The Recombinant Sea Urchin Immune Effector Protein, rSpTransformer-E1, Binds to Phosphatidic Acid and Deforms Membranes

Abstract

The purple sea urchin, Strongylocentrotus purpuratus, possesses a sophisticated innate immune system that functions without adaptive capabilities and responds to pathogens effectively by expressing the highly diverse SpTransformer gene family (formerly the Sp185/333 gene family). The swift gene expression response and the sequence diversity of SpTransformer cDNAs suggest that the encoded proteins have immune functions. Individual sea urchins can express up to 260 distinct SpTransformer proteins, and their diversity suggests that different versions may have different functions. Although the deduced proteins are diverse, they share an overall structure of a hydrophobic leader, a glycine-rich N-terminal region, a histidine-rich region, and a C-terminal region. Circular dichroism analysis of a recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1) demonstrates that it is intrinsically disordered and transforms to α helical in the presence of buffer additives and binding targets. Although native SpTrf proteins are associated with the membranes of perinuclear vesicles in the phagocyte class of coelomocytes and are present on the surface of small phagocytes, they have no predicted transmembrane region or conserved site for glycophosphatidylinositol linkage. To determine whether native SpTrf proteins associate with phagocyte membranes through interactions with lipids, when rSpTrf-E1 is incubated with lipid-embedded nylon strips, it binds to phosphatidic acid (PA) through both the glycine-rich region and the histidine-rich region. Synthetic liposomes composed of PA and phosphatidylcholine show binding between rSpTrf-E1 and PA by fluorescence resonance energy transfer, which is associated with leakage of luminal contents suggesting changes in lipid organization and perhaps liposome lysis. Interactions with liposomes also change membrane curvature leading to liposome budding, fusion, and invagination, which is associated with PA clustering induced by rSpTrf-E1 binding. Longer incubations result in the extraction of PA from the liposomes, which form disorganized clusters. CD shows that when rSpTrf-E1 binds to PA, it changes its secondary structure from disordered to α helical. These results provide evidence for how SpTransformer proteins may associate with molecules that have exposed phosphates including PA on cell membranes and how the characteristic of protein multimerization may drive changes in the organization of membrane lipids.

Keywords: Sp185/333; conformational plasticity; echinoderm; innate immunity; lipid clusters; liposomes.

Figure 1

Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1) and the recombinant fragments bind to lipids. (A) The protein structure of rSpTrf-E1 shows four regions; the N-terminal leader (L), the Gly-rich region, the His-rich region, and the C-terminal region. Four recombinant proteins are evaluated for their lipid binding characteristics using a lipid-embedded nylon strip (B); the full-length rSpTrf-E1 protein, the rGly-rich fragment, the C-terminal end of the Gly-rich region called rC-Gly, and the rHis-rich fragment. (B) rSpTrf-E1, the rGly-rich and the rHis-rich fragments bind to PA. The rHis-rich fragment also binds to PtdIns(4)P. The rC-Gly fragment binds only to PS. Arrows indicate the phospholipids to which the proteins bind. The nylon strip is embedded with spots of TAG, trisacylglyceride; DAG, diacylglycerol; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; CL, cardiolipin; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P₂, phosphatidylinositol 4,5 bisphosphate; PtdIns(3,4,5)P₃, phosphatidylinositol 3,4,5 triphosphate; SPH, sphingomyelin; SM4, 3-sulfogalactosylceramide; cholesterol.

Figure 2

Small unilamellar vesicles (SUVs) composed of 10% PA:PC with DiI plus recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1)-FITC generates fluorescence resonance energy transfer (FRET). (A) FRET increases with increasing concentration of rSpTrf-E1-FITC when mixed with 10% phosphatidic acid (PA):phosphatidylcholine (PC) SUVs. (B) FRET evaluation at varying percentages of PA in PC liposomes shows that 10% PA:PC SUVs exhibit optimal FRET when mixed with either 1.33 or 2.67 µM rSpTrf-E1-FITC (not statistically different). Reduced FRET for SUVs with 20% PA:PC may have been the result of self-quenching, and these data are not included in the statistical analysis. Bars represent mean and SE, and black and gray horizontal lines indicate statistical significance at p < 0.05. (C) SUVs composed of PC plus indicated percentages of PA show low levels of FRET in the presence of BSA-FITC. These levels are not different from SUVs in the presence of NA-FITC and are significantly lower than FRET with SUVs in the presence of all concentrations of rSpTrf-E1-FITC. Horizontal bars indicate significance at p < 0.05. FRET was initiated with excitation at 450 nm, and emission was recorded at 560 nm.

Figure 3

Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), induces giant unilamellar vesicles (GUVs) to bud, fuse, invaginate, leak, and disappear. (A) Confocal microscopy images show budding of two independent GUVs into two or three smaller vesicles (a–d, white and yellow arrows). Leakage of dextran-488 appears as black spaces in the lumen of two GUVs (c,d, white circles). (B) Images show GUV fusion between two GUVs (a–e, orange arrows), leakage at the convex curve of the membrane (white arrow), which is the site of invagination of the fused GUV (f–h, orange arrows). (C) Images show invagination (a–h, red arrows), lysis (a–h, blue arrows), and a slow decrease in dextran-488 fluorescence in a GUV (a–h, purple arrows) suggestive of slow leakage leading to lysis. Image acquisition is every 30 s as indicated after the addition of rSpTrf-E1. All scale bars indicate 10 μm.

Figure 4

Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), monomers and the rHis-rich fragment induce luminal content leakage from the large unilamellar vesicles (LUVs). (A) A Western blot evaluated with anti-SpTrf antisera shows dimers and monomers of rSpTrf-E1, unknown proteins from non-induced bacteria (Non-induced) that were processed following the same sample preparation as the recombinant proteins, and natSpTrf proteins isolated from two sea urchins by nickel affinity according to Sherman et al. (16). (B) A Western blot evaluated with anti-SpTrf antisera shows the rGly-rich and rHis-rich fragments expressed in E. coli and isolated by nickel affinity. The rHis-rich fragment shows partial degradation from 20 to 15 kDa as reported previously (12). (C) LUVs incubated with rSpTrf-E1 monomers and the rHis-rich fragment (both at 10 µM) induce fluorescent dye leakage. The other protein isolates are not active.

Figure 5

Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), induces leakage that plateaus at about 5 h. Three independent leakage assays with 10 µM rSpTrf-E1 show that reaching the fluorescence leakage plateau requires about 5 h. The table insert shows that the results are reproducible at 0 initial leakage rate (A₀) when rSpTrf-E1 is added to the sample with average fraction of ~0.58 (A₁) that is released with an average kinetic rate (k) of ~1.17 × 10⁻⁴ s⁻¹.

Figure 6

Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), causes NBD-PA to cluster in the lipid bilayer. Confocal microscopy images were captured 20 min after the addition of rSpTrf-E1 to giant unilamellar vesicles (GUVs) that are composed of 6% NBD-PA, 4% PA, and 90% PC (100% g/ml). (A) An NBD-PA cluster (arrow) is present at the intersection of two GUVs. Images show NBD-PA (a), DiD in the GUV membrane (b), and the merge (c). (B) The merged image shows an NBD-PA cluster (arrow) at a region of concave curvature of a GUV membrane. (C) A single cluster of NBD-PA is present in a GUV membrane. (D) A Z-stack of images (a–j) from the bottom to the top of two GUVs (white and yellow arrows) shows that each GUV has a single NBD-PA cluster. (E) A GUV without added rSpTrf-E1 shows no change in NBD-PA distribution at 20 min. Images NBD-PA (a), DiD in the GUV membrane (b), and the merge (c). (F) Two GUVs without added rSpTrf-E1 show an even distribution of NBD-PA at 20 min. All scale bars indicate 10 μm.

Figure 7

NBD-PA becomes separated from giant unilamellar vesicles (GUVs) after 2 h of incubation with recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1). (A,B) NBD-PA (arrows) forms clusters that are separated from the GUVs after 2 h of incubation with rSpTrf-E1. (C,D) GUVs in the absence of rSpTrf-E1 show an even distribution of NBD-PA and DiD at 2 h. Differences in the GUV sizes and content of NBD-PA are an outcome of GUV preparation. All images are merged for NBD-PA (blue) and DiD (red) as captured by confocal microscopy. All scale bars indicate 10 μm.

Figure 8

Secondary structure of recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), transforms from intrinsically disordered to α helical in the presence of phosphatidic acid (PA) small unilamellar vesicles (SUVs). (A) CD spectra show intrinsic disorder or random coils for 0.25 mM rSpTrf-E1 in 10 mM sodium phosphate buffer in the absence of PA or in the presence of 100% phosphatidylcholine SUVs (PC). rSpTrf-E1 transforms to α helical secondary structure in the presence of 10% PA:PC SUVs or 100% PA SUVs (PA). θ is the mean residue ellipticity with standard units of degrees × cm² × dmol⁻¹ as described (17). (B) The percentage of α helical structure for rSpTrf-E1 is 1.57% in the absence of lipids and 2.1% in the presence of PC. However, in the presence of 100% PA SUVs or 10% PA/PC SUVs, the α helical structure of rSpTrf-E1 is 69.6 and 71.8%, respectively. The percentage of secondary structure for rSpTrf-E1 in the presence of PA are based the deconvolution of the spectra data using the DichroWeb server. The R values [ellipticity ratios: R = θ₂₂₂/θ₂₀₇ shown in panel (A)] are indicated for the CD analysis of rSpTrf-E1 with 10% PA:PC and for 100% PA (17).

Figure 9

A schematic representation of a proposed process of how recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), may cause phosphatidic acid (PA) clustering and PA extraction from liposomes. (A) The positively charged amino acids (red+) in the Gly-rich region (orange) and the His-rich region (blue) of rSpTrf-E1 interact with the negatively charged (red−) phosphate head group of PA (blue cone-shaped lipid) through initial electrostatic attractions. Phosphatidylcholine (PC) (red rectangular lipid) is 90% of the lipids in the liposomes. (B) The positively charged amino acids from both the Gly-rich and His-rich regions of rSpTrf-E1 each bind to the phosphate head group on PA. The C terminal region of the Gly-rich (C-Gly) region (red) does not bind to PA. (C) Binding between rSpTrf-E1 and PA causes the protein to undergo a structural transformation from disordered to α helical. (D) The C-Gly region of α helical rSpTrf-E1 interacts with other C-Gly regions in other rSpTrf-E1 proteins causing protein multimerization or aggregation that brings PA into clusters. Clusters of cone-shaped PA induce liposome membrane curvature that leads to budding, invagination and fusion (not shown). (E) The C-Gly region continues to multimerize rSpTrf-E1 proteins into larger aggregates that extract PA from the liposomes and result in disordered PA clusters that are separated from the liposomes.