Abstract
Zeamatin is a 22-kDa protein isolated from Zea mays that has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect α-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human α-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.
Zeamatin is a 22-kDa protein isolated from corn (Zea mays) seeds and meal (7, 10, 11) that has significant amino acid homology to thaumatin and to thaumatin-like proteins, including pathogenesis-related group 5 (PR-5) proteins (10). Zeamatin has potent antifungal activity in vitro against a number of human and plant pathogens (7, 8, 10, 11). Recently, zeamatin, in synergy with nikkomycin Z and fluconazole, was shown to be effective in vivo in both a systemic murine candidosis model (8) and a vaginal murine candidosis model (8; D. Stevens et al., submitted for publication). Zeamatin has the potential to be used not only as a human therapeutic agent but also in transgenic plants to increase in planta resistance to pathogens. To use zeamatin as a therapeutic agent, it is important to understand its properties, including potential inhibition of mammalian enzymes.
Zeamatin binds β-1,3-glucans (9) and has antifungal activity because it can permeabilize fungal cells, leading to cell death (7). Zeamatin is identical to a previously isolated bifunctional α-amylase and trypsin activity inhibitor (6). However, thaumatin and several other thaumatin-like proteins with significant homology to zeamatin, e.g., PR-R and PR-S, do not inhibit trypsin or α-amylase activity (3, 5).
To determine if zeamatin is both an α-amylase and trypsin inhibitor and an antifungal protein, we tested highly purified zeamatin for inhibition of α-amylase and trypsin activities. We extracted zeamatin from corn (Zea mays) meal and purified it by two reversed-phase chromatography steps to apparent homogeneity (12). Tribolium castaneum larvae were provided by Sue Haas (Kansas State University, Manhattan); Bacillus species, human saliva, porcine pancreas, and barley malt α-amylases were obtained from Sigma-Aldrich (St. Louis, Mo.). Tribolium α-amylase was prepared by lysing larvae in ice-cold buffer (20 mM NaH2PO4, pH 6.0, containing 6 mM NaCl) using a Dounce homogenizer; the supernatant from a 1,000 × g, 10 min, 4°C centrifugation was used as a source of α-amylase activity. The amount of α-amylase protein in the lysate was estimated to be ∼12% of the total protein by densitometry of Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (data not shown). Zeamatin, an α-amylase inhibitor from a biological source (A-1520; Sigma-Aldrich) or buffer (20 mM NaH2PO4, pH 6.0, containing 6 mM NaCl) was incubated with the various α-amylases at 25°C for 20 min, and α-amylase activity was assayed by the method of Bernfeld (2). Consistent with the results of Blanco-Labra and Iturbe-Chiñas (4), we found that zeamatin strongly inhibited (95%) α-amylase activity from T. castaneum, slightly inhibited (30%) the activity of Bacillus α-amylase, but did not inhibit the α-amylase activity from human saliva, porcine pancreas, or barley malt (Table 1). Thaumatin at a 120:1 molar ratio to Tribolium α-amylase and a 1,800:1 molar ratio to porcine pancreas α-amylase did not inhibit α-amylase activity (data not shown). Since zeamatin did not inhibit the activity of any of the commercially available α-amylases except Bacillus sp. α-amylase, and that only slightly and at a high molar ratio, it is not surprising that researchers have not seen α-amylase inhibition by zeamatin.
TABLE 1.
Inhibition of α-amylase activity from various sources by zeamatin
| α-Amylase source | Zeamatin concn (μM) | Molar ratio of zeamatin to α-amylasea | % Inhibitionb |
|---|---|---|---|
| Tribolium castaneum | 160 | 120:1 | 95 ± 0 |
| 16 | 13:1 | 86 ± 7 | |
| 5 | 4:1 | 40 ± 9 | |
| Bacillus sp. | 160 | 2,300:1 | 31 ± 11 |
| Human saliva | 160 | 1,600:1 | 1 ± 2 |
| Porcine pancreas | 160 | 1,800:1 | 0 ± 41 |
| Barley malt | 160 | —c | 13 ± 19 |
The number of moles of α-amylase varied so that approximately 1 U of α-amylase was used in each assay.
Each value represents the average of at least two separate experiments, except for porcine pancreas (n = 7) and the Tribolium amylase-to-zeamatin ratios of 13:1 (n = 3) and 4:1 (n = 6) ± the standard deviation.
—, the number of milligrams of α-amylase protein per milligram of solid in this commercial preparation of barley malt enzyme is unknown. The number of units of α-amylase activity used in the assay was equivalent to that of the other four α-amylases.
Purified zeamatin also was incubated with trypsin, and trypsin activity was assayed using a spectrophotometric assay (1) which measures trypsin digestion of N-α-benzyol-l-arginine ethyl ester (BAEE). In a total volume of 200 μl, porcine pancreas trypsin (0.15 nmol in 1 mM HCl) was mixed with either buffer (67 mM NaH2PO4, pH 7.6), a commercially available trypsin inhibitor (Sigma T9003) (0.15 or 1.5 nmol), zeamatin (0.15, 1.5, 4.5, or 15 nmol), thaumatin (15 nmol) (Sigma-Aldrich), bovine serum albumin (15 nmol), or lysozyme (Sigma L-6876) (15 nmol). To these mixtures, 1 ml of BAEE (0.25 mM BAEE in buffer; Aldrich Chemical Co, Milwaukee, Wis.) was added and the A253 was measured over time against a blank containing the identical components except 1 mM HCl in place of trypsin. Zeamatin at a 100:1 molar ratio to trypsin inhibited trypsin activity by 62% ± 4% (average ± standard deviation) and slightly inhibited trypsin activity (29% ± 9%) at a 30:1 mole ratio. Zeamatin had no effect on trypsin activity at a 10:1 or 1:1 molar ratio (1% ± 9% and 7% ± 9% inhibition, respectively). In contrast, a 100:1 molar ratio of thaumatin, bovine serum albumin, or lysozyme to trypsin did not inhibit trypsin activity (4% ± 7%, 3% ± 1%, and 11% ± 8% inhibition, respectively). In comparison, the trypsin inhibitor from Sigma inhibited trypsin activity at both a 1:10 and a 1:1 molar ratio (93% ± 7% and 44% ± 5%, respectively). Again, not surprisingly, inhibition of trypsin activity by zeamatin has not been observed by other researchers since high molar ratios of zeamatin to trypsin are required. Importantly, inhibition of insect α-amylase and mammalian trypsin appears to be unique to zeamatin and not shared by thaumatin and other PR-5 proteins.
In conclusion, in addition to its antifungal activity, zeamatin can inhibit the activities of Tribolium α-amylase and porcine pancreas trypsin, in agreement with the results originally reported by Richardson et al. (6) and Blanco-Labra and Iturbe-Chiñas (4). Since zeamatin did not inhibit fungal α-amylase (4) and fungi do not contain trypsin, zeamatin's antifungal activity is not the result of inhibition of these enzymes. Zeamatin did not inhibit mammalian α-amylase and inhibited trypsin activity only at high molar ratios; this effect is not likely to lead to clinically relevant toxicity, even at high oral or intravaginal doses of zeamatin.
Acknowledgments
We thank Shelly Wilson for purifying zeamatin. We also especially thank Sue Haas at Kansas State University for generously providing T. castaneum larvae.
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