Hyper-acidic fusion minipeptides escort the intrinsic antioxidative ability of the pattern recognition receptor CRP in non-animal organisms

Abstract

C-reactive protein (CRP) is widely used as a biomarker of inflammation. It plays important roles in innate immunity response as a member of pattern recognition receptors, by binding oxidation-specific epitopes including some intermediates of lipid oxidative chain reaction. The inferred antioxidative ability of CRP was ever demonstrated by only few in vitro evidences, and needs to be clarified especially in vivo. Herein, we expressed human CRP in three representative non-animal organisms (Escherichia coli, Saccharomyces cerevisiae, and tobacco) inherently lacking the milieu for CRP signalling, and found CRP did possess an intrinsic antioxidative ability. Heterologous CRP could confer increased oxidative resistance in its recombinant E. coli and yeast cells and transgenic tobaccos. We also revealed a positive correlation between the antioxidative effect of CRP and its solubility. Only soluble CRP could exhibit distinct antioxidative activity, while the CRP aggregates might be instead toxic (probably pro-oxidative) to cells. Moreover, fusion with hyper-acidic minipeptides could remarkably improve CRP solubility, and meanwhile guarantee or enhance CRP antioxidative ability. These results not only provide a new insight for understanding the etiology of CRP-involved inflammations and diseases, and also endorse a potential of CRP biotechnological applications in developing new pharmaceutical therapies and improving plant oxidative resistance.

Figure 1

Dot-plating test with serial dilutions (1-, 2-, 4-, 8- fold) to compare the colony growth of E. coli recombinant strains of pET(CRP), pET(CRPm), pET30s(CRP), pET30s(t-CRP), pET30s(a-CRP) and the control strain of pET30s, under normal condition (CK) and diverse oxidative stresses of 1.1 mM H₂O₂, 0.5 mM PQ, and 5 mM CuSO₄. (A,B) E. coli cells with pre-induction. (C) E. coli cells without pre-induction.

Figure 2

Protein expression in E. coli of various CRP recombinant vectors including pET(CRP) (A), pET30s(CRP) (B), pET30s(t-CRP) (C) and pET30s(a-CRP) (D), analyzed by SDS-PAGE. M: protein molecular weight marker; UI: crude lysate of uninduced bacterial cells; T: crude lysate of induced bacterial cells; P, S: the pellet and supernatant fractions of induced bacterial cell lysate (T) after centrifugation, respectively. Full-length gels are presented in Supplementary Fig. 9.

Figure 3

Dot-plating test with serial dilutions (1-, 10-, 100-, 1000-, 10000- fold) to compare the colony growth of yeast cells with pre-induction among the recombinant strains of pYES2(CRP), pYES2(t-CRP), pYES2(a-CRP) and the control strain of pYES2 under normal condition (CK) and different oxidative stresses of 30 mM H₂O₂, 0.2 mM PQ, and 4 mM CuSO₄.

Figure 4

Preliminary antioxidation analysis of various CRP transgenic tobaccos of T₀ generation. For a chlorosis test (A), leaf discs of 4-week-old WT and transgenic tobaccos (CRP, t-CRP, a-CRP) growing in greenhouse were submerged into deionized water (CK) or 20 µM PQ solution for 7 d in a growth chamber. After photo-recording, their chlorophyll contents were measured (B). Statistical significance was determined by Student’s t test (n = 3, two replicates per experiment, data are the means ± SD, ***P < 0.001).

Figure 5

Seed germination test of various CRP transgenic tobaccos under moderate oxidative stress. Sterile seeds of WT and transgenic tobaccos (a-CRP, t-CRP, CRP) were germinated in a growth chamber on MS medium containing 0.2 μM PQ for 10 d, and then assessed for the germination profile (A), germination rate (B), seedling status (C), and stem-length of seedlings (D). Statistical significance was determined by Student’s t test (n = 3, >80 seeds per replicate experiment, data are the means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Leaf spray test and histochemical staining of various CRP transgenic tobaccos with a oxidative treatment. (A) Small plants (1-week growth in greenhouse after 1-month sterile cultivation of seedlings) of WT and transgenic tobaccos (a-CRP, t-CRP, CRP) were daily leaf-sprayed with deionized water (CK) or 20 µM PQ solution for 7 d, then the injury spots on leaves were assessed. (B) Intact leaves (daily sprayed with 20 µM PQ for 7 d) of WT and transgenic tobaccos (a-CRP, t-CRP, CRP) were detected for dead cells by Trypan Blue staining (indigo mark), for H₂O₂ by DAB staining (dark brown mark), and for O₂⁻ by NBT staining (blue mark), respectively.

Figure 7

Oxidation-associated physiological changes in various CRP transgenic tobaccos after oxidative treatment. Leaves (daily sprayed with 20 µM PQ or deionized water (CK) for 7 d) of WT and transgenic tobaccos (a-CRP, t-CRP, CRP) were measured for MDA content (A), electrolyte leakage (B), protein carbonyl content (C), and H₂O₂ content (D). Statistical significance was determined by Student’s t test (n = 3, two replicates per experiment, data are the means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 8

Changes in the expression of CRP and its fusions at protein level in their transgenic tobaccos upon oxidative treatment. Leaves (daily sprayed with 20 µM PQ or deionized water (CK) for 7 d) of WT and transgenic tobaccos (CRP, t-CRP, a-CRP) were subjected for protein extraction plus fractionation into ‘S’, ‘P’ samples and subsequent immunoblotting with CRP polyclonal antibody. Actin (‘S’ fraction) was used as the internal reference (A) to calibrate the total expression (‘S’ + ‘P’) of CRP protein or its fusions t/a-CRP (B) in various transgenic tobaccos by membrane-band grey densitometric estimation. The lowest ratio was standardized as 1 to obtain the relative protein levels of CRP and its fusions for comparison (C). Data represent the means ± SD for three separate experiments. Full-length blots are presented in Supplementary Fig. 10.