Wheat phytase can alleviate the cellular toxic and inflammatory effects of lipopolysaccharide

Wheat phytase (Sigma-Aldrich, Saint Louis, MO, USA) was reconstituted in endotoxin-free water (Sigma-Aldrich). The phosphatase activity of the enzyme (28.6 mU/mL) against the substrate, LPS (100 μg/mL) was determined in acetate buffer (pH 5.0) at 37°C for the given duration (15 min or 1 h) in the presence or absence of inhibitors (10 mM L-phenylalanine or L-homoarginine). In addition, LPS (100 μg/mL) was treated with different units of wheat phytase (14.3 and 57.2 mU/mL) in acetate buffer (pH 5.0) at 37°C for 1 h and the phosphatase activity of the enzyme against LPS was assayed with different concentrations (5 and 20 mM) of the inhibitors. The inorganic phosphate release was measured at optical density (OD) 635 nm using the malachite green-based PiColor Lock gold phosphate detection kit (Innova Biosciences, Cambridge, UK), according to the manufacturer’s instructions.

LPS (20 ng/mL) was hydrolyzed with wheat phytase (11.44 × 10⁻³ mU/mL) for 1–3 h, respectively and different levels of LPS (10 and 40 ng/mL) were treated with the enzyme for 3 h. The residual toxicities of the LPS were assayed at OD 545 nm using ToxinSensor chromogenic LAL (limulus amebocyte lysate)-based endotoxin assay kit (GenScript, Piscataway, NJ, USA), according to the manufacturer’s instructions.

Human aortic endothelial (HAE) cell and human colorectal adenocarcinoma HT-29 cell were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These two cells were used as well-established systems to measure LPS cytotoxicity and gut-induced IL-8 secretion, respectively. The former was maintained in cascade biologics medium 200 (Gibco Life technologies, Carlsbad, CA, USA) containing low serum growth supplement kit (Gibco Life technologies), and the latter in McCoy’s 5A medium (Gibco Life technologies) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Gibco Life technologies). These cells were cultured at 37°C in a humidified incubator with 95% air and 5% CO₂.

HAE cells were initially seeded onto a 96-well plate at a concentration of 2 × 10⁴ cells per well and cultured until 80% confluency. LPS (2 mg/mL) was incubated at 37°C for 1 h with or without wheat phytase (28.6 mU/mL). Aliquots (10 μL) of the reaction mixtures were added to the cells with final concentration of 200 μg/mL LPS at 37°C for 24 h in the CO₂ incubator. In addition, aliquots of the reaction mixtures were applied to the cells with final concentration of LPS (100, 200, and 400 μg/mL), respectively. The cell viabilities were measured at OD 450 nm using EZ-CYTOX kit (DogenBio, Seoul, Korea), according to the manufacturer’s instructions.

HT-29 cells were initially seeded onto a 96-well plate at a concentration of 2 × 10⁴ cells per well and cultured until 80% confluency. LPS (100 μg/mL) was incubated at 37°C for 2 h with or without wheat phytase (286 mU/mL) and aliquots (10 μL) of the reaction mixtures were added to the cells with final concentration of 10 μg/mL LPS at 37°C for 24 h in the CO₂ incubator. In addition, aliquots of the reaction mixtures were applied to the cells with final concentration of LPS (5, 10, and 20 μg/mL), respectively. The levels of IL-8 secreted into the culture media were assayed at OD 450 nm using Cymax™ human IL-8 ELISA kit (Ab FRONTIER, Seoul, Korea), according to the manufacturer’s instructions.

Statistical significance between groups was determined by a one-way analysis of variance using PROC GLM (SAS 9.4, SAS Institute, Cary, NC, USA) followed by Duncan’s multiple range test or by Student’s t-test. A p-value < 0.05 was considered as statistically significant difference.

As shown in Fig. 1A, wheat phytase hydrolyzed LPS, significantly releasing inorganic phosphate for 1 h (p < 0.05). In addition, the enzyme dephosphorylated LPS in a dose-dependent fashion (p < 0.05) (Fig. 1B). Previously, alkaline phosphatase was regarded as a sole enzyme that degraded LPS, whether it is a tissue-specific or tissue-non-specific type [10,11,15]. Nevertheless, our novel finding is remarkable considering that wheat phytase has highly strict substrate specificity for phytate with little or no phosphatase activity against other physiologically-relevant phosphorylated conjugates such as simple sugar phosphates, ATP, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate and phosphoenolpyruvate [13]. Furthermore, the degradation of LPS by wheat phytase was nearly unaffected by the addition of L-phenylalanine, the inhibitor of tissue-specific alkaline phosphatase or L-homoarginine, the inhibitor of tissue-non-specific alkaline phosphatase (Figs. 2A and 2B). Indeed, bovine intestinal alkaline phosphatase inhibited the dephosphorylation of LPS in the presence of L-phenylalanine, but not L-homoarginine [16], while murine uterine alkaline phosphatase inhibited the dephosphorylation of LPS in the presence of L-homoarginine, but not L-phenylalanine [15]. This present result suggests that the catalytic mechanism of LPS hydrolysis by wheat phytase may vary from that of alkaline phosphatases.

Fig. 1. Phosphatase activity of wheat phytase against LPS at different enzyme reaction times (A) or enzyme units (B). (A) LPS (100 μg/mL) was treated with wheat phytase (28.6 mU/mL) in acetate buffer (pH 5.0) at 37°C for the given duration. I: absence of enzyme reaction, II: enzyme reaction for 15 min, III: enzyme reaction for 1 h. Data were presented as mean and standard errors from three experiments. ^a,bMeans lacking common superscripts differ significantly (p < 0.05). (B) LPS (100 μg/mL) was treated with different units of wheat phytase in acetate buffer (pH 5.0) at 37°C for 1 h. I: absence of enzyme reaction, II: the enzyme (14.3 mU/mL), III: the enzyme (57.2 mU/mL). Data were presented as mean and standard errors from three experiments. ^a–cMeans lacking common superscripts differ significantly (p < 0.05). LPS, lipopolysaccharide.

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Fig. 2. Effect of enzyme inhibitors (10 mM) such as L-phenylalanine and L-homoarginine on dephosphorylation of LPS (100 μg/mL) by wheat phytase (28.6 mU/mL) (A) and the phosphatase activity of the enzyme against the substrate with different concentrations (5 and 20 mM) of the inhibitors (B). (A) I: absence of enzyme reaction, II: enzyme reaction for 1 h in the absence of enzyme inhibitors, III: enzyme reaction for 1 h in the presence of L-phenylalanine, IV: enzyme reaction for 1 h in the presence of L-homoarginine. Data were presented as mean and standard errors from three experiments. ^a,bMeans lacking common superscripts differ significantly (p < 0.05). (B) I: enzyme reaction for 1 h in the absence of enzyme inhibitors, II: enzyme reaction for 1 h in the presence of 5 mM L-phenylalanine, III: enzyme reaction for 1 h in the presence of 20 mM L-phenylalanine, IV: enzyme reaction for 1 h in the presence of 5 mM L-homoarginine, V: enzyme reaction for 1 h in the presence of 20 mM L-homoarginine. Data were presented as mean and standard errors from three experiments. ^a,bMeans lacking common superscripts differ significantly (p < 0.05). LPS, lipopolysaccharide.

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As shown in Figs. 3A and 3B, wheat phytase effectively reduced the in vitro toxicity of LPS, and 63% and 54% of its initial toxicity was retained after 1 h and 3 h of the enzyme reaction, respectively (p < 0.05) (Fig. 3A). In this regard, Koyama et al. [10] demonstrated that the structural destruction of LPS by enzymatic dephosphorylation was closely associated with LPS detoxification, due to defective binding to the effector cells.

Fig. 3. Effect of wheat phytase (11.44 × 10⁻³ mU/mL) on in vitro toxicity of LPS (20 ng/mL) (A) and the change of the toxicity at different levels of LPS (10 and 40 ng/mL) treated with the enzyme (B). (A) I: absence of enzymatic dephosphorylation of LPS, II: enzymatic dephosphorylation of LPS for 1 h, III: enzymatic dephosphorylation of LPS for 3 h. Data were presented as mean and standard errors from three experiments. ^a–cMeans lacking common superscripts differ significantly (p < 0.05). (B) I: (closed bar); absence of enzymatic dephosphorylation of LPS (10 ng/mL), (open bar); enzymatic dephosphorylation of LPS (10 ng/mL) for 3 h, II: (closed bar); absence of enzymatic dephosphorylation of LPS (40 ng/mL), (open bar); Enzymatic dephosphorylation of LPS (40 ng/mL) for 3 h. Data were presented as mean and standard errors from three experiments. ^a–cMeans lacking common superscripts differ significantly (p < 0.05). LPS, lipopolysaccharide.

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As shown in Figs. 4A and 4B, intact LPS clearly decreased the cell viability of HAE cells with unusual sensitivity [17]. However, the LPS dephosphorylated by wheat phytase counteracted the inhibitory effect on cell viability. Meanwhile, it was reported that the cell viability of HAE cells was enhanced following exposure to LPS and was attributed to the presence of alkaline phosphatase induced by IL-6 [10,18].

Fig. 4. Cell viability of HAE cells exposed to LPS treated with wheat phytase (A) and the change of cell viabilities at different levels of LPS treated with the enzyme (B). (A) I: no addition, II: addition of intact LPS (200 μg/mL), III: addition of LPS (200 μg/mL) dephosphorylated by wheat phytase (28.6 mU/mL). Data were presented as mean and standard errors from three experiments. ^a,bMeans lacking common superscripts differ significantly (p < 0.05). (B) I: no addition, II: (closed bar); addition of intact LPS (100 μg/mL), (open bar); addition of LPS (100 μg/mL) dephosphorylated by wheat phytase (28.6 mU/mL), III: (closed bar); addition of intact LPS (200 μg/mL), (open bar); addition of LPS (200 μg/mL) dephosphorylated by wheat phytase (28.6 mU/mL), IV: (closed bar); addition of intact LPS (400 μg/mL), (open bar); addition of LPS (400 μg/mL) dephosphorylated by wheat phytase (28.6 mU/mL). Data were presented as mean and standard errors from three experiments. ^a–dMeans lacking common superscripts differ significantly (p < 0.05). HAE, human aortic endothelial; LPS, lipopolysaccharide.

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LPS treated with wheat phytase decreased IL-8 secretion from intestinal epithelial cell line, HT-29 cells to 14% (p < 0.05) when compared with intact LPS (Fig. 5A). In particular, the decrease of IL-8 secretion from the cells was effective at 20 μg/mL of LPS treated with the enzyme (Fig. 5B). It was known that LPS specifically exerts its inflammatory effects via IL-8 in HT-29 cell [19], via appropriate interaction of phosphorylated lipid A moiety of LPS with toll-like receptor 4 (TLR4)-MD2 complex on the cell surface induced by interferon gamma (IFN γ) [20,21]. Thus, the dephosphorylation of LPS by wheat phytase may suppress the side-effects of excessive inflammation induced by IL-8 over-expression [22].

Fig. 5. Effect of LPS treated with wheat phytase on IL-8 secretion in HT-29 cells (A) and IL-8 release from the cells exposed at different levels of LPS treated with the enzyme (B). (A) I: addition of intact LPS (10 μg/mL), II: addition of LPS (10 μg/mL) dephosphorylated by wheat phytase (286 mU/mL). Data were presented as mean and standard errors from three experiments. (*p < 0.05: Student’s t-test). (B) I: (closed bar); addition of intact LPS (5 μg/mL), (open bar); addition of LPS (5 μg/mL) dephosphorylated by wheat phytase (286 mU/mL), II: (closed bar); addition of intact LPS (10 μg/mL), (open bar); addition of LPS (10 μg/mL) dephosphorylated by wheat phytase (286 mU/mL), III: (closed bar); addition of intact LPS (20 μg/mL), (open bar); addition of LPS (20 μg/mL) dephosphorylated by wheat phytase (286 mU/mL). Data were presented as mean and standard errors from three experiments. ^a–cMeans lacking common superscripts differ significantly (p < 0.05). LPS, lipopolysaccharide.

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