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A xylanase from roots of sanchi ginseng (Panax notoginseng) with inhibitory effects on human immunodeficiency virus-1 reverse transcriptase
Life Sciences 70 (2002) 3049 – 3058
A xylanase from roots of sanchi ginseng (Panax notoginseng) with inhibitory effects on human immunodeficiency virus-1 reverse transcriptase S.K. Lam, T.B. Ng* Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 29 June 2001; accepted 19 December 2001
Abstract A xylanase with a molecular weight of 15 kDa, which is lower than those of previously reported xylanases, was isolated from the roots of the medicinal herb Panax notoginseng. The xylanase exhibits a temperature optimum of 50 C and a pH optimum between 5 and 6. It inhibits HIV-1 reverse transcriptase with an IC50 of 10 mM, but does not affect translation in a cell-free rabbit reticulocyte system when tested up to 70 mM. The enzyme is adsorbed on CM-cellulose, Affi-gel blue gel and Mono S. Previously xylanases have been isolated from seeds and not from roots, and have not been demonstrated to inhibit HIV-1 reverse transcriptase. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Xylanase; Roots; Medicinal herb; Panax notoginseng
Introduction It is well documented that Panax notoginseng exerts beneficial effects on the cardiovascular, nervous, immune and hepatobiliary systems. Administration of the total saponins of P. notoginseng results in an improvement of myocardial relaxation function [1], decrement of plasma lipids [2], lowering of mean blood pressure and cerebrovascular resistance [3],
* Corresponding author. Tel.: +852-2609-6875; fax: +852-2603-5123. E-mail address: [email protected] (T.B. Ng). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 5 7 - 6
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suppression of proliferation of aortic smooth muscle cells induced by hypercholesterolemic serum [4], improvements in the early postburn cardiac function [5] and scopolamine-induced amnesia learning and memory deficit in rats [6], protective effects on ischemic brain damage [7], and moderate hepatoprotective effects [8]. It can also bring about anti-inflammatory [9], anti-carcinogenic [10] and immunostimulating [11] actions. Not much information is available in the literature regarding the existence of xylanases in roots. It is reported herein that a xylanase, possessing an N-terminal amino acid sequence displaying similarities to that of Cellvibrio mixtus xylanase, could be isolated from P. notoginseng roots. This xylanase is interesting in that it possesses a molecular weight (15 kDa) lower than the range of molecular weights (17–340 kDa) reported for other xylanases. Furthermore, it displays an inhibitory action on HIV-1 reverse transcriptase.
Materials and Methods Isolation of xylanase The roots of Panax notoginseng (sanchi ginseng) from a local vendor were extracted in 10 mM NH4OAc (pH 4.6). After centrifugation the supernatant was loaded on a column of CM-cellulose (5.5 25 cm). Following removal of unadsorbed proteins (CM1) with the same buffer, adsorbed proteins (CM2) were desorbed using 50 mM NH4OAc (pH 7). CM2 was dialyzed prior to affinity chromatography on Affi-gel blue gel in 10 mM Tris–HCl buffer (pH 7.2). After elution of unadsorbed proteins (BG1), adsorbed proteins (BG2) were eluted with 1.5 M NaCl in 10 mM Tris–HCl buffer (pH 7.2). BG2 was dialyzed and then subjected to ion exchange chromatography on Mono S in 10 mM Tris–HC1 (pH 6.8) by fast protein liquid chromatography. Unadsorbed proteins were eluted with the buffer while the purified xylanase was eluted with a linear NaCl concentration gradient (0–0.25 M) in the buffer. The xylanase was then applied to an FPLC–Superdex 75 column which had been calibrated with molecular weight marker proteins. The eluting buffer was 200 mM NH4OAc (pH 7.0). Assay for xylanase activity The assay for xylanase activity was carried out using Somogyi’s reagent [12] and Nelson’s reagent [13] as follows. A xylan solution was freshly prepared by adding 175 mg oat xylan (Sigma) to 12.5 ml 60 mM potassium phosphate buffer (pH 7) which had been autoclaved. Deionized water was added to the supernatant resulting after centrifugation to make the volume up to 35 ml. Solution A was prepared by mixing 2.86 ml xylan solution, 1.07 ml 60 mM phosphate buffer and 870 ml deoinized water. Solution A (156 ml) in a test tube was pre-incubated at 40 C for 5 minutes. Different concentrations of xylose were used as standards. The sample to be tested for xylanase activity (10 ml) was then added. Diethyl pyrocarbonate-H2O was added in control and standards. At 30 minutes 167 ml Somogyi’s reagent were added. The reaction was stopped by placing the reaction mixture in a 100 C water bath for 10 minutes. The tubes were cooled down to room temperature before addition
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of Nelson’s reagent (167 ml) and deionized water (0.5 ml). Absorbance was measured at 720 nm against the blank after centrifugation at 4500 rpm for 15 minutes. Assay for translation-inhibiting activity The assay for translation-inhibiting activity using a cell-free reticulocyte lysate system was conducted as previously described [14]. Rabbit reticulocyte lysate was prepared from the blood of rabbits rendered anemic by phenylhydrazine injections. An assay based on the rabbit reticulocyte lysate system [15] was used. The test sample (10 ml) was added to 10 ml of radioactive mixture (500 mM KC1, 5 mM MgC12, 130 mM phosphocreatine and 1 mCi [4,5] leucine) and 30 ml working rabbit reticulocyte lysate containing 0.1 mM hemin and 5 ml creatine kinase. Incubation proceeded at 37 C for 30 min before addition of 330 ml 1 M NaOH and 1.2% H2O2. Further incubation for 10 min allowed decolorization and tRNA digestion. An equal volume of the reaction mixture was then added to 40% trichloroacetic acid with 2% casein hydrolyzate in a 96-well plate to precipitate radioactively labeled protein. The precipitate was collected on a glass fiber Whatman GF/A filter, washed and dried with absolute alcohol passing through a cell harvester attached to a vacuum pump. The filter was suspended in scintillant and counted in an LS6500 Beckman liquid scintillation counter. Assay for HIV-1 reverse transcriptase inhibitory activity The assay for HIV reverse transcriptase inhibitory activity was carried out as described by Ng and Wang [15] and Wang and Ng [16] using a non-radioactive kit from Boehringer Mannheim (Germany). The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(A) oligo (dT)15. In place of radio-labeled nucleotides, digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into one and the same DNA molecule, which is freshly synthesized by the reverse transcriptase (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol: Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigeninlabeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be determined using microtiter plate (ELISA) reader and is directly correlated to the level of RT activity. A fixed amount (4–6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of Panax notoginseng xylanase was calculated as percent inhibition as compared to a control without the protein.
Results When the extract of Panax notoginseng roots was chromatographed on an ion exchange column of CM-cellulose, a large unadsorbed fraction and a smaller adsorbed fraction were
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Fig. 1. Ion exchange chromatography of a crude extract of Panax notoginseng on a CM-cellulose column (5.5 25 cm). Eluent: 10 mM NH4OAc (pH 4.6). The eluent was changed to 50 mM NH4OAc (pH 7) when the elution volume was 8 L.
obtained (Fig. 1). Subsequent affinity chromatography of the adsorbed fraction on Affi-gel blue gel yielded a large unadsorbed peak and a much smaller adsorbed peak (Fig. 2). The
Fig. 2. Affi-gel blue gel chromatography of the fraction of crude P. notoginseng extract adsorbed on CM-cellulose. Eluent: 10 mM Tris – HCl (pH 7.2). The eluent was changed to 1.5 M NaCl in 10 mM Tris – HCl (pH 7.2) when the elution volume was 400 ml.
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Fig. 3. Ion exchange chromatography on Mono S. The fraction adsorbed on Affi-gel blue gel was applied to the FPLC Mono S column in 10 mM NaOAc (pH 6.8). After elution of unadsorbed protein the column was eluted with a linear NaCl concentration gradient, as indicated by the slanting line across the chromatogram. Protein in the first adsorbed peak represents purified xylanase.
adsorbed peak from Affi-gel blue gel was then subjected to FPLC on Mono S. After elution of a small unadsorbed peak, the adsorbed material was fractionated into five peaks using a
Fig. 4. Sodium dedecyl sulfate – polyacrylamide gel electrophoresis of purified xylanase. Lane 1 shows Pharmacia molecular weight markers, from top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin (14.4 kDa). Lane 2 represents purified xylanase from Panax notoginseng.
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Fig. 5. The effect of pH on activity of Panax notoginseng xylanase. (Data represent means ± SD, n = 3).
linear salt gradient (Fig. 3). The first two-thirds of the first adsorbed peak were collected and found to contain purified xylanase which appeared as a single peak with a molecular weight of 15 kDa upon gel filtration on a Superdex 75 column which had been calibrated with
Fig. 6. The effect of temperature on activity of Panax notoginseng xylanase. (Data represent means ± SD, n = 3).
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Fig. 7. Inhibitory effect of Panax notoginseng xylanase on activity of HIV-1 reverse transcriptase. (Data represent means ± SD, n = 3).
molecular weight markers from Amersham Pharmacia (data not shown). One band corresponding to a molecular weight of 15 kDa was seen in SDS–PAGE (Fig. 4). The xylanase activity of the isolated protein was maximal at pH 5–6 (Fig. 5) and 50 C (Fig. 6). The protein exerted an inhibitory effect on the activity of HIV-1 reverse transcriptase with an IC50 of 10 mM (Fig. 7). The xylanase exhibited substantial homology to Cellvibrio mixtus xylanase when a comparison of their N-terminal sequences was made (Table 1). From 1.2 kg Panax notoginseng roots 2 g extract, 208 mg protein adsorbed on CM-cellulose, 111 mg protein adsorbed on Affi-gel blue gel and eventually 0.6 mg xylanase were obtained. Xylanase activity was undetectable in the crude extract and fraction adsorbed on CM-cellulose,
Table 1 Comparison of N-terminal Sequence of Panax notoginseng Xylanase with Those of Cellvibrio mixtus Xylanase (Results of a Blast search) and Other Xylanases Residues Panax notoginseng xylanase Cellvibrio mixtus xylanase Streptomyces xylanase Cex XYLA
1 26 1 1 1
GDNNNRVANN GDNNNSSANN AESTLGAAAA A-TTLKDAAD GLASLADFPI
Residue
% Identity
10 35 10 9 10
100 80 – – –
Residues identical to corresponding residues in the Panax notoginseng xylanase are underlined. Panax notoginseng xylanase shows no sequence similarity to other xylanases including XYLA (xylanase A from Pseudomonas fluorescens) [27] Cex (b-1,4-glycanase Cex from Cellulomonas fimi) [28].
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and 1.87 and 25.2 mmol xylose per mg protein in the fraction adsorbed on Affi-gel blue gel and the final purified preparation respectively.
Discussion Xylanases (1,4-b-D-xylan xylanohydrolase) catalyze the random hydrolysis of the xylan backbone of heteroxylans. As a result the cellulose fibrils are exposed and susceptible to attack of side-chain cleaving enzymes such as a-arabinofuranosidase and acetylxylanase. Xylanases occur in diverse organisms including bacteria, fungi, algae, protozoans, crustaceans, insects, snails and seeds [17]. Bacterial and fungal xylanases are produced inductively or constitutively in response to the carbon source on which they are grown. For instance, xylanase production by the Antarctic yeast Cryptococcus adeliae was increased by addition of xylan into the culture medium [18]. The leguminous plant Stylosanthes humilis is unique in that it contains large amounts of arabinoxylan in its seeds [19] and that xylanase activity is very high during germination [20]. Xylanase gene expression in germinated barley grains is largely confined to the aleurone layer, and no mRNA transcripts are detectable in young vegetable tissues [21]. In maize, highly substituted glucuronoarabino xylans are localized mainly in unlignified walls and low-branched xylan in lignified walls [22]. Due to the nature of the assay for HIV-1 reverse transcriptase inhibitory activity, it is not possible to test if this activity of Panax notoginseng demonstrates temperature and pH dependence similar to its xylanase activity. The homogeneity of the xylanase preparation in FPLC-gel filtration suggests that the anti-HIV-1 reverse transcriptase and xylanase activities are attributed to the same protein. The novel aspects of the results of the present investigation comprise the isolation of a lowmolecular-weight xylanase from the roots of a medicinal plant with suppressive effects on HIV-1 reverse transcriptase. Hitherto xylanases have been reported from seeds but not from roots. The physiological role of xylanase in the root remains to be elucidated. In the case of bacterial and fungal xylanases, xylan induces its own metabolizing enzyme. It is noteworthy that Panax notoginseng xylanase can be isolated from uninduced tissue. The previously isolated xylanases have not been tested for other biological activities. The present finding that Panax notoginseng xylanase manifests anti-HIV-1 reverse transcriptase activity is interesting. It has been observed that some enzymes, e.g. chitinases, inhibit HIV-1 reverse transcriptase [23–25]. The xylanase from Panax notoginseng can be developed into an anti-HIV drug because some of the natural products inhibit HIV-1 reverse transcriptase with a much lower potency than the xylanase [26]. The mechanism of its inhibitory action on HIV-1 reverse transcriptase awaits elucidation.
Acknowledgments This work was supported by an earmarked grant from Hong Kong Research Grants Council. The excellent secretarial assistance of Miss Fion Yung is gratefully acknowledged.
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References 1. Feng PF, Qin NP, Qiao P. Clinical and experimental study of improving left ventricular diastolic function by total saponins of Panax notoginseng. Zhongguo Zhong Xi Yi Jie He Za Zhe 1997;17:714 – 7. 2. Xu Q, Zhao Y, Cheng GR. Blood-lipid decreasing action of total saponins of Panax notoginseng (Burk.) F.H. Chen. Zhongguo Zhong Yao Za Zhi 1993;18:367 – 8. 3. Wu JX, Sun JJ. Comparative effects of Panax notoginseng saponins, verapamil and norepinephrine on cerebral circulation in aresthetized rats and rabbits. Zhongguo Yao Li Xue Bao 1992;13:520 – 3. 4. Lin SG, Zheng XL, Chen QY, Sun JJ. Effect of Panax notoginseng saponins on increased proliferation of cultural aortic smooth muscle cells stimulated by hypercholesterolemic serum. Zhongguo Yao Li Xue Bao 1993;14:314 – 6. 5. Huang YS, Yang ZC, Yan BG, Hu XC, Li AN, Crowther RS. Improvement of early postburn cardiac function by use of Panax notoginseng and immediate total eschar excision in one operation. Burns 1999;25:35 – 41. 6. Hsieh MT, Peng WH, Wu CR, Wang WH. The ameliorating effects of the cognitive-enhancing Chinese herbs on scopolamine-induced amnesia in rats. Phytother Res 2000;14:375 – 7. 7. Han JA, Hu WY. Progress in the study on protective effect of saponins of Panax notoginseng on ischemic brain damage. Zhongguo Zhong Xi Yi Jie He Za Zhi 1996;16:506 – 7. 8. Liu J, Liu Y, Klaassen CD. The effect of Chinese hepatoprotective medicines on experimental liver injury in mice. J Ethnopharmacol 1994;42:183 – 91. 9. Li SH, Chu Y. Anti-inflammatory effects of total saponins of Panax notoginseng. Zhongguo Yao Li Xue Bao 1999;20:551 – 4. 10. Konoshima T, Takasaki M, Tokuda H. Anti-carcinogenic activity of the roots of Panax notoginseng. Biol Pharm Bull 1999;22:1150 – 2. 11. Gao H, Wang F, Lien EJ, Trousdale MD. Immunostimulating polysaccharides from Panax notoginseng. Pharm Res 1996;13:1196 – 200. 12. Somogyi M. Notes on sugar determination. J Biol Chem 1952;195:19 – 23. 13. Nelson N. A photometric adaptation of Somogyi method for determination of glucose. J Biol Chem 1944;153:375 – 80. 14. Lam SSL, Wang HX, Ng TB. Purification and characterization of novel ribosome inactivating proteins, alphaand beta-pisavins, from seeds of the garden pea Pisum sativum. Biochem Biophys Res Commun 1998;253: 135 – 42. 15. Ng TB, Wang HX. Panaxagin, a new protein from Chinese ginseng possesses anti-fungal, anti-viral, translation-inhibiting and ribonuclease activities. Life Sci 2001;68:739 – 49. 16. Wang HX, Ng TB. Quinqueginsin, a novel protein with anti-human immunodeficiency virus, antifungal, ribonuclease and cell-free translation-inhibitory activities from Amercian ginseng roots. Biochem Biophys Res Commun 2000;269:203 – 8. 17. Sunna A, Autranikian G. Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 1997;17:39 – 67. 18. Gomes J, Gomes I, Steiner W. Thermoliable xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties. Extremophiles 2000;4:227 – 35. 19. Blake JD, Richards GN. An examination of some methods for fractionation of plant hemicelluloses. Carbohydr Res 1971;17:253 – 68. 20. Dekker RF, Richards GN. Hemicellulases: their occurrence, purification, properties and mode of action. Adv Carbohydr Chem Biochem 1976;32:277 – 352. 21. Banik M, Garrett TP, Fincher GB. Molecular cloning of cDNAs encoding (1!4)-beta-xylan endohydrolases from the aleurone layer of germinated barley (Hordeum vulgare). Plant Mol Biol 1996;31:1163 – 72. 22. Suzuki K, Kitamura S, Kato Y, Itoh T. Highly substituted glucuronoarabinoxylans (hsGAXs) and lowbranched xylans show a distinct localization pattern in the tissues of Zea mays L. Plant Cell Physiol 2000; 41:948 – 59. 23. Lam YW, Wang HX, Ng TB. A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochem Biophys Res Commun 2000;279:74 – 80.
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24. Ye XY, Wang HX, Ng TB. Dolichin, a new chitinase-like antifungal protein isolated from field beans (Dolichos lablab). Biochem Biophys Res Commun 2000;269:155 – 9. 25. Ye XY, Wang HX, Ng TB. Structurally dissimilar proteins with antiviral and antifungal potency from cowpea (Vigna unguiculata) seeds. Life Sci 2000;67:3199 – 208. 26. Ng TB, Fong WP, Wan DCC, Yeung HW. Anti-HIV natural products with special emphasis on HIV reverse transcriptase inhibitors. Life Sci 1997;61:933 – 49. 27. Harris GW, Jenkins JA, Connection I, Pickersgill RW. Refined crystal structure of the catalytic domain of xylanase A from Pseudomonas fluorescens at 1.8 A resolution. Acta Crystallog Sect D 1996;52:393 – 401. 28. White A, Withers SG, Gilkes NR, Rose DR. Crystal structure of the catalytic domain of the – 1,4-glycanase Cex from Cellulomonas fimi. Biochemistry 1994;33:12546 – 52.
A xylanase from roots of sanchi ginseng (Panax notoginseng) with inhibitory effects on human immunodeficiency virus-1 reverse transcriptase S.K. Lam, T.B. Ng* Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 29 June 2001; accepted 19 December 2001
Abstract A xylanase with a molecular weight of 15 kDa, which is lower than those of previously reported xylanases, was isolated from the roots of the medicinal herb Panax notoginseng. The xylanase exhibits a temperature optimum of 50 C and a pH optimum between 5 and 6. It inhibits HIV-1 reverse transcriptase with an IC50 of 10 mM, but does not affect translation in a cell-free rabbit reticulocyte system when tested up to 70 mM. The enzyme is adsorbed on CM-cellulose, Affi-gel blue gel and Mono S. Previously xylanases have been isolated from seeds and not from roots, and have not been demonstrated to inhibit HIV-1 reverse transcriptase. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Xylanase; Roots; Medicinal herb; Panax notoginseng
Introduction It is well documented that Panax notoginseng exerts beneficial effects on the cardiovascular, nervous, immune and hepatobiliary systems. Administration of the total saponins of P. notoginseng results in an improvement of myocardial relaxation function [1], decrement of plasma lipids [2], lowering of mean blood pressure and cerebrovascular resistance [3],
* Corresponding author. Tel.: +852-2609-6875; fax: +852-2603-5123. E-mail address: [email protected] (T.B. Ng). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 5 7 - 6
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suppression of proliferation of aortic smooth muscle cells induced by hypercholesterolemic serum [4], improvements in the early postburn cardiac function [5] and scopolamine-induced amnesia learning and memory deficit in rats [6], protective effects on ischemic brain damage [7], and moderate hepatoprotective effects [8]. It can also bring about anti-inflammatory [9], anti-carcinogenic [10] and immunostimulating [11] actions. Not much information is available in the literature regarding the existence of xylanases in roots. It is reported herein that a xylanase, possessing an N-terminal amino acid sequence displaying similarities to that of Cellvibrio mixtus xylanase, could be isolated from P. notoginseng roots. This xylanase is interesting in that it possesses a molecular weight (15 kDa) lower than the range of molecular weights (17–340 kDa) reported for other xylanases. Furthermore, it displays an inhibitory action on HIV-1 reverse transcriptase.
Materials and Methods Isolation of xylanase The roots of Panax notoginseng (sanchi ginseng) from a local vendor were extracted in 10 mM NH4OAc (pH 4.6). After centrifugation the supernatant was loaded on a column of CM-cellulose (5.5 25 cm). Following removal of unadsorbed proteins (CM1) with the same buffer, adsorbed proteins (CM2) were desorbed using 50 mM NH4OAc (pH 7). CM2 was dialyzed prior to affinity chromatography on Affi-gel blue gel in 10 mM Tris–HCl buffer (pH 7.2). After elution of unadsorbed proteins (BG1), adsorbed proteins (BG2) were eluted with 1.5 M NaCl in 10 mM Tris–HCl buffer (pH 7.2). BG2 was dialyzed and then subjected to ion exchange chromatography on Mono S in 10 mM Tris–HC1 (pH 6.8) by fast protein liquid chromatography. Unadsorbed proteins were eluted with the buffer while the purified xylanase was eluted with a linear NaCl concentration gradient (0–0.25 M) in the buffer. The xylanase was then applied to an FPLC–Superdex 75 column which had been calibrated with molecular weight marker proteins. The eluting buffer was 200 mM NH4OAc (pH 7.0). Assay for xylanase activity The assay for xylanase activity was carried out using Somogyi’s reagent [12] and Nelson’s reagent [13] as follows. A xylan solution was freshly prepared by adding 175 mg oat xylan (Sigma) to 12.5 ml 60 mM potassium phosphate buffer (pH 7) which had been autoclaved. Deionized water was added to the supernatant resulting after centrifugation to make the volume up to 35 ml. Solution A was prepared by mixing 2.86 ml xylan solution, 1.07 ml 60 mM phosphate buffer and 870 ml deoinized water. Solution A (156 ml) in a test tube was pre-incubated at 40 C for 5 minutes. Different concentrations of xylose were used as standards. The sample to be tested for xylanase activity (10 ml) was then added. Diethyl pyrocarbonate-H2O was added in control and standards. At 30 minutes 167 ml Somogyi’s reagent were added. The reaction was stopped by placing the reaction mixture in a 100 C water bath for 10 minutes. The tubes were cooled down to room temperature before addition
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of Nelson’s reagent (167 ml) and deionized water (0.5 ml). Absorbance was measured at 720 nm against the blank after centrifugation at 4500 rpm for 15 minutes. Assay for translation-inhibiting activity The assay for translation-inhibiting activity using a cell-free reticulocyte lysate system was conducted as previously described [14]. Rabbit reticulocyte lysate was prepared from the blood of rabbits rendered anemic by phenylhydrazine injections. An assay based on the rabbit reticulocyte lysate system [15] was used. The test sample (10 ml) was added to 10 ml of radioactive mixture (500 mM KC1, 5 mM MgC12, 130 mM phosphocreatine and 1 mCi [4,5] leucine) and 30 ml working rabbit reticulocyte lysate containing 0.1 mM hemin and 5 ml creatine kinase. Incubation proceeded at 37 C for 30 min before addition of 330 ml 1 M NaOH and 1.2% H2O2. Further incubation for 10 min allowed decolorization and tRNA digestion. An equal volume of the reaction mixture was then added to 40% trichloroacetic acid with 2% casein hydrolyzate in a 96-well plate to precipitate radioactively labeled protein. The precipitate was collected on a glass fiber Whatman GF/A filter, washed and dried with absolute alcohol passing through a cell harvester attached to a vacuum pump. The filter was suspended in scintillant and counted in an LS6500 Beckman liquid scintillation counter. Assay for HIV-1 reverse transcriptase inhibitory activity The assay for HIV reverse transcriptase inhibitory activity was carried out as described by Ng and Wang [15] and Wang and Ng [16] using a non-radioactive kit from Boehringer Mannheim (Germany). The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(A) oligo (dT)15. In place of radio-labeled nucleotides, digoxigenin- and biotin-labeled nucleotides in an optimized ratio are incorporated into one and the same DNA molecule, which is freshly synthesized by the reverse transcriptase (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich ELISA protocol: Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. In the next step, an antibody to digoxigenin, conjugated to peroxidase, binds to the digoxigeninlabeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be determined using microtiter plate (ELISA) reader and is directly correlated to the level of RT activity. A fixed amount (4–6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of Panax notoginseng xylanase was calculated as percent inhibition as compared to a control without the protein.
Results When the extract of Panax notoginseng roots was chromatographed on an ion exchange column of CM-cellulose, a large unadsorbed fraction and a smaller adsorbed fraction were
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Fig. 1. Ion exchange chromatography of a crude extract of Panax notoginseng on a CM-cellulose column (5.5 25 cm). Eluent: 10 mM NH4OAc (pH 4.6). The eluent was changed to 50 mM NH4OAc (pH 7) when the elution volume was 8 L.
obtained (Fig. 1). Subsequent affinity chromatography of the adsorbed fraction on Affi-gel blue gel yielded a large unadsorbed peak and a much smaller adsorbed peak (Fig. 2). The
Fig. 2. Affi-gel blue gel chromatography of the fraction of crude P. notoginseng extract adsorbed on CM-cellulose. Eluent: 10 mM Tris – HCl (pH 7.2). The eluent was changed to 1.5 M NaCl in 10 mM Tris – HCl (pH 7.2) when the elution volume was 400 ml.
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Fig. 3. Ion exchange chromatography on Mono S. The fraction adsorbed on Affi-gel blue gel was applied to the FPLC Mono S column in 10 mM NaOAc (pH 6.8). After elution of unadsorbed protein the column was eluted with a linear NaCl concentration gradient, as indicated by the slanting line across the chromatogram. Protein in the first adsorbed peak represents purified xylanase.
adsorbed peak from Affi-gel blue gel was then subjected to FPLC on Mono S. After elution of a small unadsorbed peak, the adsorbed material was fractionated into five peaks using a
Fig. 4. Sodium dedecyl sulfate – polyacrylamide gel electrophoresis of purified xylanase. Lane 1 shows Pharmacia molecular weight markers, from top downward: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and a-lactalbumin (14.4 kDa). Lane 2 represents purified xylanase from Panax notoginseng.
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Fig. 5. The effect of pH on activity of Panax notoginseng xylanase. (Data represent means ± SD, n = 3).
linear salt gradient (Fig. 3). The first two-thirds of the first adsorbed peak were collected and found to contain purified xylanase which appeared as a single peak with a molecular weight of 15 kDa upon gel filtration on a Superdex 75 column which had been calibrated with
Fig. 6. The effect of temperature on activity of Panax notoginseng xylanase. (Data represent means ± SD, n = 3).
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Fig. 7. Inhibitory effect of Panax notoginseng xylanase on activity of HIV-1 reverse transcriptase. (Data represent means ± SD, n = 3).
molecular weight markers from Amersham Pharmacia (data not shown). One band corresponding to a molecular weight of 15 kDa was seen in SDS–PAGE (Fig. 4). The xylanase activity of the isolated protein was maximal at pH 5–6 (Fig. 5) and 50 C (Fig. 6). The protein exerted an inhibitory effect on the activity of HIV-1 reverse transcriptase with an IC50 of 10 mM (Fig. 7). The xylanase exhibited substantial homology to Cellvibrio mixtus xylanase when a comparison of their N-terminal sequences was made (Table 1). From 1.2 kg Panax notoginseng roots 2 g extract, 208 mg protein adsorbed on CM-cellulose, 111 mg protein adsorbed on Affi-gel blue gel and eventually 0.6 mg xylanase were obtained. Xylanase activity was undetectable in the crude extract and fraction adsorbed on CM-cellulose,
Table 1 Comparison of N-terminal Sequence of Panax notoginseng Xylanase with Those of Cellvibrio mixtus Xylanase (Results of a Blast search) and Other Xylanases Residues Panax notoginseng xylanase Cellvibrio mixtus xylanase Streptomyces xylanase Cex XYLA
1 26 1 1 1
GDNNNRVANN GDNNNSSANN AESTLGAAAA A-TTLKDAAD GLASLADFPI
Residue
% Identity
10 35 10 9 10
100 80 – – –
Residues identical to corresponding residues in the Panax notoginseng xylanase are underlined. Panax notoginseng xylanase shows no sequence similarity to other xylanases including XYLA (xylanase A from Pseudomonas fluorescens) [27] Cex (b-1,4-glycanase Cex from Cellulomonas fimi) [28].
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and 1.87 and 25.2 mmol xylose per mg protein in the fraction adsorbed on Affi-gel blue gel and the final purified preparation respectively.
Discussion Xylanases (1,4-b-D-xylan xylanohydrolase) catalyze the random hydrolysis of the xylan backbone of heteroxylans. As a result the cellulose fibrils are exposed and susceptible to attack of side-chain cleaving enzymes such as a-arabinofuranosidase and acetylxylanase. Xylanases occur in diverse organisms including bacteria, fungi, algae, protozoans, crustaceans, insects, snails and seeds [17]. Bacterial and fungal xylanases are produced inductively or constitutively in response to the carbon source on which they are grown. For instance, xylanase production by the Antarctic yeast Cryptococcus adeliae was increased by addition of xylan into the culture medium [18]. The leguminous plant Stylosanthes humilis is unique in that it contains large amounts of arabinoxylan in its seeds [19] and that xylanase activity is very high during germination [20]. Xylanase gene expression in germinated barley grains is largely confined to the aleurone layer, and no mRNA transcripts are detectable in young vegetable tissues [21]. In maize, highly substituted glucuronoarabino xylans are localized mainly in unlignified walls and low-branched xylan in lignified walls [22]. Due to the nature of the assay for HIV-1 reverse transcriptase inhibitory activity, it is not possible to test if this activity of Panax notoginseng demonstrates temperature and pH dependence similar to its xylanase activity. The homogeneity of the xylanase preparation in FPLC-gel filtration suggests that the anti-HIV-1 reverse transcriptase and xylanase activities are attributed to the same protein. The novel aspects of the results of the present investigation comprise the isolation of a lowmolecular-weight xylanase from the roots of a medicinal plant with suppressive effects on HIV-1 reverse transcriptase. Hitherto xylanases have been reported from seeds but not from roots. The physiological role of xylanase in the root remains to be elucidated. In the case of bacterial and fungal xylanases, xylan induces its own metabolizing enzyme. It is noteworthy that Panax notoginseng xylanase can be isolated from uninduced tissue. The previously isolated xylanases have not been tested for other biological activities. The present finding that Panax notoginseng xylanase manifests anti-HIV-1 reverse transcriptase activity is interesting. It has been observed that some enzymes, e.g. chitinases, inhibit HIV-1 reverse transcriptase [23–25]. The xylanase from Panax notoginseng can be developed into an anti-HIV drug because some of the natural products inhibit HIV-1 reverse transcriptase with a much lower potency than the xylanase [26]. The mechanism of its inhibitory action on HIV-1 reverse transcriptase awaits elucidation.
Acknowledgments This work was supported by an earmarked grant from Hong Kong Research Grants Council. The excellent secretarial assistance of Miss Fion Yung is gratefully acknowledged.
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