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.

The SpTransformer Gene Family (Formerly Sp185/333) in the Purple Sea Urchin and the Functional Diversity of the Anti-Pathogen rSpTransformer-E1 Protein

“…(4, 5) . PO 4 ,10 mM phosphate buffer pH = 7.4; SDS, sodium dodecyl sulfate; TFE, 2,2,2-trifluoroethanol; LPS, lipopolysaccharide from Escherichia coli ; PA, phosphatidic acid in the form of small vesicles; n/d, not done; N/A, not applicable; the deconvolution to calculate the β strand percentage is not feasible for these samples (4) . b Helix tightness is estimated from the R value obtained from circular dichroism (CD) spectra and is used to infer the width of an α helical twist. A standard helix has an R value of 1.…”

“…Consequently, when tested for lipid binding, rSpTrf-E1, the rGly-rich, and the rHis-rich fragments all bind to PA, the rHis-rich fragment also binds weakly to phosphatidylinositol 4 phosphate, and rC-Gly binds weakly to phosphatidylserine (5). PA has a similar amphipathic structure as SDS except it has a phosphate head group, which is the likely binding site as none of the proteins bind to diacylglycerol.…”

SpTransformer proteins from the purple sea urchin opsonize bacteria, augment phagocytosis, and retard bacterial growth

“…The range of cellular targets and PAMPs to which rSpTrf-E1 binds, and those to which it does not bind, is unexpected for an anti-pathogen protein and suggests unusual and flexible binding mechanisms. rSpTrf-E1 is an intrinsically disordered protein (IDP) based on bioinformatic predictions and results from circular dichroism, however the secondary structure transforms from disordered to α helical in the presence of binding targets such as LPS, the phospholipid, phosphatidic acid (PA), and sodium dodecyl sulfate or 2,2,2-trifluoroethanol that are typically used in circular dichroism for evaluating the secondary structure of proteins [ 55 , 56 ]. Once rSpTrf-E1 binds to a target, K d values are consistent with an off-rate that is below measurable levels, and tight binding may be the outcome of both initial target association followed by structural transformation.…”

“…The proteins are localized to the membranes of perinuclear vesicles of all phagocyte types and are associated with the outer surface of the plasma membrane on small phagocytes [ 46 , 47 , 60 ]. This localization may be based on interactions between the SpTrf proteins and theoretical cell surface receptors or the histidine-rich region association with negatively charged molecules as predicted from rSpTrf-E1 binding activities [ 49 , 56 ]…”

Echinochrome A Release by Red Spherule Cells Is an Iron-Withholding Strategy of Sea Urchin Innate Immunity

“…Cell-derived immunity in sea urchins is provided by the coelomocytes -a heterogeneous population consisting of 4 distinct morphotypes: phagocytes, vibratile cells, and colourless and red spherule cells. The former can be subdivided into discoidal, polygonal, and small phagocytes, which express a myriad of immune effectors belonging to the (Sp)Transformer gene family [6,7]. The phagocytes are tasked with identifying, ingesting, and destroying invading pathogens, whereas the vibratile cells are said to be involved in hemostasis [8][9][10].…”