Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

Abstract

Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity.

Multitasking Immune Sp185/333 Protein, rSpTransformer-E1, and Its Recombinant Fragments Undergo Secondary Structural Transformation upon Binding Targets

“…Similar characteristics have been noted for the He185/333 genes and proteins from a different sea urchin species, Heliocidaris erythrogramma (20). Two-dimensional Western blots show up to 260 Sp185/333 + spots with a wide range of molecular masses and isoelectric points that illustrate the level of protein diversity among animals (13,21). Despite the sequence diversity, the predicted Sp185/333 proteins share a common structure composed of an N-terminal hydrophobic leader peptide, a Glyrich region with an arginine-glycine-aspartic acid motif, a His-rich region, and a C-terminal region ( Fig.…”

“…The Sp185/333 gene family has an estimated ∼50 6 10 genes per genome that encode a vastly diverse repertoire of putative immune response proteins (6)(7)(8)(9)(10). Each gene has two exons that encode the leader peptide and the mature protein (9)(10)(11)(12)(13). The deduced mature proteins have tandem and interspersed repeats, as well as subsets of 25-27 elements, which are recognizable blocks of sequence defined by large artificial gaps that optimize two possible alignments (6).…”

“…We have proposed previously that each phagocyte type may be subcategorized based on expression of the Sp185/333 genes and proteins and expression of the complement C3 homolog in subsets of these cell types (7,18). The sizes of deduced Sp185/333 proteins range from 4 to 55 kDa based on cDNA sequences (19); however, the native proteins isolated from individual sea urchins show unexpectedly large sizes that contradict predictions (7,13). Similar characteristics have been noted for the He185/333 genes and proteins from a different sea urchin species, Heliocidaris erythrogramma (20).…”

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

“…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].…”

“…1a) [20, 25]. Although the mechanism by which the mosaic element patterns are generated is unknown, this unique modular structure in the second exon results in great sequence diversity among the different genes and the encoded proteins [23, 27, 30], while maintaining a consistent general structure (Fig. 1b) [20, 29].…”