Báo cáo Y học: Functional expression of human liver cytosolic b-glucosidase in Pichia pastoris Insights into its role in the metabolism of dietary glucosides ppt

Functional expression of human liver cytosolic b-glucosidase in Pichia pastoris Insights into its role in the metabolism of dietary glucosides Jean-Guy Berrin 1,2 , W. Russell McLauchlan 1 , Paul Needs 1 , Gary Williamson 1 , Antoine Puigserver 2 , Paul A. Kroon 1 and Nathalie Juge 1,2 1 Nutrition, Health and Consumer Sciences Division, Institute of Food Research, Norwich, UK; 2 Institut Me  diterrane  en de Recherche en Nutrition, Faculte  des Sciences et Techniques de Saint-Je  ro à me, Marseilles, France Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic b-glucosidase (CBG) that hydrolyses var ious b- D -glycosides, but whose physiological function is not known. Here, we describe the ®rst hetero- logous expression of human CBG, a system that facili- tated a detailed a ssessment of the enzyme speci®city towards dietary glycosides. A full-length CBG c DNA (cbg-1) w as cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of  10 mgáL )1 . The recombinant CBG (reCBG) was puri®ed from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and speci®c activity towards 4-nitro- phenyl-b- D -glucoside. Furthermore, the r eCBG displayed a broad speci®city with respect to the g lycone moiety of various aryl-glycosides (b- D -fucosides, a- L -arabinosides, b- D -glucosides, b- D -galactosides, b- L -xylosides, b- D -arabino- sides), similar to the native enzyme. For the ®rst time, we show that the human enzyme has signi®cant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, ¯avonoids, s imple phenolics and cyanogens with higher apparent anities (K m ) and speci®cities (k cat /K m ) for d ietary xenobiotics than f or other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides a nd may play a critical role in xenobiotic metabolism. Keywords: heterologous expression; xenobiotic metabolism, ¯avonoids; iso¯avones; ®rst-pass metabolism. b-Glucosidases (b- D -glucoside glucohydrolase; EC 3.2.1.21) are members of glycosyl hydrolase families 1 and 3 [1,2]. b-Glucosidases h ydrolyse O-glycosidic bonds at the t ermi- nal, nonreducing end of carbohydrates with retention of anomeric con®guration. They are widely present in nature where they demonstrate catalytic activity against a broad range of b- D -glycosides. In humans, sev eral b-glucosidases have been described and for most of them, the role and physiological substrates are known. For example, the lysosomal b-glucosidase (Ôacid b-glucosidaseÕ) hydrolyses glucocerebrosides (glycosphingo- lipids) present in the lysosomal membranes, a nd a lack of this enzyme is the cause of the various form s of Gaucher's disease, one of the hereditary lysosomal storage disorders [3]. Lactase-phlorizin hyd rolase (LPH) is anchored in the mucosal membrane in the brush-border of the small intestine, where it hydrolyses lactose present in milk. A de®ciency of LPH is the cause of lactose intolerance that is common except in Northern European adults and a few small ethnic populations [4]. Another human b-glucosidase is speci®c for the hydrolysis of pyridoxine 5 ¢-b- D -glucopyra- noside, a common dietary form of vitamin B 6 , and has been ascribed a role in vitamin B 6 bioavailability [ 5]. A putative protein, pr edicted from the klotho (kl ) g ene, shows homol- ogy t o family 1 g lycosyl hydrolase and is also predicted to occur in the cytosol of certain human cells [6,7] where it might have a role in human aging [6]. Finally, a b-glucosidase, termed cytosolic b-glucosidase, is present in the liver, kidney, intestine and spleen of humans. This c ytosolic b-glucosidase (CBG) h as been puri®ed from human liver and partially characterized [8±10]. It is a 53-kDa monomeric protein with a pI of  4.7, a broad and near-neutral pH optimum, and a broad speci®city w ith respect to the g lycone moiety of substrates. Human CBG hydrolyses synthetic aryl glycosides (including 4-nitrophenyl and 4-methylumbelliferyl monoglycosides) [9], but no physiological substrate h as been found and the function in vivo has yet to be determined. However, during our research into the mechanisms underlying the absorption and metabolism of dietary ¯avonoids and iso¯avones, we demonstrated that crude protein e xtracts derived from human liver and small intestine tissues ef®ciently hydrolysed a range of foo d-borne phytochemical (¯avonoid and iso¯avone) glucosides [11]. The effects of s peci®c enzyme inhibitors appeared to indicate that the majority of Correspondence to P. A. Kroon, Nutrition, Health & Consumer Sci- ences Division, Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK. Fax: + 44 1603 255038, Tel.: + 44 1603 255236, E-mail: paul.kroon@bbsrc.ac.uk Abbreviations: AOX1, a lcohol oxidase; BMGY, bu ered minimal glycerol-complex medium; BMMY, buered minimal methanol- complex medium; ESI, electrospray ionization; CBG, cytosolic b-glucosidase; cbg-1, cDNA encoding CBG; reCBG, recombinant CBG; LPH, lactase-phlorizin hydrolase; 4NP, 4-nitrophenol; YNB, yeast nitrogen base; YPD, yeast extract peptone d extrose. (Received 12 October 2001, accepted 30 October 2001) Eur. J. Biochem. 269, 249±258 (2002) Ó FEBS 2002 hydrolytic activity was due to human CBG [11]. CBGs obtained from o ther mammals have been shown to hydrolyse some glycosides of plant origin including phen- olic, pyrimidine, and cyanogenic glycosides [12±14]. We demonstrated that CBG isolated f rom pig liver hydrolysed various ¯avonoid glycosides with reasonable turnover numbers and micromolar K m values [14]. Furthermore the localization of human CBG in metabolic tissues such as the intestine, liver, k idney and spleen indicate that CBG is exposed to orally ingested xenobio tic glycosides. The broad speci®city of the CBG distinguishes this mammalian b-glucosidase from all o thers and has led to the suggestion that it is involved in the primary stage of xenobiotic metabolism [15], but this hypothesis r emained t o b e t ested using the pure human enzyme. Isolation of CBG from human tissues is not easy due to dif®culties associated with obtaining appropriate amounts of suitable tissues, large variations in activity between tissues obtained from different individuals [9,16; P. A. Kroon, unpublished d ata], a nd the need for a multistep fractionation procedure to obtain pure protein [9,17]. In order to fac ilitate biochemical and molecular studies on the signi®cance of human CBG in xenobiotic metabolism, we isolated a human CBG cDNA (cbg-1) and successfully expressed it heterologously in the yeast Pichia pastoris. This organism possesses a number of attributes that renders it an attractive host for the expression and production of CBG: it can b e grown conveniently to high den sity levels in a simple and inexpensive medium; it is able to carry out certain post-translational modi®cation events such as proteolytic maturation, glycosylation and disul®de bond formation; under the co ntrol of t he ef®cient and highly regulated promoter of the alcohol oxidase gene, AOX1, it c an secrete p roteins to very h igh levels [18±20]. In this report, we show that puri®ed recombinant CBG possesses similar physical and enzymatic properties to CBG isolated from human liver. Furthermore, we investigated the speci®city of the human CBG with r espect to the glycone and aglycone moieties, and in particular characterized the ef®ciency of the enzyme in hydrolysing a broad r ange of dietary xenobiotic glycosides. The potential role for human CBG in xenobiotic metabolism and uptake is also discussed. MATERIALS AND METHODS Materials and strains The Zero Blunt TM TOPO TM PCR cloning vector and the pHIL-S1 shuttle vector [32] were purchased from Invitrogen (San Diego, CA, USA). Restriction endonucleases and DNA modifying enzymes were purchased from Promega (Madison WI, USA) and used according to the manufac- turer's recommendation. Escherichia c oli DH5 (supE44, hsdR17, recA1, endA 1, gyrA96, th i-1, relA1) and TOP10 (F ± mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacX74 recA1 deoR araD139 D(ara-leu)7697 gal U galK rpsL(Str R ) endA1 nupG) were used for DNA manipulation. Oligonu- cleotides were s ynthesized by PerkinElmer Applied B iosys- tems (Warrington, UK). Quercetin-3-xyloside (Q3Xyl; isolated from apple skins), quercetin-3,4¢-diglucoside (Q3,4¢Glc) and malonylated quercetin-3-glucoside (Q3Glc- Mal; both isolated from onions) w ere kind gifts from Keith Price (IFR, Norwich, UK). Kaempferol-3-glucuronide (K3GlA; isolated from lettuce) was a kind gift from S. DuPont (IFR, Norwich, UK). Quercetin-7-glucoside (Q7Glc) was synthesized as described below. Quercetin glucuronides (quercetin-3-glucuronide, quercetin-7-glucuro- nide, quercetin-4¢-glucuronide and quercetin-3¢-glucuro- nide) were b iosynthesized using pig liver microsomes as a source of UDP-glucuronosyl transferase (UDP-GT) activ- ity, UDP-glucuronic acid, UDP-glucosylamine and querce- tin (all obtained from Sigma Aldrich) as donor, cofactor and acceptor, respectively, and were puri®ed using s olid-phase extraction on polyamide followed by preparative HPLC using a reversed-phase LUNA C-18 column (4.6 ´ 25 mm, 5 lm; Phenomonex, Maccles®eld, UK). Other ¯avonoids and their conjugates were purchased in the purest form available from Extrasynthe Á se (ZI Lyon Nord, BP 62, 69730 Genay, France) or Apin Chemicals Ltd (Milton Park, Abingdon, Oxford, UK). Mandelonitrile-b- D -glucopyrano- side (prunasin), mandelonitrile-b- D -gentiobioside (amyg- dalin), 1,4-benzenediol-b- D -glucopyranoside (arbutin), guiacol-b- D -glucopyranoside (salicin), 2,4-dinitrophenyl-2- ¯uoro-2-deoxy-b- D -glucopyranoside, and the nitrophenyl glycosyl derivatives were obtained from Sigma Aldrich (Poole, Dorset, U K). Synthesis of quercetin-7- O -b- D -glucopyranoside (Q7Glc) 3¢,4¢,4,5-Tetrabenzoylquercetin [21] (100 mg, 139 lmol), 2,3,4,6-tetra-O-acetyl-a- D -glucopyranosyl b romide (170 mg, 3eq.),Ag 2 CO 3 (115 mg,3 eq.),3 A Ê sieves (250 mg) and dry CH 2 Cl 2 (10 mL) and collidine (55 lL, 3 eq.) w ere stirred under Ar, in the dark, for 3 days. After ® ltration, combined ®ltrate and washings (5% MeOH/CH 2 Cl 2, 100 mL) were washed with 1 M HCl (50 mL), H 2 O (50 mL), 0.1 M Na 2 S 2 O 3 (50 mL), H 2 O (50 mL), saturated NaHCO 3 (50 mL), and H 2 O ( 50 mL), and t hen dried (MgSO 4 ). The evaporated residue was stirred into 1 M NaOH ( 50 mL) under Ar (0°, 9 0 m in), warmed to room temperture, heated at re¯ux (20 min), and cooled. Dowex 50 W resin (H + form, 70 mL) was added. Filtrate and washings (50% aqueous MeOH, 100 mL) were evaporated, dissolved in 10% aqueous MeOH (300 mL), and washed CH 2 Cl 2 (3 ´ 80 mL). The aqueous phase was evaporated, taken up in MeOH (2.5 m L) and puri®ed by HPLC. Yield 7 m g, 12%. 1 H-NMR (CD 3 OD): d 7.74 (d, 1 H, J 2¢,6¢ 2.0 Hz, H-2¢), 7.65 (dd, 1 H, J 6¢,5¢ 7.6 Hz, H-6¢), 6.88 (d, 1 H, H-5¢), 6.74 (d, 1H,J 8,6 2.0Hz,H-8),6.44(d,1H,H-6),5.05(d,1H,J 1¢¢,2¢¢ 7.2 Hz, H-1¢¢), 3.95 (dd, 1 H, J 6A¢¢,6¢¢B 11.9 Hz, H -6 A ¢¢), 3.73 (dd, 1H, H -6B¢¢), 3.43±3.57 (m, 3 H, H-2¢¢,H-3¢¢,H-4¢¢). ESMS: m/z 465 [M + H] + 487 [M + Na] + . Isolation of cytosolic b-glucosidase from human liver Liver samples were obtained from redundant tissue of surgical specimens f rom patients undergoing hepatic sur- gery. The patient c oncerned had given informed consent for the w ork to be performed. A sample of liver was obtained fresh, cut into pieces ( 5 g ) and snap-frozen in liquid nitrogen before use. CBG was isolated from 100 g (fresh weight) of thawed liver b y a modi®cation of a procedure described previously [14]. B rie¯y, the isolation involved homogenization, centrifugation at high speed to remove membranes and large debris, cation-exchange chromato- graphy on CM-Sephadex, af®nity chromatography using octyl-Sepharose, chromatofocussing using a Mono P HR 250 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 5/20 chromatography column (Amersham Pharmacia Bio- tech), and gel ®ltration u sing a Superdex 200 HR 10/30 gel ®ltration chromatography column (Amersham Pharmacia Biotech). Fraction s containing CBG activity were pooled, mixedwithanequivalentvolumeofethyleneglycoland stored at )20 °C. Isolation, sequencing and analysis of cbg-1 from a human cDNA library The full length cDNA encoding for human CBG w as isolated from a human liver kTriplEx TM cDNA library (Clontech, Palo Alto, CA, USA) by h ybridization screening using a 900-bp o ligonucleotide probe ampli®ed from the cDNA library by PCR using d egenerate primers designed against conserved regions in domains II I and IV of human LPH [22] and guinea pig CBG [23]. The sequence of forward primer HCG/F2 was 5¢-TAYCGNTTYTCNATHTCN TGG-3¢. The sequence o f the reverse p rimer HCG/R3 was 5¢-NCCNTTYTCNGTRATRTA-3¢. PCR was p erformed using 1 lL of library lysate, 20 p mol of primers HCG/F2 and HCG/R3, 0.2 m M dNTPs, 2.5 U of Taq polymerase (Amersham Pharmacia B iotech) 1 0 m M Tris/HCl, pH 9 .0, 50 m M KCl, 3.5 m M MgCl 2 on a PerkinElmer Gene Amp 2400 thermal cycler (PE Biosystems, Foster City, CA, USA) at 94 °C for 2 min followed by 30 cycles of 94 °Cfor1min, 42.0 °C for 1 min, 72.0 °C for 2 min. The ampli®cation was completed with a ®nal extension at 72.0 °C for 5 min. T he probe w as gel-puri®ed using a QIAquick gel extraction k it (Qiagen Ltd, Crawley, UK) and labelled with horserad- ish peroxidase using an ECL TM direct nucleic acid labelling and detection k it (Amersham P harmacia Biotech). The library was plated out on 20 cm ´ 20 cm bioassay plate ( Nalge Nunc Intern ational, N aperville, USA) for the primary hybridization screen according to the manufactur- ers protocol. Plaques were transferred to a nylon membrane (Hybond N + , Amersham Pharmacia Biotech) and cross- linked using UV irradiation (Stratalinker 2400, Stratagene, La Jolla, California, USA). Enhanced chemiluminescence signal generation was carried out using the direct nucleic acid labelling and detection kit and autoradiography. Positive colonies from the primary screen were taken through a secondary screen as described above, except o n 150 mm p lates a t a density of 200±1000 plaques p er pla te. Single well-isolated positive plaques from the secondary screen were converted from kTriplEx clones to pTriplEx clones by in vivo excision and circularization according to the protocol in the library users manual. The clones were sequenced on both strands using the ABI Prism BigDye TM Terminator Cycle Sequencing kit and an ABI 373 DNA sequencer. Sequence analysis was carried out using the Wisconsin GCG V 10.1 software package (Genetics Computer Group, Madison, Wisconsin, USA) and sequence align- ments using BLAST v2.0 [24]. Construction of the pHIL-S1/ cbg-1 expression plasmid The pHIL-S1-derived expression plasmid w ith the cDNA insert encoding human CBG is shown in Fig. 1. The DNA manipulations were carried out using standard procedures [25]. The cDNA fragment (1407 bp) containing the cbg-1 coding region was ampli®ed by PCR from the TriplEx clone by using Pfu DNA polymerase (Stratagene) and the upstream primer (5¢-TTTTTT CTCGAGAAGCTTTCC CTGCAGGAT-3¢) and downstream primer (5¢-TTTTT T GGATCCCTACAGATGTGCTTCAAGGCC-3¢), thus introducing XhoIandBamHI sites, respectively (underlined) at each end of the gene. The 5¢ terminus of this construct was designed to introduce t he Pichia phosphatase sign al sequence cleavage site (Ala-Arg) in frame with the cbg-1 coding sequence (Fig. 1). As the native PHO1 signal sequence cleavage site contains a g lutamate re sidue imme- diately a fter the Ala-Arg residues, a glutamate codon (GAA) was included in the primer to preserve the phosphatase's native context. DNA ampli®cation was carried out through 25 cycles of denaturation (1 min at 94 °C), annealing (0.5 min at 61 °C), and extension (1.5 min at 7 2 °C) in a DNA thermocycler (PerkinElmer). The resulting PCR product (1430 bp) was puri®ed using the Qiaquick PCR puri®cation kit ( Qiagen), subcloned into Fig. 1. Nucleotide and amino acid sequences in the cleavage region between the leader peptide and mature reCBG. Th e construction of the vector is detailed under Mate rials and meth- ods. ss, PHO1 secretion signal seq uence; 5¢AOX1 (Pro), P. pastoris alcohol oxidase promoter region; 3¢AOX1 (TT), P. pastoris AOX1 transcriptional terminating sequence. * i s the N-terminal residue o f the native human cytosolic be ta-glucosidase. Ó FEBS 2002 Xenobiotic metabolism by a human b-glucosidase (Eur. J. Biochem. 269) 251 the T OPO vector and subjected to DNA sequencing using the ABI prism Big Dye TM Terminator Cycle Sequencing kit to con®rm t hat n o e rrors wer e generated during the PCR. The positive clone was d igested by a c ombination of XhoI and BamHI, and subsequently the cDNA insert was puri®ed using the Qiaquick PCR puri®cation kit and ligated into the XhoIandBamHI sites of the pHIL-S1 vector, i n phase with the PHO1 signal sequence. E. coli strain DH5 was trans- formed according to the procedures described in Sambrook et al. [25]. Transformants were grown in liquid bacterial cultures, recombinant plasmids isolated using Q iagen col- umns (Mini-Prep kit), and identity c hecked by restriction mapping to yield pHIL-S1/cbg-1. Transformation of Pichia pastoris and selection of a recombinant clone Transformation of the P. pa storis strain (his4)/GS115 [26] and screening were achieved using the spheroplast proce- dure [27], modi®ed as described previously [28]. Brie¯y, pHIL-S1/cbg-1 ( 1 lg) as well as the pHIL-S1 vector, as negative control, were digested with BgIII prior to trans- formation by the spheroplast method. After screening f or methanol sensitive clones, Mut s colonies were used to inoculate 10 mL BMGY pH 6 . After 2 d ays with shaking at 250 r.p.m., 30 °C, the cells were pelleted a nd resuspended in 2 mL BMMY. Following another 5 days at 30 °C, the culture w as centrifuged and the amount of reCBG in the supernatant w as estim ated b y activity measurement ass ays using 4NPGlc a s substrate. Expression of cbg-1 in P. pastoris and isolation of reCBG Large-scale expression was achieved using 250 mL cultures in 1 L baf¯ed ¯as ks. Cells grown i n B MGY a t 30 °Ctoa density of D 600  20±25 were harvested, resuspended in 50 mL of BMMY and incubated with shaking (250 r.p.m.) in ®ve 5 0 m L loosely cap tubes at 30 °C. The culture was continued for a total of 5 d ays with aliquots of the supernatant removed at various time points in order to monitor p roduction of reCBG. Puri®cation of reCBG was achieved in a single step using af®nity chromatog- raphy. Supernatant (50 mL) was loaded onto a column (1.5 ´ 5 cm) of octyl sepharose previously equilibrated w ith 20 m M sodium p hosphate buffer (pH 6.5) containing 1 m M EDTA, the column was washed with 20% ethylene glycol in sodium phosphate buffer and unbound material discar ded. Bound material was eluted with ethylene glycol (50% v/v) at a ¯ow rate of 0.5 mLámin )1 over 1 h . b-Glucosidase- containing fractions were pooled and checked for purity by SDS/PAGE. Enzyme assays Fractions g enerated during isolation of CBG from human liver were assayed for CBG a ctivity u sing a spectrophoto- metric assay where the release of 4-nitrophenol (4NP) from 4-nitrophenyl-b- D -glucopyranoside (4NPGlc; 10 m M )in 50 m M sodium-phosphate buffer (pH 6.5) at 37 °Cis determined at 400 n m using the molar extinction coef®cient for 4NP of 18 300 M )1 ácm )1 . The p H optimum for r eCBG was determined b y m easuring the b-glucosidase activity in 50 m M sodium phosphate (pH range 2.8±7.6). The thermal stability of CBG was assessed by measuring the residual b- glucosidase activity (4NPGlc as substrate) follow ing incu- bation (30 m in) of CBG samples at various temperatures (23±70 °C). The activity of puri®ed CBG towards various nitrophenyl glycosides (a- D -glucopyranoside, a- D -glucopyr- anoside, a- D -galactopyranoside, a- L -arabinopyranoside, b- L -arabinopyranoside, a- L -arabino-furanoside, a- D -man- nopyranoside, a- D -mannopyranoside, a- D -fucopyranoside, a- D -xylopyranoside, a- L -rhamnopyranoside) w as deter- mined u sing the same m ethod. Activities towards phenolic or mandelonitrile glycosides were determined by measuring the amount of aglycone released from the substrate (10± 5000 l M in 50 m M sodium-phosphate buffer), with p artic- ular care taken to ensure complete solubility of substrates as described previously [14]. Brie¯y, pure phenolic/mandelo- nitrile glycosides were dissolved in a small volume of dimethylsulfoxide prior to dilution with assay buffer (50 m M NaCl/P i , pH 6.5; ®nal c oncentration d imethylsulf- oxide < 2%, v/v), equilibrated at 37 °C , and reactions started with the addition of enzyme (0.1±1 lgin10lL) in a ®nal volume o f 100 lL. Reactions were terminated by the addition of acetonitrile/1% aqueous tri¯uoroacetic acid (50 : 50 , v/v; 100 lL), ®ltered and analysed by reversed- phase HPLC with online diode-array detection using a LUNA C-18 co lumn (4.6 ´ 25 mm, 5 lm; Phenomonex, Maccles®eld, UK) with an injection volume of 20 lL. Solvents A (water/tetrahydrofuran/tri¯uoroacetic acid, 98 : 2 : 0.1 v/v), B (acetonitrile), C (water/tri¯uoroacetic acid, 99.9 : 0.1), and D (methanol/tri¯uoroacetic acid, 99.9 : 0.1) were run at a ¯ow rate of 1 mLámin )1 .The following gradients were used: incubations containing arbutin or salicin as substrate; 100% C initial, i ncreasing D t o 2 0% (10 min), 50% ( 15 min), 100% (5 min), held at 100% (5 min); cyanodin glycosides; 5% B/95% A initial (5 min), increasing B to 20% (10 m in), 90% (10 min), held at 90% (5 m in); iso¯avonoid, mandelonitrile and dihydr- ochalcone glycosides, 17% A/83% B initial (1 min), increasing B to 90% (10 min), held at 90% (4 min). The column was re-equilibrated (5 m in) in the appropriate starting solvent conditions following gradient development. Standard curves were constructed using HPLC grade aglycones from which response factors were calculated and used t o estimate t he amount of product released in test incubations. For estimations of the apparent af®nity (K m ) and k cat , steady-state rates were determined over a range of substrate concentrations (at least 0.2±5.0 ´ K m where possible) and k inetic constants e stimated using a nonlinear weighted least-squares regression analysis method [29]. The concentration of phenolic and mandelonitrile glycosides present in solution at the higher concentrations of substrate used was con®rmed b y HPLC analysis of t he supernatant obtained following centrifugation (13 000 g,10min). Inhibition of reCBG with 2,4-dinitrophenyl-2-¯uoro- 2-deoxy-b- D -glucopyranoside Inhibition studies were performed by incubating reCBG (100 lL) with 2,4-dinitrophenyl-2-¯uoro-2-deoxy- b- D - glucopyranoside (100 lL) at ®nal inh ibitor concentrations of 1 and 5 l M ([E]/[I] ratio s of 1 : 3 and 1 : 15, respectively) at 37 °C. The b-glucosidase activity remaining after various incubation periods (see Fig. 3) was determined by adding 252 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 20 lL of the enzyme/inhibitor mixture to 180 lLof substrate (4NPGlc), incubating for 30 min at 37 °C, and measuring the rel ease of 4NP. Protein assays and protein sequencing Total protein in crude and semipuri®ed samples was estimated using the Pierce Protein Assay Reagent with BSA as standard. For puri®ed reCBG, total protein was calculated using an e xtinction coef®cient at 2 80 nm (122 120 M )1 ácm )1 ) derived fro m the a mino-acid composi- tion for the primary s tructure for reCBG. Protein sequenc- ing was performed at the Protein Sequencing & Peptide Synthesis Facility (John Innes C entre, Norwich, UK) using an ABI 4 91 Procise sequencer. Gel electrophoresis SDS/PAGE was routinely p erformed using 12% homoge- neous Tris/glycine gels (Novex, Frankfurt, Germany) according to the manufacturer's instructions, a nd stained with Coomassie Blue. Molecular m asses were estimated from plots of log(M r ) vs. migration for a series of known standard proteins (LMW Marker Kit; Amersham Pharma- cia Biotech). Isoelectric focusing w as performed using 5% homogeneous polyacrylamide gels for the pH range 3±7 (Novex) according to t he manufacturer's i nstructions, and stained with C oomassie Blue. Values for pI w ere estimated from plots o f pI vs. distance from the anode for a series of known protein standards (Low pI Kit; Amersham Phar- macia Biotech). RESULTS Isolation and characterization of cytosolic b-glucosidase from human liver Human liver was chosen as a source of CBG a s this o rgan is potentially a rich source of the enzyme and disease-free tissue can be obtained fresh during relatively routine surgical procedures. Isolation of CBG from human liver has b een described and involves a fairly long series of fractionation procedures [9,17]. The isolation u sed here involved cation-exchange chromatography (CBG does not bind at pH 5.5), hydrophobic i nteraction chromatography using octyl sepharose (behaves as an af® nity column for mammalian CBG [17]), chromatofocusing, and removal o f ampholines by gel ®ltration chromatography. Starting with 100 g fresh liver tissue, this procedure resulted in a small amount ( 50 lg) of electrophoretically pure protein with a speci®c activity towards 4NPGlc o f 12.8 lmolámin )1 ámg protein )1 , an apparent molecular m ass (by SDS/PAGE) o f 51.9 kDa and a pI o f  4.7. These values are in good agreement with previously published values for mamma- lian liver CBGs [9,12,14,23,30]. We were unable to obtain an N-terminal sequence for the puri®ed enzyme probably because, as with guinea-p ig CBG [23], the N-terminus was blocked. Human liver cbg-1 cDNA cloning and sequence analysis A human liver cDNA library was screened b y a c onven- tional a pproach using a 900-bp 32 P-labelled DNA fragment from human CBG. This DNA probe was ampli®ed by PCR from the c DNA library using two degenerate oligonucleo- tide primers d esigned against consensus sequences from the coding regions of domains III and IV of human lactase phlorizin hydrolase (LPH) [22] and guinea pig cytosolic b-glucosidase [23]. Five cDNA clones were isolated and sequenced. The largest clone was found t o contain an ORF of 1407 nucleotides encoding a p rotein of 496 a mino acids with a calculated molecular mass of 53.7 kDa. A single putative glycosylation site was located at N47 of the deduced amino-acid sequence within the motif KNQT. No signal sequence was apparent which indicates, as expected, CBG is located in the cytosol. The nucleotide a nd amin o- acid sequence has been submitted to the GenBank seq uence data bank and is available under a ccession number AF317840. The primary sequence for human CBG s hared extensive sequence homology with other mammalian b-glucosidases. CBG shared 79% nucleotide similarity and 83.6 % ami no- acid similarity with gu inea pig C BG, and showed homol- ogy w ith domains III and I V o f mammalian LPH (56 and 57% amino-acid similarity, respectively) and with the putative cytosolic and membrane-bound forms of human klotho (42 and 32% amino acid similarity, respectively). Highly conserved regions were identi®ed including those surrounding the putative catalytic glutamates, character- ized by the sequence motifs VKQWITINEA (residues 157± 166) and IYITENG (residues 369±375) found in all family 1 b-glycosidases [31±34]. Alignment o f the cbg-1 cDNA sequence with the other available sequences for human CBG [ 35±37] allowed us to identify s everal nucleotide differences, some of which lead to changes in the p rotein primary structure. We are con®dent these are not due to errors in the cbg-1 sequence as it was derived from a full-length cDNA iso lated using a radiolabelled cDNA probe. The observed differences may be genuine and re¯ect genetic polymorphism. It w as therefore impor- tant to clone, express, and characterize the product arising from a single gene. Expression of cbg-1 in P. pastoris The cDNA sequence encoding the entire human liver cbg-1 cDNA was inserted into the expression vector pHIL-S1 i n frame with the P. pa storis phosphatase signal sequence (Fig. 1). The resulting expression plasmid was used to transform P. pastoris and the transformants screened for the best expression performances. Mut s transformants were grown under noninduced conditions (MGY or BMGY) and then transferred to medium containing methanol (MMY or BMMY). Routine activity assays against pNP-b- D -gluco- pyranoside were u sed for the selection of clones with h igh b-glucosidase productivity. b-Glucosidase activ ity was found only when rich m edium (BMGY/BMMY) was used for induction of CBG expression. However, as P. p ast oris secretes endogenous b-glucosidase activity into the medium, although at very low level, it was important to discriminate between the recombinant and endogenous activities. This was achieved using the ¯avonol glucoside Q4¢Glc, which is a substrate for human liver CBG (Table 2) but not for P. pastoris endogenous b-glucosidase, as demonstrated using media from P. pastoris transformedwithpHIL-S1 lacking the CBG cDNA insertion (data not shown). Hence, Ó FEBS 2002 Xenobiotic metabolism by a human b-glucosidase (Eur. J. Biochem. 269) 253 although both the Pichia endogenous b-glucosidase and the human reCBG hydrolysed 4NPGlc, the use of Q4¢Glc con®rmed that the increased level of b-glucosidase activ ity was due to the secretion of the human recombinant enzyme. A representative His + Mut s transformant was selected for production of recombinant CBG (reCBG) in shake-¯ask cultures with secretion yields up to 10 mgáL )1 after 5 days of culture. When cells were transformed with the pHIL-S1/ cbg-1 vector and induced with methanol, a single major protein band of  53 kDa was identi®ed following SDS/ PAGE analysis of the culture supernatant, and only trace amounts of other proteins were visible a s faint bands (data not shown). The 53-kDa protein was absent from the medium of cells transformed with the vector alone. Puri®cation and characterization of reCBG A single puri®cation step using octyl-Sepharose se parated the reCBG from Pichia endogenous b-glucosidase, and gave an e lectrophoretically pure protein (M r  53 kDa; Fig. 2A) with a speci®c activity on 4NPGlc of 10.0 lmolámin )1 ámg protein )1 . Eighty-two percent of the total b-glucosidase activity in the culture supernatant was recovered in a single peak (chromatogram not shown). No bands other than the 53-kDa band were visible even following silver staining, indicating a very h igh level of purity. The small discrepancy b etween the speci®c activities for human liver CBG a nd reCBG w as shown to be due to the different methods used to estimate total p rotein. Isoelectric focusing of puri®ed reCBG gave t wo bands at pI 4.7 and 4.8 (Fig. 2B), in good agreement with that obtained for CBG isolated from human liver. Conventional Edman sequencing of reCBG indicated a single N-terminal sequence (REAFP) demonstrating that there had been correct processing of the PHO1 signal sequence (Fig. 1). The b-glucosidase inhibitor, 2,4-dinitroph enyl-2-¯uoro- 2-deoxy-b- D -glucopyranoside, was a potent inhibitor of reCBG (Fig. 3). Incubation of reCBG (0.35 l M ®nal concentration) in the presence of 1 and 5 l M inhibitor reduced the b-glucosidase activity in a time-dependent manner; 36 and 70% of the b-glucosidase activity remain ed following 30 and 50 min incubation with 1 and 5 l M inhibitor, respectively. b-Glucosidase activity was not recovered following extensive dialysis of the inhibited enzyme, indicating that inhibition was essentially irrevers- ible. The highest rates for hydrolysis of 4NPGlc over 10 min were obtained at 50 °C, 2.3-fold faster than at 37 °Cand 4-fold faster than at 58 °C (re¯ecting thermal inactivation). The enzyme was relatively stable at 37 °C as more than 80% activity remained after 24 h at this temperature. The pH optimum for b oth r eCBG and human liver CBG w as 6.5, with ³ 70% o f optimum activity maintained over the pH range 5.0±7.5, but < 4% at pH 4.0. Furthermore, we examined the s peci®city o f reCBG with respect to the glycone moiety using a series of NP derivatives. The enzyme catalysed the release of 4NP from six of the 11 4-substituted substrates tested an d the kinetic parameters f or these are presented in Table 1. We detecte d no measureable release of NP using 4NP-a- D -glucopyranoside, 4NP-a- L - arabinofuranoside, 4NP- a- L -rhamnopyranoside, 4NP- a- D - mannopyranoside or 4NP-b- D -mannopyrano-side. The activity towards 2NP-galactopyranoside (10 m M )was  10-fold lower than observed for the 4NP-derivative (data not shown). K inetic analysis under steady-state conditions indicated that the speci®city (k cat /K m )ofreCBGfor4NP- glycosides was b- D -fucopyranoside > a- L -arabinopyrano- side > b- D -glucopyranoside > b- D -galactopyranoside > b- D -xylopyranoside > b- L -arabinopyranoside. These data are in general agreement with those obtained by Daniels et al. [9] and con®rm that CBG has a broad speci®city that can accommodate several glycones in the active site, including b- D -linked pentose and hexose s ugars and a- L - or b- L -linked arabinopyranosides, although several other a-linked sugar derivatives (pentose and hexose) are not hydrolysed by reCBG. Although we detected no me asure- able release of 4NP from 4NP-b- D -mannopyranoside, we were able to con®rm [16] that this compound was an Fig. 2. Gel electrophoresis of reCBG. (A) Reducing SDS/PAGE: Desalted samples (5 lg of puri®ed reCBG) were mixed with 15 lLof 2 ´ SDSsamplebuerandheatedat100°Cfor5minbeforeelec- trophoresis on a 12% homogeneous Tris/glycine polyacrylamide gel. The molecular masses of the marker proteins are shown to the right. Gel was stained with Coomassie blue. (B) Isoelectric focusing: desalted samples were mixed with 2 ´ sample buer and focused o n a 5% homogeneous polyacrylamide gel containing ampholines covering the pH range 3±7. Proteins were stained with Coomassie Blue. Fig. 3. Time-dependent irreversible inactivation of reCBG by 2 ,4-dini- trophenyl-2-¯uoro-2-deoxy-b- D -glucopyranoside. The enzyme (0.35 l M ) was incubated at 37 °C for the indicated period time with 1 l M inhibitor ( ,), 5 l M inhibitor (h) and withou t inhibitor ( s), an d then assayed for beta-glucosidase activity at 37 °C for 30 min. 254 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 effective in hibitor of reCBG [the hydrolysis of 4NP-b- D - glucopyranoside (10 m M ) was reduced by 98% in the presence of 4NP-b- D -mannopyranoside (10 m M )]. Hydrolysis of xenobiotic glycosides by reCBG The a bility of CBG to hydrolyse a variety of glucosides was assessed using a wide variety of aglycone structures that were linked to sugars through various positions on the aglycone (Tables 2 and 3, Fig. 4 ). The analysis was performed in order to (a) assess the capacity of CBG to hydrolyse a variety of plant-derived glycosides which are commonly ingested by humans, and (b) determine some relationships between aglycone structure a nd CBG speci- ®city. CBG hydrolysed e f®ciently many of t he compounds tested, demonstrating lower apparent af®nities (K m )and higher speci®city constants (k cat /K m ) than those obtained using various nitrophenyl glycosides (compare with data in Table 1 ). b- D -Glucosides of ¯avones, iso¯avones and ¯avonols were hydrolysed p articularly ef®ciently. For example, the estimates of apparent af®nity and speci®city constant obtained using the ¯avone glucoside luteolin-4¢- Glcassubstrate(10l M and 117 m M )1 ás )1 , respectively) were 176-fold l ower and 1 7-fold greater, respect ively, than those obtained using 4NPGlc as substrate (Table 2 ). Flavanone glucosides were hydrolysed less ef®ciently (due to higher K m values) compared to glucosides of iso¯avones, ¯avones and ¯avonols. Hydrolysis of cyanogenic glycosides Table 1. The glycone speci®city of reCBG. The hydrolysis of 4NP-glycosides was determined in 50 m M sodium phosphate buer (pH 6.5) at 37 °C by estimating the release of 4NP spectrophotometric ally at 400 nm. For each substrate, d ata were obtained at various concentrations under steady- state conditions, and the data ®tted to the Michaelis±Menten eq uation in orde r to obtain estimates for the kinetic constants ( k cat , K m ). Substrate k cat (s )1 ) K m (m M ) k cat / K m (m M )1 ás )1 ) 4NP-b- D -fucopyranoside 10.7  0.0 0.37  0.01 28.9 4NP-a- L -arabinopyranoside 5.97  0.45 0.57  0.08 10.4 4NP-b- D -glucopyranoside 12.1  0.3 1.76  0.15 6.9 4NP-b- D -galactopyranoside 17.6  0.3 3.14  0.15 5.6 4NP-b- D -xylopyranoside 0.75  0.02 1.58  0.14 0.48 4NP-b- L -arabinopyranoside 0.66  0.09 52.6  8.4 0.013 Table 2. Hydrolysis of xenobiotic glycosides by reCBG. Incubations were performed a t 37 °Cin50m M sodium pho sphate bu er ( pH 6.5). T he release of aglyc one was estimated using re versed-phase HPLC with reference to standard curves constructed using appropriate pure compounds. Where signi® cant rates of h ydrolysis were observed, steady-state rates were obtained for a r ange of initial substrate concentrations and the data ®tted to the Michaelis±Menten equation in order to obtain estimates for K m and k cat . Glc, glucoside; diGlc, diglucoside; MalGlc, malonylglucoside; General, ge ntiobioside; GlA, glucu ronide; GlcRha, rut inoside (1,6-linked rham noglucoside). ND, n ot determined. Substrate Speci®c activity a (lmolámin )1 ámg )1 ) k cat (s )1 ) K m (l M ) k cat /K m (m M )1 ás )1 ) Simple Phenolics Salicyl alcohol-Glc (salicin) 0.171 ND ND ND Hydroquinone-Glc (arbutin) 0.015 ND ND ND Iso¯avones (phytoestrogens) Genistein-7-Glc (genistin) 1.73 1.53  0.04 35  2.9 44 Daidzein-7-Glc (daidzin) 2.75 3.55  0.16 118  11 30 Daidzein-7-MalGlc 0.038 0.24  0.01 3230  130 0.075 Flavonols Quercetin-4¢-Glc (spiraeoside) 1.19 1.08  0.02 31.8  2.9 34 Quercetin-7-Glc 0.77 0.69  0.02 42.2  3.2 16 Quercetin-3,4¢-diGlc 0.21 b 0.30  0.01 274  21 1.1 Flavones Apigenin-7-Glc (apigetrin) 1.30 1.53  0.05 21.5  1.6 71 Luteolin-4¢-Glc 1.30 1.17  0.01 10  0.06 117 Luteolin-7-Glc 2.85 3.05  0.07 50  3.2 61 Luteolin-3¢,7-diGlc 1.46 c ND ND ND Flavanones Naringenin-7-Glc 0.93 2.60  0.01 432  33 6.0 Eriodictyol-7-Glc 0.90 1.26  0.03 253  13 5.0 Cyanogenic glycosides Mandelonitrile-General (amygdalin) 0.100 ND ND ND Mandelonitrile-Glc (prunasin) 0.184 ND ND ND a Speci®c activities are mean data (n ³ 2) and were determined with substrate at a concentration of 500 l M , except for apigenin-7-Glc, which was determined at 200 l M . b,c Rate calculations were based on appearance of quercetin-3-Glc and luteolin aglycone, respectively. Ó FEBS 2002 Xenobiotic metabolism by a human b-glucosidase (Eur. J. Biochem. 269) 255 (prunasin, amygdalin) and glucosides of simple phenolics (salicin, arbutin) occurred at  10% and 1% of the average rate observed for (iso)¯avonoid monoglucosides, respec- tively. Malonylation of the glucose in daidzin (malonyl daidzin) decreased the speci®city 400-fold compared to daidzin due to increases in K m (30-fold) and decreases in k cat (15-fold) (Table 2 ). No activity was detected using gluco- sides of d ihydrochalcones (phlorizin), anthocyanins (e.g. kuromanin) or secoiridoids (oleuropein). Rutinosides (1,6- linked rhamnoglucosides) and glucuronides were not hydrolysed regardless of the conjugation position or aglycone structure ( Table 3). CBG demonstrated remarkable speci®city with respect to the position of glycosylation. For example, although gluco- sides formed in the 4¢- and 7-position of quercetin were ef®ciently hydrolysed, the 3-glucoside was not a substrate f or the enzyme. Indeed, no activity could b e detected on any of the glucosides c onjugated at the 3-position in the C-ring of ¯avonoids (Table 3). CBG was most active on substrates conjugated at the 4¢-compared to the 7-position as evidenced by a lower K m and a higher k cat /K m . It was possible t o determine the relative effects of aglycone structure o n the apparent af®nity and speci®city constant using (iso)¯avo- noids conjugated in the 7 -position. Values for K m varied 20-fold, k cat 5-fold and k cat /K m 14-fold. Some of the differences could be ascribed to s ingle substitution differ- ences between otherwise s imilar aglycones, for example t he presence of a C-5 hydroxyl in genistin reduces the K m  4-fold and i ncreases k cat /K m 1.5-fold compared to daidzin, which lacks a C-5 hydroxyl in the aglycone moiety. However, the major differenc es were observed b etween aglycones containing variations in the C-ring, wh ich de®ne the ¯avonoid subclasses. In particular, saturation of the C-ring to give a ¯avanone (e.g. naringenin, eriodictyol) rather than ¯avone (e.g. apigenin, luteolin) resulted i n large increases in K m and decreases in k cat /K m (19- and 12-fold average, respectively). Quercetin (a ¯avonol) differs from luteolin (a ¯avone) only i n t hat it i s hydroxylated at the 3- position, but the effect is to reduce k cat /K m 4-fold, largely through an increase in k cat (Table 2). CBG was tested for activity against a series of ¯avonol glycosides that differed only in the glycone moiety [Q3Glu, Q3Gal, Q3Xyl, Q3Ara, Q3GlA, Q3Rha and Q3GlcMal; K3Glc, K3GlA and k3(pCA)Glc]. However, we were not able to assess the effects of the glycone moiety in this way as none of these compounds were substrates. These data indicate that ¯avonoid-3-glycosides are not substrates for C BG. DISCUSSION The m echanism by w hich xenobiotics are metabolized and absorbed in humans has re ceived much attention due to the high levels of plant-derived compounds that are ingested orally and bioactive, or which g enerate potentially t oxic or bene®cial metabolites [38±41]. The vast majority of t hese compounds are in the form of b-glycosides (most commonly b- D -glucosides) and hydrolysis to release the relatively more hydrophobic aglycone is, almost without exception, a prerequisite to metabolism, conjugation and excretion. It has been commonly thought that hydrolysis of ingested glycosides occurs only in the colon, facilitated by microbial b-glucosidases. However, there is clear evidence to show that uptake via the c olon is not the only route for dietary xenobiotics to enter the general circulation. Firstly, phenyl- glycosides can be actively transported i nto small intestinal enterocytes by hexose transporters such as the sodium- dependent glucose transporter (SGLT1 [42±45]). Secondly, pharmacokinetic data indicate that absorption of many xenobiotic glycosides occurs very rapidly f ollowing inges- tion, with uptake clearly occurring before compounds have reached the colon [46,47]. Furthermore, it has been dem- onstrated that the bioavailability of some xenobiotics is dependent mainly on small intestinal uptake [46±49]. Taken together these ®ndings suggest that the mechanisms by which xenobiotics are metabolized and absorbed in humans involve endogenous human enzymes (rather than those produced by the colon micro¯ora) that able to hydrolyse glycosides to release the (bioactive) aglycone in the small intestine. The purpose of this study was to determine whether the human cytosolic b-glucosidase could function to deglycosylate dietary ¯avonoid and iso ¯avone glycosides during ®rst pass metabolism. In order to assess this, cbg-1 Table 3. Xenobiotic glycosides not hydrolysed by r eCBG. In cubation s were performed for 2 h at 37 °Cin50m M sodium phosphate buer (pH 6.5). Glc, glucoside; diGlc, diglucoside; GlA, glucuronide; GlcRha, rutinoside (1,6-linked rhamnoglucoside). Class Compound Flavonols Quercetin-3-Glc (isoquercitrin) Quercetin-3-GlcRha (rutin) Quercetin-4¢-GlA Kaempferol-3-Glc Isorhamnetin-3-Glc Flavanones Naringenin-7-GlcRha (naringin) Hesperetin-7-GlcRha (hesperidin) Dihydrochalcones Phloretin-7-Glc (phlorizin) Secoiridoids Oleuropein Anthocyanidins Cyanidin-3-Glc (kuromanin) Cyanidin-3,5-diGlc Fig. 4. Structure s of the xenobiotic aglycones, potential substrates for cytosolic b-glucosidase. (A) quercetin (R1, OH; R2, OH), ap igenin (R1, H, R2, H), luteolin (R1, H, R2, OH); (B) naringenin (R, H ), eriodictyol (R, OH); (C) daidzein (R, H), genistein (R, OH); (D) h ydroquinone; (E) salicyl alcohol; (F) mandelonitrile. 256 J G. Berrin et al. (Eur. J. Biochem. 269) Ó FEBS 2002 cDNA was c loned f rom a human liver c DNA library and expressed heterologously. The recombinant protein, produced in P. pastoris,was very similar to CBG isolated from human liver according to various criteria (electrophoretic mobility, isoelectric point, speci®c activity towards 4NPGlc). Furthermore, reCBG hydrolysed various aryl-glycosides ef®ciently and was inhibited in a time-dependent manner by 2,4-dinitrophe- nyl-2-¯uoro-2-deoxy-b- D -glucopyranoside ( a known mech- anism-based b-glucosidase inhibitor). This is the ®rst re port describing expression of a b-glucosidase gene in the methylotrophic yeast P. pastoris ,anorganismthathas shown great potential for heterologous protein expression [19,20,50]. Expression facilitated the puri®cation of CBG and allowed characterization in s ome detail, especially with respect to its glycone/aglycone speci®city a nd ability to catalyse the hydrolysis of dietary xenobiotic glycosides. This is also the ®rst report describing heterologous expression of a mammalian CBG, and will facilitate identi®cation of putative endogenous substrate(s). CBG f ul®ls many of the criteria required f or an enzyme involved in xenobiotic metabolism [ 15,51]: (a) af®nity for amphipathic xenobiotics due to the presence o f polar and apolar regions involved in su bstrate binding [10,17]; (b) a broad speci®city with regard t o t he glycone moiety and t o some extent the aglycone moiety; and (c) it is found in signi®cant concentrations in the liver and intestine [13]. I n this report, we show for the ®rst time that human CBG hydrolyses many xenobiotic glycosides that are c ommonly ingested as part of the diet, including p hytoestrogens (abundant in soya products), ¯avonols (onions, endive, green beans, broccoli, tomatoes, black grapes, berries, apples skins, tea, leeks, grapefruit), ¯avones (artichokes, parsley, celery, olive, red pepper, lemon), ¯avanones (citrus fruits and juices) a nd cyanogens such as mandelonitrile (cassava) (see Table 2). However, CBG did not hydrolyse all the xenobiotic s tested, and was inactive on dihydroch- alcones such as phlorizin (abu ndant in apple skins), anthocyanodins such as kuromanin (red wine, g rape skins and seeds, berries, raspberries, strawberries) and secoirid- oids such as oleuropein (olives). The fourth general property of enzymes involved in transformation of xenobiotics is increased enzyme levels in the presence of xenobiotic substrates, i.e. inducibility. Cloning the human cbg- 1 gene (i.e. including the 5¢-and3¢-¯anking regions) will facili- tate future studies co ncerned with controls of expression for CBG. In conclusion, a human cDNA encoding CBG has been cloned and expressed in t he yeast P. pastoris and the recombinant protein extensively c haracterized. We show that human CBG hydrolyses a number of xenobiotic glycosides at appreciable rates and with micromolar af®nity constants, and have suggested a role for this enzyme in xenobiotic metabolism. ACKNOWLEDGEMENTS The a uthors thank Dr N. Lambe rt for assistance with the puri®cation of CBG, Dr M. J . Naldrett (Jo hn I nnes C entre, Norwic h, UK) for protein sequ encing, J . Eagle s for mass spectro scopy, S . D upont and K. O'Leary for kind gifts of ¯avonoid glycosides and quercetin glucuronides, respectively, Dr A.J. Day for useful discussions, and the Anatomic Gift Fo undation (Maryland, USA) for the sample of human liver. 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The potential role for human CBG in xenobiotic metabolism