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.
Highlights
The purple sea urchin, Strongylocentrotus purpuratus, is a member of the echinoderm phylum this is a sister group to the chordates
The repertoire of Ni-Sp185/333 proteins differs i) before vs. after immune challenge in individual sea urchins, ii) among individual sea urchins following immune challenge with the same bacteria, and iii) following multiple immune challenges among individual sea urchins. These results suggest that the Sp185/333 system in echinoids produces a highly diverse array of immune response proteins in the CF of sea urchins that may impart effective host protection, which is flexible for responding to the wide range of potential microbial pathogens that are present in the marine habitat
Animals were immune activated with sets of injections of either heatkilled Vibrio diazotrophicus, E. coli, or LPS to induce or increase the amount of Sp185/333 proteins in the whole coelomic fluid (wCF). wCF lysates were loaded onto Ni-affinity columns and Western blots of wash and elution fractions demonstrated that suites of Ni-Sp185/333 proteins could be isolated successfully with this method
Summary
The purple sea urchin, Strongylocentrotus purpuratus, is a member of the echinoderm phylum this is a sister group to the chordates. We have speculated that Sp185/333 proteins may have an expanded repertoire of immune responsiveness through six putative diversification mechanisms that include i) the presence of the large gene family both within individuals and within the population, which encodes a wide variety of protein isoforms, ii) putative variations in gene family size and gene sequence among individuals, iii) genomic instability within the Sp185/333 gene family that may promote unequal crossovers, gene conversion, gene duplication/deletion and paralogous misspairing to promote sequence diversification, iv) variations in gene expression from different Sp185/333 genes, v) RNA editing, perhaps by deaminases, or error prone transcription perhaps by low fidelity RNA polymerases, such as Polμ, that changes the amino acid sequence, alters the reading frame and inserts missense sequence, and changes single nucleotide changes that insert early stops leading to truncated proteins, and vi) a broad array of putative modifications to the proteins either during or after translation [2,3,9,11,12,13] The outcome of these multiple layers of diversification is an array of the proteins that is much greater than expected from a gene family of up to 60 members. This wide range in proteins may enable swift and flexible activity against a wide range of pathogens, which is likely to be very effective in host protection in the marine system
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