Expression profiling and binding properties of fibrinogen-related proteins (FREPs), plasma proteins from the schistosome snail host Biomphalaria glabrata - PubMed (original) (raw)

Si-Ming Zhang et al. Innate Immun. 2008 Jun.

Abstract

A growing body of evidence suggests an important role for fibrinogen-like proteins in innate immunity in both vertebrates and invertebrates. It has been shown that fibrinogen-related proteins (FREPs), plasma proteins present in the freshwater snail Biomphalaria glabrata, the intermediate host for the human blood fluke Schistosoma mansoni, are diverse and involved in snail innate defense responses. To gain further insight into the functions of FREPs, recombinant FREP proteins (rFREPs) were produced in Escherichia coli and antibodies (Abs) were raised against the corresponding rFREPs. We first show that most FREP proteins exist in their native conformation in snail hemolymph as multimeric proteins. Western blot analyses reveal that expression of multiple FREPs including FREP4 in plasma from M line and BS-90 snails, which are susceptible and resistant to S. mansoni infection, respectively, is up-regulated significantly after infection with the trematode Echinostoma paraensei. Moreover, our assays demonstrate that FREPs are able to bind E. paraensei sporocysts and their secretory/excretory products (SEPs), and a variety of microbes (Gram-positive and Gram-negative bacteria and yeast). Furthermore, this binding capability shows evidence of specificity with respect to pathogen type; for example, 65-75-kDa FREPs (mainly FREP4) bind to E. paraensei sporocysts and their SEPs whereas 95-kDa and 125-kDa FREPs bind the microbes assayed. Our results suggest that FREPs can recognize a wide range of pathogens, from prokaryotes to eukaryotes, and different categories of FREPs seem to exhibit functional specialization with respect to the pathogen encountered.

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Figures

Fig. 1

Fig. 1

In vitro expression of full-length and truncated forms of FREPs. (A) Schematic diagram of gene products of FREPs with one IgSF domain (FREPs 2 and 4) or two IgSF domains (FREP3). SP, signal peptide; ICR, interceding region; IgSF, immunoglobin superfamily; FBG, fibrinogen. The regions underlined for each FREP were cloned into vectors and expressed in E. coli. The number above the line represents the number of the gel lane in (B). The diagrams are not drawn to scale. (B) Western blot showing expression of 10 rFREP proteins including truncated forms. Blots were probed with anti-6xhis monoclonal antibody because all rFREPs are tagged with 6 histamines at the C-terminus. Mk, protein marker; Lane 1 is loaded with control bacteria lacking expression constructs. Lanes are numbered as follows: (2) rIgSF-FREP2 (142 aa; pETbF2Ig-F+pETbF2Ig-R), (3) rFBG-FREP2 (210 aa; pETbF2FG-F+pETbF2FG-R), (4) rFREP2 (374 aa; pETbF2Ig-F+pETbF2FG-R), (5) rIgSF1-FREP3 (141 aa; pETbF3Ig1-F+pETbF2Ig1-R), (6) rIgSF1+2-FREP3 (301 aa; pETbF3Ig1-F+pETbF3Ig2-R), (7) rFBG-FREP3 (212 aa; pETbF3FG-F+pETbF3FG-R), (8) rFREP3 (648 aa; pETbF3Ig1-F+pETbF3FG-R), (9) rIgSF-FREP4 (141 aa; pETbF4Ig-F+pETbF4Ig-R), (10) rFBG-FREP4 (266 aa; pETbF4FG-F+pETbF4FG-R), and (11) rREPF4 (408 aa; pETbF4Ig-F+ pETbF4FG-R). The size of the peptide generated and the primer combination used for generation of the peptide are provided in parentheses. Primer sequences are listed in Table 1. (C) SDS-PAGE gel showing the expression and purification of IgSF and FBG domains of FREP4 from E. coli stained by Coomassie blue. Lane Mk is protein size markers; Lanes 1 and 4 are proteins expressed by E. coli lacking expression vectors; Lanes 2 and 5 are proteins expressed by bacteria transformed with expression vectors (lane 2, IgSF vector; lane 5, FBG vector); Lanes 3 and 6 show the purified rIgSF and rFBG proteins obtained using the nickel column.

Fig. 2

Fig. 2

Detection of multiple FREPs and FREP4 under reducing and non-reducing conditions. Western blots shown were probed with anti-rFBG Ab (A) and anti-rFREP4 Ab (B). Lanes R and NR show the plasma from M line or BS-90 snails at 4-days post-exposure to E. paraensei under reducing and non-reducing conditions, respectively.

Fig. 3

Fig. 3

As detected by probing with anti-rFBG, expression of putative multiple FREP proteins in the plasma of M line (A) and BS-90 snails (B) in unexposed control snails (Con) or after exposure to E. paraensei at different days post-exposure (dpe). The left panel shows the Western blots from relatively few random samples; the days post-exposure for each lane is indicated by a solid line under the lane. The right panel shows the expression of FREPs relative to the non-exposed snails based on a statistical analysis of densitometric scans. The Y-axis shows the relative expression level and the X-axis shows the different days post-exposure (below line). The numbers indicated above the line are the numbers of all snail plasma samples that were scanned and used for the statistical analysis at each time point. The asterisks indicate statistical significance (P < 0.05) as compared to unexposed control snails. The numbers above the arrowed line show the total number of random samples used for the Western blots as shown in the right panel and those samples were also used for the statistical analysis.

Fig. 4

Fig. 4

As detected by probing with anti-rFREP4 Ab, expression of putative FREP4 proteins in plasma of M line (A) and BS-90 snails (B) after exposure to E. paraensei. Abbreviations, lane designations and statistical analysis are as described in the caption to Figure 3.

Fig. 5

Fig. 5

Use of anti-rFBG antibody to detect the binding of putative multiple FREPs from two snail strains (M line and BS-90) to E. paraensei sporocysts (A, B) and E. paraensei sporocyst SEPs (C, D). (A) Binding of M line plasma to E. paraensei sporocysts. Lane 1 is loaded with solubilized sporocysts. Lanes 2–8 are loaded with M line plasma components that bound sporocysts, from non-exposed controls snails (lane 2); snails exposed to E. paraensei (lane 3) or S. mansoni (lane 4); or snails injected with PBS (lane 5), Staph. aureus (lane 6), E. coli (lane 7) or S. cerevisiae (lane 8). All plasma was collected at 4 days post-infection or post-injection for all binding studies throughout this work. (B) Binding of BS-90 plasma to E. paraensei sporocysts. Lane 1 is loaded with sporocyst antigens only. Plasma components binding SEPs are derived from non-exposed control snails (lane 2), or snails exposed to E. paraensei (lane 3) or S. mansoni (lane 4). (C) Binding of M line plasma to SEPs. All SEPs used in this study was derived from E. paraensei sporocysts at 2–4 days post-transformation in vitro. Lane 1 received SEPs only. Lane 2 was loaded with half-strength 199-medium. Treatments for lanes 3–9 are the same, and in the same order, as described for lanes 2–8 in Figure 6A. (D) Binding of BS-90 plasma to SEPs. Lanes 1 and 2 were loaded the same as lanes 1 and 2 as described in the caption to Figure 5C. Treatments for lanes 3 to 5 are the same, and in the same order, as lanes 2 to 4 in Figure 5B.

Fig. 6

Fig. 6

Use of anti-rFBG antibody to examine the binding of putative multiple FREPs from two snail strains to Staph. aureus (A, B), E. coli (C, D) and S. cerevisiae (E, F). The left and right panels show binding of plasma components from M line and BS-90 snails, respectively. Lane 1 was loaded with putative antigen that was tested for binding assay. The M line plasma treatments for lanes 2–8 shown in the left panels are the same as lanes 2–8 in Figure 5A and plasma from lanes 2 to 4 in (BS-90 snails: on the right) are same as lanes 2 to 4 in Figure 5B.

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