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.
Highlights
The genome of the California purple sea urchin (Strongylocentrotus purpuratus) has a number of large immune gene families that are quite complex [1,2,3]
small unilamellar vesicle (SUV) composed of 20% PA:PC showed signs of self-quenching and produced lower net fluorescence resonance energy transfer (FRET) (Figure 2B) likely because the higher concentration of NeutrAvidin-fluorescein isothiocyanate (NA-FITC) in the controls interfered with emission detection [34]
Results with 20% PA:PC SUVs were not included in the statistical analyses and were not evaluated further. rSpTrf-E1-FITC binding to SUVs containing PA appeared to be specific, because increasing concentrations of bovine serum albumin (BSA)-FITC did not show significant changes in net FRET when evaluated with SUVs with various percentages of PA (Figure 2C)
Summary
The genome of the California purple sea urchin (Strongylocentrotus purpuratus) has a number of large immune gene families that are quite complex [1,2,3]. The SpTrf gene family has been estimated to have ~50 members, and the genes have two exons that encode the leader and the mature protein [4,5,6,7,8,9] that respond with swift increases in expression upon challenges from microbes and pathogen-associated molecular patterns (PAMPs) [4, 10, 11]. Increases in size are likely the result of multimerization of natSpTrf proteins that is induced by isolation and processing, which can be induced for a recombinant (r)SpTrf-E1 protein (originally called rSp0032) after isolation from E. coli and in the absence of other sea urchin proteins [12, 14]. Recombinant SpTransformer protein, rSpTransformer-E1 (rSpTrf-E1), binds tightly to lipopolysaccharide (LPS), β-1,3-glucan, and flagellin and once bound cannot be dissociated from one PAMP for rebinding to another
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