rSpTrf proteins to the same coelomocytes (black dashed line; p ≤ 0.05; Pearson’s correlation test). Differences exist among the rSpTrf proteins that were tested. The high level of binding by rSpTrf-A6 (A, B), has an inverse relationship between the level of natSpTrf proteins on the surface of coelomocytes tested for rSpTrf-A6 binding. rSpTrf-E1 has a low level of binding (A, B) and has an inverse relationship with natSpTrf on cells. The result for rSpTrf-E2-3 is intermediate.
	Figure 7. The rSpTrf proteins show specific binding to phagocytes. Binding specificity by rSpTrf proteins to fixed cells in wells is evaluated by self-competition and cross-competition with other rSpTrf proteins. (A) Each rSpTrf protein competes with itself for binding to phagocytes. B-SpTrf proteins (20 nM) mixed with increasing concentrations of the same but unlabeled rSpTrf protein were incubated with coelomocytes. Cell binding by the biotinylated proteins was quantified with streptavidin-HRP using In-cell ELISA. Results were normalized to protein binding in the absence of a competitor, which was set to 100%. Each protein shows decreased binding in self-competition assays. (B) Cell binding by B-SpTrf-E2-4 is competed by each of the unlabeled rSpTrf proteins. B-rSpTrfE2-4 (20 nM) mixed with each of the rSpTrf proteins (80 nM) or BSA (80 nM) was evaluated for cell binding with streptavidin-HRP using In-cell ELISA. Cross competition binding results were normalized to the competition with the irrelevant control protein, BSA, which was set to 100%. All rSpTrf proteins compete and reduce significantly binding of B-rSpTrf-E2-4 to cell surfaces. (C) Cell binding by B-SpTrf-01 is competed by each of the unlabeled rSpTrf proteins. Cross competition was repeated as described for panel (B) using B-rSpTrf-01. Results were similar; all rSpTrf proteins compete with B-rSpTrf-01 for binding to cells. Significant reductions (p < 0.01) in binding shown in (B, C) were determined using Dunnett’s test. ***, p < 0.001.
	Figure 8. Phagocytes with bound rSpTrf proteins modulate expression of the SpTrf gene family and SpIL17-9. Soluble rSpTrf proteins incubated with coelomocytes from at least six sea urchins in short term cultures maintain low expression of the SpTrf genes and SpIL17-9, but not SpEchinoidin. Coelomocytes were either processed immediately (initial) for total RNA isolation, or were incubated with each of the rSpTrf proteins, with rCSF-1 (protein control), or without added proteins (negative control). Total RNA was isolated after 4 hours of incubation, processed, and evaluated for expression of selected genes by qRT-PCR. Results were normalized to the housekeeping gene SpL8 which encodes the large ribosomal protein 8 (71). Fold changes in gene expression were compared to the negative control, without added protein, and evaluated for significant differences (p ≤ 0.01, Dunnet’s test). (A) Each rSpTrf protein maintains low expression of the SpTrf gene family. Cells incubated with the rSpTrf proteins maintain low expression of the SpTrf gene family that is similar to the initial level of expression of the cells. In the absence of added protein or with rCSF-1, SpTrf expression is elevated. (B) A subset of rSpTrf proteins maintain low expression of SpIL17-9. Cells incubated with rSpTrf-01, -C1, -A6, and -01 maintain low expression of the SpIL17-9 that is similar to the initial level of expression. Expression after incubation with the other rSpTrf proteins is more random among cells from different sea urchins and is not different from the negative control or incubation with rCSF-1. (C) The rSpTrf proteins do not impact expression of SpEchinoidin. Cells incubated with the rSpTrf proteins do not show significant changes in the expression of SpEchinoidin compared to the initial expression level, to cells incubated with rCSF-1, or to the negative control cells incubated without protein. The colors of each dot in each of the panels indicates results for cells collected from individual sea urchins. , p < 0.01; *, p < 0.001.