A recombinant Sp185/333 protein from the purple sea urchin has multitasking binding activities towards certain microbes and PAMPs

Abstract

The purple sea urchin, Strongylocentrotus purpuratus, possesses a sophisticated innate immune system that responds to microbes effectively by swift expression of the highly diverse Sp185/333 gene family. The Sp185/333 proteins are predicted to have anti-pathogen functions based on inducible gene expression and their significant sequence diversity. Sp185/333 proteins are all predicted to be intrinsically disordered and do not exhibit sequence similarities to other known proteins. To test the anti-pathogen hypothesis, a recombinant Sp185/333 protein, rSp0032, was evaluated and found to exhibit specific binding to marine Vibrio diazotrophicus and to Saccharomyces cerevisiae, but not to two Bacillus species. rSp0032 also binds to LPS, β-1,3-glucan and flagellin but not to peptidoglycan. rSp0032 binding to LPS can be competed by LPS, β-1,3-glucan and flagellin but not by peptidoglycan. We speculate that the predicted intrinsically disordered structure of rSp0032 may adapt to different conformations in binding to a limited number of PAMPs and pathogens. Given that rSp0032 binds to a range of targets, and that up to 260 different Sp185/333 proteins can be expressed per individual sea urchin, this family of immune response proteins may facilitate effective host protection against a broad array of potential pathogens encountered in the marine environment.

Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family

“…Clues may be the unique positioning of the STRs [21] that flank genes and segmental duplications, and their locations at putative gene deletion sites. When the concepts of a dynamically changing Sp185/333 gene family are combined with the notion that the encoded proteins may all have multitasking, anti-pathogen binding capabilities [28], the outcome is an innate immune system in the sea urchin that is highly sophisticated, complex and flexible. The dynamics of this gene family including rapid changes in its genomic organization and coding sequence may provide a significant evolutionary advantage to these invertebrates for their survival over millennia in their marine habitat.…”

“…Echinoid genomes also accommodate a unique immune response gene family, the 185/333 genes that have been partially characterized in two sea urchin species, Strongylocentrotus purpuratus ( Sp185/333 ; [20, 21]) and Heliocidaris erythrogramma ( He185/333 ; [22]). The Sp185/333 genes function in the immune effector arm of sea urchin immunity and are strongly upregulated in response to different types of pathogens and PAMPs [23–28]. The Sp185/333 genes have two exons, of which the first encodes the signal sequence, and the second of variable size among genes encodes the mature protein [20].…”

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

“…(8)]. Despite the sequence diversity of the genes and transcripts, the encoded proteins have a generic structure that is composed of an N-terminal leader, a glycine-rich (Gly-rich) region with an arginine–glycine–aspartic acid motif, a histidine-rich (His-rich) region, and a C-terminal region (Figure 1A) (4, 12). The diversity of element patterns, plus putative editing of the SpTrf mRNAs (13) that introduces missense sequence and early stop codons produces a wide range of deduced SpTrf proteins of 4–55 kDa (4, 11).…”

“…An evaluation of the native (nat)SpTrf proteins in one sea urchin suggests that it can express up to 260 different variants and that the native proteins appear unexpectedly large on Western blots relative to the deduced protein size predictions (14–16). Increases in size are likely the result of multimerization of natSpTrf proteins that is induced by isolation and processing, which can also 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). Once multimerized, SpTrf proteins, whether native or recombinant, cannot be separated to monomers [see supplemental materials in Ref.…”

“…Once multimerized, SpTrf proteins, whether native or recombinant, cannot be separated to monomers [see supplemental materials in Ref. (16)] and once bound cannot be dissociated from the marine bacteria, Vibrio diazotrophicus , or Baker’s yeast, Saccharomyces cerevisiae (12). Furthermore, 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.…”