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Fig 1. Sp185/333 protein structure and nickel affinity.(A) Sp185/333 protein structure. The Sp185/333 proteins have diverse sequences, however, their overall structure is conserved and includes a signal sequence (SS), which is likely cleaved during protein processing, a glycine-rich region including an arginine/glycine/aspartic acid (RGD) motif (star), a histidine-rich region, and a C terminal region (C). Three polyclonal rabbit anti-Sp185/333 sera against conserved peptides correspond to different regions of the protein (10). Anti-Sp185/333-66 (a-66) is specific for a region near the N terminus, anti-Sp185/333-68 (a-68) is specific for the region that includes the RGD motif, and anti-Sp185/333-71 (a-71) is specific for a moderately conserved region near the C terminus [9,10]. (B) Sp185/333 proteins can be isolated by affinity to Ni-His60 resin (ClonTech Laboratories, Inc.) based on the manufacturer’s instructions. A composite Western blot of elution fractions from eight sea urchins (designated by number at the top) are evaluated with a mixture of the three anti-Sp185/333 sera (1:15,000 dilution each), and several animals share proteins of the same size (see Table 2 for a list of all protein sizes in all samples). The 38 kDa band from animal 13 is likely the only monomer based on predictions from cDNA sequences [7]. (C) Sp185/333 proteins are eluted from nickel columns with low quantities of other coelomic fluid proteins. Elution fractions from animal 8 contain many protein bands when evaluated by Coomassie (C) and silver stain (SS). Analysis by Western blot shows that these elution fractions contain Sp185/333 proteins but are negative when probed for the sea urchin homologue of complement C3 with anti-SpC3 (C3, 1:8000 dilution [16]) or for the profilin homologue using anti-SpProfilin (Prof, 1:12000 dilution [17]).
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Fig 2.
Sp185/333 mRNA editing decreases in response to immune challenge.
Sp185/333 cDNA sequences reported by Terwilliger et al. [7] were evaluated for the change in the number sequences encoding truncated vs. full-length Sp185/333 proteins post-challenge compared to pre-challenge with LPS, β,1–3,glucan (glucan), double stranded RNA (dsRNA), or artificial coelomic fluid (aCF, buffer, sham injection control). Bars that appear to be missing indicate 0 change. Nine sea urchins (indicated by number) were used in the analysis and animal 2 was evaluated for multiple types of challenges.
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Fig 3. Ni-Sp185/333 proteins of a single MW by 1DE/Western blots resolve to multiple spots and trains in the basic pI range by 2DE/Western blots.The sizes of the Ni-Sp185/333 proteins from sea urchin 101 and that were isolated under optimized protocols (see S1 Protocol) and analyzed by 2DE/Western blot, range in MW from ~40 kDa to over 200 kDa. Most MW sizes are composed of multiple pI variants of which most have a basic pI. (A) A complex of variants with different pI are present within the pI range of 3 to 10, and appear as multiple spots and trains of proteins of ~60 to 80 kDa (white arrows) and >150 kDa (black arrows). (B) The same sample is evaluated in basic pI range of 7 to 10, which is the location on 2DE gels to which most of the Sp185/333 charge variants migrate. Protein trains of similar size and pI as that in A are identified by corresponding arrows.
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Fig 4. Some sea urchins do not express many Ni-Sp185/333 proteins.A small repertoire of Ni-Sp185/333 proteins is expressed by sea urchin 102 before (A) and after (B) challenge with V. diazotrophicus. White arrows indicate short trains of proteins with the same MW that are present both before and after challenge. A limited array of more basic proteins appears after challenge (Box in B). These images were cropped on the bottom because they do not show spots of less than 40 kDa.
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Fig 5. A wide repertoire of Ni-Sp185/333 proteins may be present in some sea urchins both before and after challenge.(A) Sea urchin 103 shows a broad repertoire of Ni-Sp185/333 proteins prior to immune challenge. (B) After the first challenge with Vibrio diazotrophicus, the train of ~150 to 250 kDa proteins shifts to much more basic (white box) as does the train of ~90 kDa proteins (white arrow). Proteins of ~60 kDa (black arrow) decrease in intensity compared to those of similar sizes and pI in A. (C) After the second challenge with V. diazotrophicus, the train of ~150 to 250 kDa proteins is more evenly distributed along the pI gradient (white box), the ~90 kDa proteins extend into the more acidic range (white arrow), and the ~60 kDa proteins decrease in intensity (black arrow). Proteins of new MW/pI appear, which may be monomers (black box, gray arrow). These images were cropped on the bottom because they do not show spots of less than 40 kDa.
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Fig 6. A few Ni-Sp185/333 proteins shift in abundance following immune challenge.In response to immune challenge, sea urchin 104 changes the abundances of some Ni-Sp185/333 proteins before (A) compared to after (B) challenge with V. diazotrophicus. Although the larger proteins do not change in intensity (white arrow), there is an increase in the size and intensity of the Sp185/333+ spot at ~60 kDa and pI ~8 (black arrow) after challenge. These images were cropped on the bottom because they do not show spots of less than 40 kDa.
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Fig 7. Immune challenge increases the intensity of some Ni-Sp185/333 proteins and decreases the intensity of others.(A) Prior to immune challenge, sea urchin 105 shows a range of Ni-Sp185/333 proteins ranging from ~35 kDa (likely monomers, black arrow) to ~150 to 250 kDa that are all within the pI range of ~7 to 10 (black boxes). (B) After challenge with V. diazotrophicus, larger MW proteins decrease in intensity (compare white arrows in A and B), whereas the monomers increase in intensity (compare black arrows in A and B).
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Fig 8. Major changes in the Ni-Sp185/333 protein repertoire may not occur until after the second challenge with V. diazotrophicus.(A) Sea urchin 106 has a limited array of Ni-Sp185/333 proteins prior to immune challenge, with a few high MW proteins of basic pI (arrow). (B) After the first challenge with V. diazotrophicus, the Ni-Sp185/333 proteins appear as more acidic (arrows) plus proteins of ~60 kDa appear (arrow head). (C) After the second challenge with V. diazotrophicus, a significant change in the repertoire of Ni-Sp185/333 proteins shows a shift to more basic pI for both the large MW proteins (arrows), and the ~60 kDa proteins (arrowhead). These images were cropped on the bottom because they do not show spots of less than 50 kDa.
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Fig 9. A wide range of changes in the MW/pI of Ni-Sp185/333 proteins can occur over time and in response to multiple challenges with different types of microbes.(A) Prior to immune challenge, sea urchin 107 shows two major trains of Ni-Sp185/333 proteins of ~80 and ~150 kDa (arrows). (B) After the first challenge with V. diazotrophicus, the majority of the proteins in the trains shift to more basic (arrows), with an expansion of acidic proteins within the same trains (black arrowheads). (C) Prior to challenge with Bacillus sp, sea urchin 107 shows a wide repertoire of Sp185/333 proteins, particularly those of ~60 to 80 kDa and pI of ~7 to 8 (white box). (D) After challenge with Bacillus sp, there is a slight decrease in the intensity of the Ni-Sp185/333 proteins, particularly those of ~60 to 80 kDa/pI ~6 to 8 (white box). (E) The second challenge with Bacillus sp results in a decrease in the intensity of the repertoire of Sp185/333 proteins. (F) The third challenge with Bacillus sp further decreases the repertoire and intensity of the Ni-Sp185/333 proteins. (G) Two weeks after challenge with Bacillus sp the Ni-Sp185/333 protein repertoire shows a further decrease in the number of spots and their intensity. (H) Subsequent challenge with V. diazotrophicus does not induce an increase in the Ni-Sp185/333 protein repertoire, but shows further decreases in diversity. Images (A, B, E-H) were cropped at the bottom because they do not show spots of less than 40 kDa.
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Fig 10. Challenge with Bacillus sp does not induce increased expression of Ni-Sp185/333 proteins.(A) Sea urchin 108 shows a repertoire of Ni-Sp185/333 proteins that range in size from ~45 to 200 kDa prior to challenge. (B) After challenge with Bacillus sp there is a decrease in the array and intensity of the Ni-Sp185/333 proteins, with only three short trains (arrow heads) matching in size and pI to those observed prior to challenge.
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