Crow RS et al. (2024), Recombinant SpTransformer proteins are function...

ECB-ART-53117

Front Immunol 2024 Jan 01;15:1372904. doi: 10.3389/fimmu.2024.1372904.

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Recombinant SpTransformer proteins are functionally diverse for binding and phagocytosis by three subtypes of sea urchin phagocytes.

Crow RS , Shaw CG , Grayfer L , Smith LC .

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INTRODUCTION: The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. METHODS: To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. RESULTS: The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. CONCLUSION: These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.

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	Figure 1. The alignment illustrates the mosaic element patterns in the rSpTrf proteins chosen for expression in insect cells. Elements are recognizable blocks of sequence that are slightly variable, and are defined by gaps in an alignment of all known SpTrf protein sequences deduced from cDNA and gene sequences (30, 31). Elements are depicted as colored rectangles. All possible elements are numbered at the top of the alignment. There are no SpTrf sequences that include all elements. Both tandem and interspersed repeats are shown below the alignment. This figure is modified from (32).
	Figure 2. rSpTrf proteins are expressed in Sf9 insect cell cultures. (A) A Coomassie stained protein gel shows the rSpTrf proteins isolated from insect cells. (B) A Western blot shows the rSpTrf protein bands are based on recognition by rabbit-anti-V5-HRP. Broad Range protein standards (BioRad) are shown on the left. (C) The rSpTrf proteins are glycosylated with N-linked oligosaccharides. Each of the proteins was incubated with PNGaseF (F) to remove N-linked oligosaccharides, and a combination of neuraminidase and O-glycosidase (O) to remove O-linked oligosaccharides. Size changes were identified based on comparison to untreated controls (-). (D) CF containing natSpTrf proteins was treated with the glycosidases, which shows size changes with PNGaseF (F), but not with neuraminidase and O-glycosidase (O), compared to untreated controls (-). Rabbit-anti-natSpTrf antibodies (37) identify the natSpTrf proteins in the CF.
	Figure 3. All rSpTrf proteins cross-linked to beads augment the association with phagocytes. (A) When phagocytes bound to slides are incubated with rSpTrf::beads, a greater percentage of phagocytes (, p < 0.05) are associated with beads compared to the control BSA::beads. There are no significant differences (ns) among the rSpTrf proteins relative to the association of beads with phagocytes. (B) Pre-incubation of rSpTrf-E1::beads and rSpTrf-E2-3::beads with three rabbit-anti-natSpTrf antibodies reduces the percentage of phagocytes associated with rSpTrf-E1::beads (, p < 0.05), but has no significant effect on rSpTrf-E2-3::beads (ns). (C) Deglycosylation of the rSpTrf::beads prior to blocking with rabbit-anti-natSpTrf antibodies decreases (**, p < 0.05) the percentage of phagocytes with associated rSpTrf-E1::beads and rSpTrf-E2-3::beads compared to beads that are not pre-incubated with the antibodies. Each dot represents the results for phagocytes collected from a single sea urchin. For each protein at least six animals were tested, and at least 500 cells per animal were counted for bead association. The protease inhibitor, HALT (1%), was added to all proteins. The box plots show the average and interquartile range of each bead treatment associated with phagocytes. A Tukey ANOVA test was used to evaluate the significance in bead association among cells incubated with the different rSpTrf proteins cross-linked to beads.
	Figure 4. Phagocytes spun onto glass slides prior to incubation with rSpTrf::beads do not phagocytose surface bound beads. Spun cells with associated beads were incubated with rabbit-anti-V5-549 at 0°C followed by fixation and permeabilization for mouse-anti-actin and goat-anti-mouse-Ig-488 incubation. All beads were bound by rabbit-anti-V5-549 and appear red identifying only surface bound beads. No unlabeled beads are identified indicating that none are phagocytosed. (A) Bright field shows the location of all beads. (B) rSpTrf::beads are identified by rabbit-anti-V5-549 by UV epifluorescence. (C) Actin labeling of the cytoskeleton defines the types of phagocytes in the field; polygonal (P) and discoidal (D) phagocytes. (D) The merged image shows that all beads are labeled red indicating that no beads are internalized. (E) A Z-stack analysis by confocal microscopy shows that all beads are positioned above the cell surface and that none are positioned within the actin cytoskeleton. This indicates that the rSpTrf::beads are only surface bound. The DNA is labeled with DAPI. (F, G) Scanning electron microscopy confirms that spun phagocytes do not take up surface bound rSpTrf-E2-3::beads (false red color). Spun phagocytes are extremely flat, which decreases the vertical cytosolic space so that 1 µm beads are not internalized. The comparison between the beads bound to the cell surface vs. those bound to the slide surface suggests cellular recognition of rSpTrf-E2-3::beads.
	Figure 5. Coelomocytes incubated with rSpTrf-E2-3:beads in solution result in bead phagocytosis. When coelomocytes are incubated with beads in solution, they are capable of phagocytosis. Beads on the outside of the cells are labeled with rabbit-anti-V5-549 (red) before fixation and permeabilization, which blocks antibody access to the internalized beads (black). (A, B) Two polygonal phagocytes take up rSpTrf-E2-3::beads. The bright field image shows red beads that are located on the cell surface and unlabeled black beads that are phagocytosed. (C, D) Merged images show phagocytes with red beads on the surface and black beads that have been phagocytosed.
	Figure 6. The rSpTrf::beads show significant differences in the number of beads bound and phagocytosed by phagocytes. The numbers of beads per cell were evaluated for phagocytes based on beads bound to the cell surface and beads phagocytosed. The box plots show the average and interquartile range of each rSpTrf::bead treatment associated with phagocytes. (A) Differences in the number of rSpTrf::beads bound to the cell surface is based on the rSpTrf protein. (B) Differences in the number of phagocytosed rSpTrf::beads by phagocytes resolves to those with specific phagocytosis and those that are not different from the control BSA::beads. Data analysis was based on a total of 600 cells counted for each rSpTrf::bead and cells collected from six different sea urchins. Statistical analysis employed Tukey ANOVA and significance was set to p < 0.05. The letters above the boxplots indicate significant differences or similarities among groups. See also Table 2 for the different levels of binding and phagocytosis of the rSpTrf::beads based on statistical results using the Dunnet test. (C) The number of surface bound beads (S) compared to the number of phagocytosed beads (P) shows, for most proteins, that there are significantly more rSpTrf::beads bound to the cell surface than are phagocytosed (*, paired t-test, p < 0.05). There are no significant differences (ns) between surface bound and phagocytosed rSpTrf-E2-4::beads and rSpTrf-A6::beads.
	Figure 7. More phagocytes are associated with a subset of rSpTrf::beads compared to the control BSA::beads. Beads cross-linked to rSpTrf-E2-3, -E2-4, -A6, -C1, and -01 are associated with significantly more phagocytes (p < 0.05) than beads cross-linked with rSpTrf-D1, -E1, and the BSA control. (A) The box plots show the average and interquartile ranges for the average percentage of phagocytes with at least one bound or phagocytosed bead for the different rSpTrf::beads tested. Each dot represents the results for phagocytes collected from a single sea urchin. For each rSpTrf::bead, cells from at least six animals were tested, and at least 100 cells per animal were counted for bead association. Statistical analysis employed Tukey ANOVA and significance was set to p < 0.05. The letters above the boxplots indicate significant differences or similarities among groups. (B) The percentage of phagocytes with at least one bead shows variations among individual sea urchins. Cells from some animals were tested with subsets of rSpTrf::beads. Dots missing for individual animals indicate treatments that were not carried out for that animal.
	Figure 8. Polygonal, discoidal, and small phagocytes bind and phagocytose rSpTrf-E2-3::beads. Cells incubated with rabbit-anti-V5-549 prior to permeabilization labeled the beads bound to the cell surfaces, whereas internalized beads were not labeled because the antibodies were blocked from penetrating the plasma membrane. Identification of phagocyte type relied on actin cytoskeleton organization and cell size (37, 53). A polygonal cell (A, B) and a discoidal cell (C, D) have beads on the surface (red) as well as internalized beads (black). A small phagocyte (E, F) is also capable of bead phagocytosis showing unlabeled black beads around the nucleus.
	Figure 9. Different types of phagocytes show differences in binding and phagocytosis of rSpTrf::beads. Differential cell counts of cells processed for rabbit-anti-V5-459 labeling, as shown in Figure 8 , are quantified for binding and phagocytosis of different rSpTrf::beads. The box plots show the average and interquartile range of each rSpTrf::bead treatment associated with phagocytes. Statistical analysis employed Tukey ANOVA and significance was set to p < 0.05. The letters above the boxplots indicate significant differences or similarities among groups. (A, B) The polygonal phagocytes show elevated levels of binding and phagocytosis per cell for a subset of the rSpTrf proteins compared to the other phagocyte types. They also show differential binding and phagocytosis based on the rSpTrf protein cross-linked to the beads. (C, D) The discoidal phagocytes bind and phagocytose fewer beads compared to the polygonal phagocytes, and do not differentiate among the rSpTrf::beads or the BSA::beads. (E, F) The small phagocytes do not bind or phagocytose many beads, likely due to their small size of about 5 µm, but they show differences in binding and phagocytosis depending on the rSpTrf cross-linked to the beads. Several levels of binding and phagocytosis for the different cell types and different rSpTrf::beads are shown in Table 2 .