Biochemistry and Molecular Biology 31 (2001) 1–5 www.elsevier.com/locate/ibmb Rapid communication Purification and characterisation of a non-plant myrosinase from the aphid (L.) A.M.E. Jones a, M. Bridges a, A.M. Bones b, R. Cole c, J.T. Rossiter a,* a Imperial College at Wye, Wye, Ashford, Kent TN25 5AH, UK b Department of Botany, Faculty of Chemistry and Biology, The Norwegian University of Science and Technology, N-7491 Trondheim, Norway c HRI Wellesbourne, Wellesbourne, Warwick, CV35 9EF, UK

Received 7 August 2000; received in revised form 15 September 2000; accepted 15 September 2000

Abstract

Plant myrosinases and constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa±500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase.  2001 Elsevier Science Ltd. All rights reserved.

Keywords: Thioglucosides (glucosinolates); Thioglucoside glucohydrolase (EC 3.2.3.1 myrosinase); Brevicoryne brassicae; Aphid

1. Introduction ised but there have been no reports so far on genes of non-plant myrosinases (Bones and Rossiter, 1996). Glucosinolates and their degradation products are There are a number of reports of myrosinase-like responsible for the characteristic taste and odour of crops activity from a number of sources including fungi such as , cabbage, and (Ohtsuru and Hata, 1973), intestinal bacteria (Tani et al., ( are responsible for the ‘bite’ and 1974), mammalian tissue (Goodman et al., 1959) and pungency) and therefore in these crops the cruciferous aphids (MacGibbon and Allison, 1968). In content is valued. 1968, McGibbon and Allison observed that goitrin (5- The responsible for the of glucosi- vinyl-2-oxazolidinethione, the hydrolysis of 2- nolates is known as myrosinase (E.C. number 3.2.3.2, hydroxy-3-butenyl glucosinolate) was liberated from also known as: β-thioglucosidase, β-thioglucoside crushed B. brassicae which had been feeding on Bras- glucohydrolase). The enzyme mediated hydrolysis of sica napus. Five other species of aphid were examined glucosinolates leads to a labile aglycone, which rapidly and displayed no myrosinase activity. The species of undergoes spontaneous rearrangement, eliminating sul- aphid examined were: Macrosiphum avanae (F.), Myzus phur, to yield a variety of toxic metabolites such as iso- persicae (Sulz.), Rhopalosiphum padi (L.), Aphid cracci- , thiocyanates, cyanoepithioalkanes and vora C. L. Koch., Macrosiphum rosae (L.). M. persicae . The reaction products depend on pH and other and M. rosae are polyphagous aphids whose diet can factors such as the presence of ferrous ions, epithiospec- include crucifers. The other aphids do not feed on gluco- ifier protein and the nature of the glucosinolate side sinolate containing plants. Further work by this group chain. Plant myrosinase genes have been well character- showed that the ‘glucosinolase’ (myrosinase) also occurs in the turnip aphid, Lipaphis erysimi and activity (in both aphids) was restricted to the head and thorax regions, * Corresponding author. Fax: +44-1233-813-140. although no specific internal organ could be associated E-mail address: [email protected] (J.T. Rossiter). with the activity. L. erysimi showed consistently lower

0965-1748/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S0965-1748(00)00157-0 2 A.M.E. Jones et al. / Insect Biochemistry and Molecular Biology 31 (2001) 1–5 levels of myrosinase activity than B. brassicae tially purified aphid myrosinase (from Resource Q) and (MacGibbon and Beuzenberg, 1978). We have set out against the crude protein extract from the 40–60% to purify and characterise the myrosinase with a view to ammonium sulphate precipitate. examining both the biological function of the myro- sinase–glucosinolate system in the aphid as well as the 2.4. Western blotting enzyme mechanism. SDS–PAGE gels were run as previously described. 2. Methods Proteins were capillary press blotted, for 2 h, on to a nitro-cellulose membrane using 20 mM Tris, 150 mM 2.1. Purification of aphid myrosinase glycine, 20% methanol (pH 8.3) as transfer buffer, at 60°C. Freeze-dried aphids (8.7 g) were ground in extraction buffer (20 mM Tris, 0.15 M NaCl, 0.02% azide, leupep- 2.5. Myrosinase micro assays tin (10 µg/ml) and 0.1 mM PMSF, pH 7.5). The extract was centrifuged at 12,000g for 30 min to remove solid An assay based on the determination of matter and the supernatant fractionated with ammonium released by the hydrolysis of 2-propenyl glucosinolate sulphate. The active fraction (40–60%) was run on a () by the aphid myrosinase was used routinely Sephacryl (S-200) gel filtration column in Tris buffer (20 to determine enzyme activity during protein purification. mM Tris, 0.15 M NaCl, pH 7.5, 0.02% sodium azide) GOD-PERID test reagents were purchased from Boehr- and active fractions pooled. The pooled fractions were inger Mannheim. mixed with 1 ml of Concanavalin A (Con A) overnight, Enzyme solution and sinigrin (1.08 mM) in 500 µlof supernatant decanted and the ConA matrix washed with sodium citrate buffer (100 mM, pH 5.5) was incubated buffer (2× 1 ml, 20 mM Tris, 0.15 M NaCl, pH 7.5, at 30°C for 20 min. The reaction was stopped by addition 0.02% sodium azide) and the washings combined with of 40 µl 3M HCl (aq) and GOD-PERID reagent (2.5 ml) the supernatant. The sample was desalted by dialysis added to the reaction mixture and incubated for 15 min against 10 mM imidazole (pH 6) for 2 h followed by 20 at 37°C. The optical density was read at 346 nm and the mM imidazole (pH 6) for a further 2 h. Ion exchange glucose concentration calculated from a calibration chromatography was carried out on a Resource Q col- graph. umn (Pharmacia). The column (1 ml) was equilibrated with 20 mM imidazole (pH 6.5) and eluted with 20 mM 2.6. Protein assay imidazole (0.5 M NaCl, pH 6.5). Active fractions were pooled and desalted against starting buffer on Bio-Rad 10 DC columns. The ‘main peak’ sample was re-run on Protein content was estimated using a Bradford based Resource Q and the pure protein was dialysed (2×) dye-binding kit purchased from Bio-Rad. against deionised and stored at Ϫ20°C. 2.7. Protease digests and separation of peptides 2.2. Gel electrophoresis Trypsin, modified, sequencing grade (EC 3.4.21.4, Polypeptides were resolved in 12% (w/v) acrylamide Boehringer Mannheim) was used at a ratio of 1:50 (1 µg vertical gel slabs according to the procedure of Laemmli trypsin to 50 µg aphid myrosinase), in 0.2 M ammonium (1970) with a Bio-Rad Mini Protean II electrophoretic bicarbonate buffer, pH 7.8. Lys C (E.C. 3.4.21.50 apparatus. Polypeptides were stained with 0.25% Coom- sequencing grade Boehringer Mannheim), was used at a assie Blue R-250. ratio of 1:50 (1 µg of Lys C to 50 µg of aphid A narrow range IEF gel (pH 2.5 to 6.5) (Ampholine myrosinase) in buffer (25 mM Tris–Cl, 1 mM EDTA, PAG precast polyacrylamide gel, Pharmacia Biotech.) pH 8.5). The resultant peptides were separated by was run and resolved with Coomassie Blue. reverse-phase HPLC on a VYDAC, reverse-phase HPLC column (C18, 2.1 mm, 15 cm) using a acetonitrile/water 2.3. Polyclonal antibody production (TFA) gradient. 35 µg of purified aphid myrosinase was injected into a New Zealand White rabbit, followed on day 16 with 2.8. Protein sequencing 60 µg. The first bleed was taken on day 30, the terminal bleed a week later. This antibody is referred to as Wye Three peptides from the trypsin digestion and two Q. The antibodies raised to aphid myrosinase were from the Lys C digests were chosen for their apparent examined for specificity by Western blotting against par- purity and sequenced by automated Edman degradation. A.M.E. Jones et al. / Insect Biochemistry and Molecular Biology 31 (2001) 1–5 3

2.9. MALDI mass spectrometry

0.5–1 µl of the pure aphid myrosinase was placed on the target disc together with 1 µl of matrix (sinapinic acid in 60% acetonitrile). The intact protein samples were calibrated against BSA, in sinapinic acid. The dou- bly protonated peak was also used in the calibration. The spectrometry was carried out on a VG analytical Tof- Spec.

3. Results and discussion

The myrosinase from freeze-dried aphids was purified in five steps (Table 1). Myrosinase was precipitated at 40–60% saturation with ammonium sulphate with no appreciable activity present in any other fractions. The gel filtration step (Table 1) yielded a four-fold purifi- cation while affinity chromatography on Concanavilin A removed glycosylated proteins resulting in further puri- fication. Aphid myrosinase did not bind to the lectin con- canavalin A indicating that either the protein is not gly- cosylated or its glycosyl component is not specific for this type of lectin. Ion exchange chromatography, on a Resource Q column gave a major and minor peak of aphid myrosinase activity which were resolved by frac- tionation and subsequent re-chromatography resulting in a single homogenous peak. Characterisation of the minor aphid myrosinase peak was not attempted as there was Fig. 1. (a) Purity of aphid myrosinase compared to BSA. Lane 1 insufficient material. Although the specific activity of the molecular mass markers:77 kDa Ovotransferin, 66.25 kDa BSA, 42.50 kDa Ovalbumin, 30 kDa Carbonic anhydrase, Lane 2 BSA and aphid sample increased total activity declined during this step. myrosinase, Lane 3 BSA, Lane 4 Aphid myrosinase. (b) Isoelectric Overall, the total purification achieved was 40-fold, focussing of aphid myrosinase. Lane 1 markers; 5.85 Carbonic anhyd- while the total yield of protein was 0.13% of the crude rase (bovine), 5.20 β-Lactoglobulin A, 4.55 Trypsin inhibitor; Lane 2 extract. The purity of the protein extract was assessed Aphid myrosinase 5 µg. by SDS–PAGE [Fig. 1(a)] and comparison with BSA and isoelectric focusing [Fig. 1(b)]. Isoelectric focusing of the purified aphid myrosinase The native molecular mass of aphid myrosinase, esti- gave two bands [Fig. 1(b)]. The isoelectric point (pI) of mated from gel filtration, was 97 kDa. The molecular these bands were 4.90 and 4.95 the latter being consider- mass of the denatured and reduced protein was 53 kDa, ably denser then the former. The less dense band estimated from SDS–PAGE [Fig. 1(a)]. The molecular observed with a pI of 4.90 is possibly the minor peak mass of the subunit was confirmed by MALDI–TOF observed in the first Resource Q ion exchange chromato- mass spectrometry, giving a value of 54 kDa±500 Da. graphy step and possibly represents an isoform of Thus aphid myrosinase appears to be a dimeric protein, aphid myrosinase. with identical subunits. The pH optima of the enzyme was found to be 5.5

Table 1 Purification of aphid myrosinase from Brevicoryne brassicae

Purification step Protein (mg) Total activity Specific activity Yield (%) Purification (µmol/min) (µmol/mg/min)

Crude extract 498.00 238.0 0.478 100.00 1.00

(NH4)2SO4 cut 132.00 97.0 0.737 26.00 1.54 S-200 20.00 36.0 1.850 4.00 3.87 Con A 10.00 44.0 4.300 2.00 9.00 Res Q (I) 1.00 13.0 13.000 0.20 27.20 Pure aphid myrosinase 0.66 13.2 20.000 0.13 41.84 4 A.M.E. Jones et al. / Insect Biochemistry and Molecular Biology 31 (2001) 1–5 compared to a previously reported pH optima of 5 from S. alba and did not show a reaction to proteins (MacGibbon and Allison, 1968) for a crude protein from other Brassica pests tested (data not shown). Anti- extract of aphid myrosinase. plant myrosinase antibodies did not cross react with B. Western blots [Fig. 2(a)] showed that the antibody brassicae proteins and anti-aphid myrosinase does not raised to aphid myrosinase (Wye Q) was highly specific cross react with plant myrosinase. The results of the to a single band in crude extracts of B. brassicae from Western blots are summarised in Table 2. SDS PAGE gels [Fig. 2(b)]. Wye Q did not cross react The intact protein was N-terminally blocked and with proteins (also using Western blotting techniques) sequence data was obtained from peptide fragments. Trypsin digestion gave three peptides. Peptide A (1LVTFGSDPNnNFNPD15) failed to match any known proteins while peptide B (1GIAYYNNLIpELIK 14) matched β- and peptide C (1GWFGHPVYK 9) matched at low astringency, an apo- protein from photosystem II and various which show some similarity with myrosinase (Manntei et al., 1988). Lys C digestion gave two peptides. Peptides D (1TTGHYLAGHT 10) and E (1ISYLK 5) did not match any known protein with any degree of probability. Thus, there appears to no similarity between the sequence of the peptides analysed and known existing plant myro- sinase sequences. The myrosinase from the turnip aphid, Lipaphis erys- imi was also partly characterised (unpublished data) and shown to cross react with the anti-aphid myrosinase anti- body with a single polypeptide of molecular mass 53,450±2000 Da. Like the cabbage aphid, the turnip aphid myrosinase was not activated by ascorbate in the concentration range 0.1–20 mM (data not shown). The apparent Km of the aphid myrosinase was 0.613 and 0.915 mM respectively for allylglucosinolate and benzyl glucosinolate indicating that the enzyme has a greater affinity for allyl glucosinolate. This compares to values of 0.25–0.4 mM for the ascorbate activated plant myro- sinase isoforms from Sinapsis alba and Brassica napus (Bjo¨rkman and Lo¨nnerdal, 1973). A non-plant myrosinase has been purified for the first time and partly characterised. Unlike the plant myro- sinase the aphid enzyme does not appear to be a glyco- protein and is not activated by ascorbic acid. The use of both anti-plant and aphid myrosinase antibodies suggest

Table 2 Summarising the results of Western blots with anti-plant-myrosinase antibodies and the anti-aphid myrosinase antibodya

Organism Antibody used

Pests Wye Q Wye E Wye D DCJ Fig. 2. (a) Western blot of anti-aphid myrosinase (Wye Q) against Brevicoryne brassicae + ϪϪϪ pure myrosinase from Sinapis alba (1), pure aphid myrosinase (2), Myzus persicae Ϫ *** crude extract of Myzus persicae (3), crude extract of Brevicoryne bras- Phedon cochleariae Ϫ + Ϫ + sicae (4). (b) Duplicate gel used for Western blot of anti-aphid myro- Peris rapae Ϫ +++ sinase (Wye Q) against pure myrosinase from Sinapsis alba (Lane 2), Peris brassicae Ϫ ++Ϫ pure aphid myrosinase (Lane 3), crude extract of Myzus persicae (Lane Plant 4), crude extract of Brevicoryne brassicae (Lane 5), Mr markers, BDH Sinapis alba Ϫ *** Mr markers, ovotransferrin (77 kDa), BSA (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (30 kDa), myoglobin (17.2 kDa) and a (+) Indicates a positive reaction. (Ϫ) Indicates a negative reaction. cytochrome C (12.3 kDa) (Lane 1). (*) Indicates that this combination was not tested. A.M.E. Jones et al. / Insect Biochemistry and Molecular Biology 31 (2001) 1–5 5 that there are no common epitopes with the plant Laemmli, U.K., 1970. Cleavage of structural proteins during the enzyme. Thus it appears that specialist aphids have assembly of the head of bacteriophage T4. Nature 227, 680. MacGibbon, D.B., Allison, R.M., 1968. A glucosinolase system in the evolved their own myrosinase system which most likely cabbage aphid Brevicoryne brassicae. New Zealand Journal of is involved in the defence against predators. Science 11, 440. MacGibbon, D.B., Beuzenberg, E.J., 1978. Location of glucosinolase in Brevicoryne brassicae and Lipaphis erysimi (Aphididae). New Zealand Journal of Science 21, 389–392. References Manntei, N., Villa, M., Enzler, T., Wacker, H., Boll, W., James, P., Hunziker, W., Semenza, G., 1988. Complete primary structure of human and rabbit -phlorizin : implications for Bones, A., Rossiter, J.T., 1996. The myrosinase-glucosinolate system, biosynthesis, membrane anchoring and evolution of the enzyme. its organisation and biochemistry. Physiologia Plantarum 97, EMBO Journal 7 (9), 2705–2713. 194–208. Ohtsuru, M., Hata, T., 1973. Studies on the activation mechanism of Bjo¨rkman, R., Lo¨nnerdal, B., 1973. Studies on myrosinases III: enzy- myrosinase by L-ascorbic acid. Agriculture and Biological Chemis- matic proerties of myrosinases from Sinapsis alba and Brassica try 37, 1971–1972. napus seeds. Biochimica et Biophysica Acta 327, 121–131. Tani, N., Ohtsura, M., Hata, T., 1974. Purification and general charac- Goodman, I., Fouts, J.R., Bresnick, E., Mengas, R., Hitchings, G.H., teristics of bacterial myrosinase produced by Enterobacter cloacae. 1959. A mammalian thioglucosidase. Science 130, 450–451. Agriculture and Biological Chemistry 38, 1623–1623.