Kinetic studies on endo-b-galactosidase by a novel colorimetric assay and synthesis of N -acetyllactosamine-repeating oligosaccharide b-glycosides using its transglycosylation activity Takeomi Murata, Takeshi Hattori, Satoshi Amarume, Akiko Koichi and Taichi Usui Department of Applied Biological Chemistry, Shizuoka University, Japan Novel chromogenic substrates for endo-b-galactosidase were designed on the basis of the structural features of keratan sulfate. Galb1-4GlcNAcb1-3Galb1-4GlcNAcb- pNP (2), which consists of two repeating units of N-acetyl- lactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl b-N-acetyllactosaminide. In a similar manner, Glc- NAcb1-3Galb1-4GlcNAcb-pNP (1), GlcNAcb1-3Galb1- 4Glcb-pNP (3), Galb1-4GlcNAcb1-3Galb1-4Glcb-pNP (4), Galb1-3GlcNAcb1-3Galb1-4Glcb-pNP (5), and Galb1- 6GlcNAcb1-3Galb1-4Glcb-pNP (6) were synthesized as analogues of 2. Endo-b-galactosidases released GlcNAcb- pNP or Glcb-pNP in an endo-manner from each substrate. A colorimetric assay for endo-b-galactosidase was devel- oped using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcb- pNP or Glcb-pNP formed by the enzyme through a coupled reaction involving b-N-acetylhexosaminidase (b-NAHase) or b- D -glucosidase. Kinetic analysis by this method showed that the value of V max /K m of 2 for Escherichia freundii endo- b-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive sub- strate for analytical use in an endo-b-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitro- phenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAc b1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (9)and GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1- 4GlcNAcb-pNP (10) as the major products, which have N-acetyllactosamine repeating units. Keywords: endo-b-galactosidase; enzyme assay; kinetics; poly (N-acetyllactosamine); transglycosylation. Endo-b-galactosidases were discovered as keratan sulfate- degrading enzymes, so-called keratanases, in culture filtrates of Escherichia freundii (glycoside hydrolase family 16) [1], Coccobacillus sp. [2], Pseudomonas sp. [3], Flavobacterium keratolyticus (glycoside hydrolase family 16) [4,5], and Bacteroides fragilis [6]. E. freundii keratanase was found to have hydrolysing activity for a wide range of nonsulfated oligosaccharides isolated from human milk and carbohy- drate moieties of glycoproteins and glycolipids [7–10]. The use of endo-b-galactosidase has been expanded to detection of poly (N-acetyllactosamine) chains in a variety of complex glycoconjugates in addition to keratan sulfate. Bacteroides fragilis endo-b-galactosidase has properties similar to those of E. freundii endo-b-galactosidase [11–13]. Therefore, the endo-b-galactosidases from E. freundii and B. fragilis have been widely used as tools for structural and functional analyses of glycans involved in glycoconjugates. An assay using keratan sulfate as a substrate has been widely used for estimation of endo-b-galactosidase activity. However, this method is not always reproducible because of lack of uniformity of the polymer. Methods using low molecular mass substrates have been preferred and recommended for accurate determination of activities of endoglycosidases such as a-amylase [14], lysozyme [15], and endo-b-N-acetylglucosaminidase [16], because the purity of the substrate and the reaction pattern can be determined exactly. This led us to develop a substrate suitable for use in the analysis of endo-b-galactosidase. A series of chromogenic substances having a partially substi- tuted unit of poly (N-acetyllactosamine) were designed as substrate analogues for this enzyme because systematic kinetic studies on the structural modification of substrates would be helpful in revealing the requirements for binding and catalytic specificity. In general, glycosidase can cause transglycosylation as well as hydrolysis as a reverse reaction [17–19]. Transglycosylation of endo-type glycosi- dase is now used for the synthesis of N-acetyllactosamine- repeating oligosaccharide. Correspondence to T. Murata, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya, Shizuoka, 422-8529, Japan. Fax and Tel.: +81 54 238 4872, E-mail: actmura@agr.shizuoka.ac.jp Abbreviations: pNP, p-nitrophenyl; b-NAHase, b-N-acetylhexos- aminidase; b4GalT, b-1,4-galactosyltransferase; b3GnT, b-1,3-N- acetylglucosaminyltransferase; HPAEC-PAD, high-performance anion exchange chromatography-pulsed amperometric detection. Enzymes: endo-b-galactosidase (EC 3.2.1.103); b- D -galactosidase (EC 3.2.1.23); b-1,4-galactosyltransferase (EC 2.4.1.22); b-1,3-N- acetylglucosaminyltransferase (EC 2.4.1.149); b-D-glucosidase (EC 3.2.1.21); b-N-acetylhexosaminidase (EC 3.2.1.52). (Received 20 December 2002, revised 11 July 2003, accepted 17 July 2003) Eur. J. Biochem. 270, 3709–3719 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03757.x In this paper, we describe the enzymatic synthesis of a novel substrate 2 and its analogues for use a colorimetric assay of endo-b-galactosidase activity and the usefulness of the resulting chromogenic substrates for kinetic studies on the enzyme. In the latter part of this paper, synthesis of N-acetyllactosamine-repeating oligosaccharide b-glycoside utilizing endo-b-galactosidase-mediated transglycosylation is described. Materials and methods Materials Endo-b-galactosidases from E. freundii and B. fragilis were from Seikagaku Corporation (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. b- D -Galactosidase from Bacillus circulans ATCC31382 [20] was a kind gift from Meiji Milk Products Co. Ltd. (Tokyo, Japan). b- D -Glucosidase from almonds was from Sigma Chemicals. b-N-acetylhexosaminidase (b-NAHase) from Amycolatopsis orientalis IFO 12806T was purified by 80% saturated ammonium sulfate precipitation followed by GlcNAc-cellulofine affinity chromatography. Bovine milk b-1,4-galactosyltransferase (b4GalT) was from Calbiochem (CA, USA). Crude b-1,3-N-acetylglucosaminyltransferase (b3GnT) from bovine serum was prepared as follows. Bovine serum was brought to 80% saturation with solid ammonium sulfate and left standing overnight at 4 °C. The precipitate was collected by centrifugation at 5000 g for 30 min, dissolved in 50 m M Tris/HCl buffer (pH 8.0), and dialysed against distilled water overnight at 4 °C. The enzyme solution was lyophilized and then used for synthesis of oligosaccharides without further purification. The crude enzyme preparation catalysed the transfer of a GlcNAc residue from UDP-GlcNAc to the OH-3¢ positions of Galb1-4Glc, Galb1-4GlcNAc, Galb1-4Glcb-pNP and Galb1-4GlcNAcb-pNP. The specific activity for Galb1- 4GlcNAcb-pNP as an acceptor substrate was 69 lUÆmg )1 . Galb1-4GlcNAc, GlcNAcb1-3Galb1-4GlcNAc, Galb1- 4Glcb-pNP, Galb1-4GlcNAcb-pNP, and GlcNAcb1- 6Galb1-4Glcb-pNP were synthesized by our previously described methods [21–24]. UDP-GlcNAc and UDP-Gal were kind gifts from Yamasa Corporation (Choshi, Japan). All other chemicals were obtained from commercial sources. Enzyme assay b- D -Galactosidase, b- D -glucosidase and b-NAHase activit- ies were assayed as follows. A mixture containing 2 m M substrate solution (Galb-pNP, Glcb-pNP, and GlcNAcb- pNP) in 0.4 mL 50 m M sodium phosphate buffer pH 6.0 and an appropriate amount of enzyme in a total volume of 0.1 mL was incubated for 10 min at 40 °C. One hundred microlitres of the reaction mixture were then added to 0.1 mL 1.0 M Na 2 CO 3 on a microplate at 2-min intervals during to stop the reaction, and the amount of liberated p-nitrophenol was determined by measuring absorbance at 405 nm using a microplate reader (Biolumin 960, Amer- sham Pharmacia). One unit of enzyme was defined as the amount releasing 1 lmol p-nitrophenolÆmin )1 .Theb3GnT assay was carried out as follows. Galb1-4GlcNAcb-pNP (5 mg) and UDP-GlcNAc (3.6 mg) were dissolved in 0.5mL50m M Tris/HCl buffer pH 8.0 containing 1.6 mg MnCl 2 and 0.5 mg ATP, followed by addition of an appropriate amount of b3GnT preparation. The reaction mixture was incubated at 37 °C for 96 h. Fifty microlitres of the reaction mixture was taken out at 24-h intervals during the reaction and boiled for 5 min. Resulting GlcNAcb1- 3Galb1-4GlcNAcb-pNP was measured by HPLC as des- cribed in the Analytical methods. Analytical methods HPLC was carried out using a Mightysil RP18ODS column (4.6 · 150 mm, Kanto Chemical Co. Ltd, Tokyo, Japan) in a Hitachi 6000-series liquid chromatograph with an L-4000 ultraviolet detector (absorbance at 300 nm). Elution of the column was performed with H 2 O/CH 3 OH (95 : 5, v/v). The flow rate was 1.0 mLÆmin )1 at 40 °C. HPAEC-PAD analysis was conducted on a DX-300 Bio-LC system equipped with a pulsed amperometric detector (Dionex, Sunnyvale, USA). Oligosaccharides were analysed by a CarboPac P-1 column (Dionex, 4 · 250 mm) at a flow rate of 1 mLÆmin )1 at room temperature. The elution was effected with 100 m M NaOH for 40 min. For NMR analysis, an appropriate oligosaccharide sample was dis- solved in 200 lLofD 2 O and filtrated with a Millipore filter (0.22 lm) and then put into a sample tube (i.d. 3 mm). 1 H- and 13 C-NMR spectra were recorded on a JEOL JNM-LA 500 spectrometer at 25 °C. Chemical shifts are expressed in d relative to sodium 3-(trimethylsilyl) propio- nate as an external standard. ESI-MS analysis was carried out in the positive-ion mode on a JEOL MS-700 (JEOL Ltd, Akishima, Japan) using 2,5-dihydroxy benzoic acid as the matrix. Determination of the amount of protein was carried out using a Bio-Rad protein assay kit. Determin- ation of total carbohydrate was carried out as follows. One hundred microliters of sample was put into a test tube (1 · 10 cm), and 100 lL of 5% (w/v) phenol and 0.5 mL concentrated sulfuric acid were immediately added. The sample mixture was vortexed and then kept for 20 min at room temperature, and absorbance was read at 490 nm. Preparation of GlcNAcb1-3Galb1-4GlcNAcb- p NP (1) and GlcNAcb1-3Galb1-4GlcNAcb- p NP (3) Galb1-4GlcNAcb-pNP (504 mg, 1 mmol) and UDP-Glc- NAc (651 mg, 1 mmol) were dissolved in 25 mL 50 m M Tris/HCl buffer pH 8.0 containing 99 mg MnCl 2 , followed by addition of 850 mU of crude b3GnT preparation from bovine serum. The mixture was incubated for 172 h at 37 °C, and the reaction was terminated by boiling for 5 min. The precipitate was removed by centrifugation (8 000 g, 20 min), and the supernatant was loaded onto a Toyopearl HW-40S column (5 · 100 cm) equilibrated with 25% methanol at a flow rate of 2 mLÆmin )1 .Afterthe chromatography, the eluate was monitored by measuring the absorbance at 300 nm (p-nitrophenyl group) and at 490 nm (phenol-sulfuric acid method) by a spectrometer. Both chromatograms showed two peaks (F-1, tubes 24–33; F-2, tubes 49–60). F-1 was combined, concentrated, and lyophilized to produce 1 (24.1 mg) in a 3.4% total yield based on the acceptor added. F-2 was recovered as 3710 T. Murata et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Galb1-4GlcNAcb-pNP (0.35 g). In the same way, com- pound 3 was prepared from Galb1-4Glcb-pNP (0.5 g) and UDP-GlcNAc (450 mg) by use of bovine serum b3GnT in a 4% total yield based on the acceptor added. 1 Hand 13 C-NMR data of 1 and 3 were almost identical to data reported previously [24]. Preparation of GlcNAcb1-3Galb1-4GlcNAcb- p NP (2) and GlcNAcb1-3Galb1-4GlcNAcb- p NP (4) Compound 1 (24.1 mg, 34.1 lmol) and UDP-Gal (42.6 mg, 68.2 lmol) were dissolved in 3.4 mL 100 m M sodium cacodylate buffer (pH 6.8) containing 67.3 mg MnCl 2 followed by addition of 0.2 U b4GalT from bovine milk. The mixture was incubated for 6 h at 37 °C and separated by a Toyopearl HW-40S column (2.5 · 80 cm) as described above. Compound 2 was obtained in an 82% total yield (24.3 mg) based on the acceptor added. In the same way, compound 4 was obtained in a 71% total yield (13.2 mg) based on the acceptor from 3 and UDP-Gal. 1 Hand 13 C-NMR data of 2 and 4 are summarized in Table 1. Preparation of GlcNAcb1-3Galb1-4GlcNAcb- p NP (5) and its positional isomer GlcNAcb1-3Galb1-4GlcNAcb- p NP (6) Compound 3 (85 mg, 128 lmol) and o-nitrophenyl b- D - galactopyranoside (Galb-oNP, 238 mg, 790 lmol) were dissolved in 5.8 mL 40 m M sodium acetate buffer pH 5.5 followed by addition of 98 mU of b- D -galactosidase from Bacillus circulans ATCC31382. The mixture was incubated for 10 h at 50 °C and was loaded onto an ODS DM1020T column (5 · 100 cm) equilibrated with 5% methanol at a flow rate of 5 mLÆmin )1 in order to remove the o-nitrophenol liberated during the reaction. The fractions showing absorbance at 300 nm were concentrated and loaded onto a Toyopearl HW-40S column as above. The chromatogram showed two peaks (F-1, 1340–1520 mL; F-2, 1640–1880 mL). F-2 contained 3 (43 mg) used as the acceptor substrate. F-1 was further separated by a Shodex Asahipak NH2P-50 column (21.5 · 300 mm) equilibrated with 80% acetonitrile at a flow rate of 5 mLÆmin )1 at 40 °C. Eluate was monitored on-line by measuring the absorbance at 300 nm. The chromatogram showed two peaks (F-1a, 327–390 mL; F-1b, 396–450 mL). These peaks were Table 1. 1 H- and 13 C-chemical shifts of transfer products in D 2 O solution. J 1,2 , coupling constants are given in Hz. Compounds Chemical Shifts (d) C-1 C-2 C-3 C-4 C-5 C-6 NHCOCOCH 3 o-ph m-ph p-ph C-O H-1 J 1,2 CH 3 2 pNP 119.3 128.9 145.5 164.4 GlcNAc 101.3 57.6 74.8 80.9 77.9 62.6 177.7 24.8 5.35 8.5 2.02 Gal 105.7 72.7 84.9 71.1 77.7 63.81 4.51 8.3 GlcNAc 105.63 58.0 75.0 80.8 77.3 62.5 177.7 24.9 4.72 8.3 2.05 Gal 105.58 73.7 75.3 71.3 78.1 63.77 4.49 8.2 4 pNP 119.3 128.9 145.5 164.5 Glc 102.0 75.2 77.9 80.7 76.9 62.7 5.31 7.7 Gal 105.8 72.8 84.9 71.2 77.7 63.7 4.486 7.7 GlcNAc 105.7 58.0 77.9 81.0 77.4 62.6 177.7 25.0 4.72 8.2 2.05 Gal 105.6 73.8 76.9 71.4 78.2 63.8 4.490 7.7 5 pNP 119.2 128.9 145.4 164.4 Glc 102.0 75.2 77.9 80.6 76.8 62.6 5.30 8.0 Gal 105.4 72.8 84.79 71.1 77.7 63.77 4.47 7.9 GlcNAc 105.7 57.5 84.83 71.2 78.0 63.3 177.8 25.0 4.73 8.5 2.02 Gal 106.3 73.5 75.3 71.3 78.1 63.82 4.44 7.7 6 pNP 119.3 128.9 145.4 164.4 Glc 102.0 75.2 77.9 80.6 76.8 62.6 5.30 8.0 Gal 105.74 72.8 84.8 71.1 77.7 63.78 4.47 7.7 GlcNAc 105.71 58.94 76.3 72.3 77.4 71.3 177.8 25.0 4.69 8.6 2.02 Gal 106.36 73.5 75.5 71.4 78.0 63.81 4.44 8.0 7 pNP 118.2 127.3 143.3 164.0 Gal 101.8 72.1 83.4 68.6 77.1 61.7 5.20 6.5 GlcNAc 103.8 57.9 75.4 70.6 78.5 62.6 171.8 24.7 4.78 8.1 2.06 8 pNP 118.2 127.5 143.2 164.2 Gal 102.3 72.2 75.1 70.4 78.4 70.1 5.22 7.6 GlcNAc 103.2 57.1 74.5 71.5 78.7 62.8 170.7 24.8 4.55 8.4 1.78 9 pNP 119.3 128.9 145.5 164.5 GlcNAc 101.3 57.6 74.8 81.0 78.0 62.7 177.7 24.9 5.34 8.3 2.01 Gal 105.8 72.8 84.9 71.2 77.7 63.8 4.49 8.0 GlcNAc 105.67 58.0 75.0 80.9 77.4 62.6 177.7 25.0 4.70 8.2 2.03 Gal 105.58 72.8 84.8 71.2 77.7 63.8 4.46 8.0 GlcNAc 105.71 58.5 76.4 72.5 78.5 63.3 177.8 25.0 4.67 8.6 2.03 Ó FEBS 2003 Kinetic studies on endo-b-galactosidase (Eur. J. Biochem. 270) 3711 combined, concentrated, and lyophilized to produce 5 (5.5 mg) and 6 (7.2 mg) in 5.2 and 6.8% yields based on the acceptor added, respectively. 1 H- and 13 C-NMR data of 5 and 6 are summarized in Table 1. Preparation of GlcNAcb1-3Galb1-4GlcNAcb- p NP (7) and GlcNAcb1-3Galb1-4GlcNAcb- p NP (8) Galb-pNP (390 mg, 1.29 mmol) and N, N¢-diacetylchito- biose (GlcANc 2 , 531 mg, 1.25 mmol) were dissolved in 7.5mL20m M sodium acetate buffer pH 5.0 followed by addition of 8.7 U of A. orientalis b-N-acetylhexosamini- dase. The mixture was incubated for 100 h at 40 °C, and the reaction was terminated by boiling for 5 min. The precipi- tate was removed by centrifugation (8 000 g, 15 min), and the supernatant was loaded onto a Toyopearl HW-40S column (5 · 100 cm) as above. Eluate was monitored by measuring the absorbance at 300 nm (p-nitrophenyl group) and at 490 nm (phenol-sulfuric acid method). The chroma- togram showed four peaks (F-1, 135–150 mL; F-2, 190– 225 mL; F-3, 275–295 mL; F-4, 420–470 mL). F-2 and F-3 were combined, concentrated, and lyophilized to produce 8 (33.8 mg) and 7 (8.6 mg), respectively, in a 6.5% total yield based on the donor added. 1 H- and 13 C-NMR data of these disaccharides are summarized in Table 1. Hydrolytic actions of endo-b-galactosidase on p -nitrophenyl b-glycosides The hydrolytic actions of endo-b-galactosidase on p-nitro- phenyl oligosaccharide b-glycosides and a reducing oligo- saccharide listed in Table 2 were investigated by incubating a mixture (50 lL) containing 1 m M of substrates in 50 m M sodium acetate buffer pH 5.8 with 1 mU of the enzymes at 37 °C for 20 min. The enzyme hydrolysates were analysed by HPLC or HPAEC-PAD as described in the Analytical method section. The reaction was linear from 5 to 15 min. Therateofattackon2 was arbitrarily set at 100. Colorimetric assay of endo-b-galactosidase activity A mixture containing 0.5 m M of each p-nitrophenyl oligo- saccharide b-glycoside and 50 mU of b- D -glucosidase or 25 mU of b-N-acetyllactosaminide in 500 mL 50 m M sodium acetate buffer pH 5.8 and an appropriate amount of the enzyme was incubated at 37 °C. Samples (each 50 lL) were taken at intervals (0, 5, 10, 15 and 20 min) during the incubation and inactivated by adding 50 lL 1.0 M Na 2 CO 3 . The amount of liberated p-nitrophenol was determined by measuring absorbance at 405 nm using a microplate reader. One unit of the enzyme was defined as the amount hydrolysing 1 lmol of 2 per min. The initial rates of the enzymatic reaction were evaluated from kinetic curves of product accumulation as described above. The parameters of Michaelis–Menten-type kinetics were evalu- ated by 1/v–1/[S] plots and the least-squares method. The substrate concentration ranges used for compounds 1, 2, 3, 4 and 5 were 0.05–0.4, 0.02–0.75, 0.1–0.8, 0.25–2.0 and 0.25– 1.5 m M , respectively. Assay of endo-b-galactosidase activity by HPLC The standard assay was carried out as follows. A reaction mixture (500 lL) containing an appropriate substrate and endo-b-galactosidase in 10 m M sodium acetate buffer (pH 5.8) was incubated at 37 °C, and samples (each 50 lL) were taken at 3-min intervals during incubation. After inactivation of each sample by adding 150 mL of 1 M acetic acid, the amount of liberated GlcNAcb-pNP was determined by HPLC as described in Analytical methods. Transglycosylation reaction of endo-b-galactosidase from E. freundii Compound 1 (16 mg, 23 lmol) was dissolved in 1.9 mL 20 m M sodium acetate buffer pH 5.8 followed by addition of 2.3 mU endo-b-galactosidase from E. freundii.The mixture was incubated for 30 days at 37 °C and was loaded onto a Sep-pak accel QMA column (2 · 4 cm) equilibrated with H 2 O at a flow rate of 1 mLÆmin )1 . Eluate was collected in 2-mL fractions and monitored by measuring the absorb- ance at 300 nm using a spectrometer. The fractions showing absorbance at 300 nm were combined, concentrated and loaded onto a Shodex Asahipak GS-220FP column (7.6 · 250 mm) equilibrated with H 2 O at a flow rate of 0.6 mLÆmin )1 at 40 °C. Eluate was monitored on-line by measuring the absorbance at 300 nm. The chromatogram showed four peaks (Fig. 1A). Peak B, which was presumed to be a transglycosylation product, was concentrated and lyophilized to produce 9 (0.8 mg) in 3.3% yield based on the substrate added. 1 H- and 13 C-NMR data of 9 are summar- ized in Table 1. Results Preparation of colorimetric substances A series of chromogenic substances were designed as substrates of endo-b-galactosidase based on the structural features of keratan sulfate, which is an alternating polymer of N-acetyllactosamine units jointed to each other by a Table 2. Relative hydrolytic rates of endo-b-galactosidases on p-nitro- phenyl oligosaccharide b-glycosides. The hydrolytic actions of endo- b-galactosidase on different substrates were investigated as described in Materials and methods. The vertical arrow indicates the point of cleavage. One mM of each substrate was used for the determination of relative hydrolytic rates. –, not hydrolyzed even in the presence of 10 mU of the enzyme. 3712 T. Murata et al. (Eur. J. Biochem. 270) Ó FEBS 2003 b-(1-3) linkage. Tetrasaccharide 2 containing two N-acetyll- actosamine repeats and its analogues were synthesized by the alternative addition of b-(1-3) linked GlcNAc and b-(1- 4) linked Gal to Galb1-4GlcNAcb-pNP and Galb1-4Glcb- pNP, respectively, using two kinds of glycosyltransferases. Thus, compounds 1 and 3 were first prepared by the regioselective transfer of GlcNAc residue from UDP- GlcNAc to Galb1-4GlcNAcb-pNP and Galb1-4Glcb-pNP by b3GnT from bovine serum. They were further converted into 2 and 4 utilizing b4GalT from bovine milk (Fig. 2A,B). The enzyme efficiently catalysed the transfer of a Gal moiety to the OH-4¢ position of the acceptors in high yields (82 and 71%) depending on the acceptor. The positional isomers 5 and 6 were prepared simultaneously by Gal transfer from Galb-oNP to the OH-3¢ and OH-6¢ positions of 3 using B. circulans b- D -galactosidase-mediated transglycosylation (Fig. 2B). The resulting products were obtained in a molar ratioof1:1.3andina12%overallyieldbasedonthe acceptor added. Compound 7 and its isomer 8 were prepared from Galb-pNP using b-NAHase-mediated trans- glycosylation (Fig. 2C). Hydrolytic actions of endo-b-galactosidases The hydrolytic actions of endo-b-galactosidases on synthetic chromogenic substances (each 1 m M ) were investigated by using enzyme preparations from E. freundii and B. fragilis. Each enzyme splits compounds 1–6 into the corresponding reducing oligosaccharides and chromogenic substances, GlcNAcb-pNP/Glcb-pNP. For example, compound 2 was completely hydrolysed in an endo-manner into Galb1- 4GlcNAcb1-3Galb and GlcNAcb-pNP. The relative hydro- lytic rates of 1 and 4 compared with that of 2 (set at 100) were 47 and 10, i.e. 2- and 10-fold differences, respectively. The hydrolytic activities toward 3, 5 and 6 were not detected under the experimental conditions as described in the Materials and methods. Furthermore, the hydrolytic rate of reducing trisaccharide GlcNAcb1-3Galb1-4GlcNAc was compared with that of its glycoside 1 in order to examine how the p-nitrophenyl group participates in the hydrolytic action. There was little progression of hydrolysis of the reducing trisaccharide under the experimental conditions, Fig. 1. HPLC analysis of the reaction mixture obtained by endo-b- galactosidase-mediated transglycosylation and time courses of the pro- duction of transglycosylation products 9 and 10 from 1 and degradation of 1. (A) HPLC analysis was performed as described in Materials and methods. (B) A reaction mixture (50 lL) containing 11.5 m M com- pound 1, 0.1% BSA and 1.5 mU E. freundii endo-b-galactosidase in 20 m M sodium acetate buffer, pH 5.8, was incubated at 37 °C. The amount of each product formed from the initial substrate was deter- mined by HPLC. s, Peak B (compound 9); d, peak A (compound 10); h, GlcNAcb-pNP; j, compound 1. Fig. 2. Summary of enzymatic synthesis of p-nitrophenyl oligosaccha- ride b-glycosides used in this work. (A) Consecutive additions of Glc- NAc and Gal to Galb1-4GlcNAcb-pNP by b3GnT and b4GalT. (B) Consecutive additions of GlcNAc and Gal to Galb1-4Glcb-pNP by b3GnT and b- D -galactosidase or b4GalT. (C) N-acetylglucosaminy- lation of Galb-pNP by b-N-acetylhexosaminidase-mediated transgly- cosylation. Ó FEBS 2003 Kinetic studies on endo-b-galactosidase (Eur. J. Biochem. 270) 3713 although the reducing trisaccharide was hydrolysed very slowly to form GlcNAcb1-3Gal and GlcNAc when a 10-fold amount of enzyme was added (Table 2). This indicates that the p-nitrophenyl group is critical for the hydrolytic action of endo-b-galactosidase. p-Nitrophenyl disaccharide b-glycosides 7, 8, Galb1-4GlcNAcb-pNP, and GlcNAcb1-6Galb1-4GlcNAcb-pNP and GlcNAcb1- 6Galb1-4Glcb-pNP did not act as substrates for the enzyme. Colorimetric assay of endo-b-galactosidase activity As shown in Fig. 3, a novel method for endo-b-galactosi- dase assay using 2 was designed on the basis of the results described above. This assay involves the colorimetric determination of p-nitrophenol liberated from the substrate by the action of the enzyme through a coupled reaction involving b-NAHase. Thus, the enzyme exclusively produ- ces GlcNAcb-pNP from 2 and then b-NAHase hydrolyses GlcNAcb-pNP to free p-nitrophenol. In this case, com- pound 1 is not suitable for this assay because the terminal GlcNAc residue is subjected to hydrolysis by b-NAHase. Compound 2 was incubated with E. freundii endo-b-galac- tosidase in the presence and absence of b-NAHase (Fig. 4A). The rate of hydrolysis was first-order with respect to the enzyme throughout the course of the determination. The reaction proceeded linearly for at least 20 min under the experimental conditions used (Fig. 4A). In this case, only 10 ng of the endo-b-galactosidase could be determined by this assay method. The dose–response plot of the coupled enzyme vs. the colour intensity of the enzyme was found to be a plateau in the range of 20–40 mU for 15 min. The addition of 20 mU of coupled enzyme to the assay system was sufficient to obtain the maximal activity of endo-b- galactosidase (Fig. 4B). In a similar manner, compound 4 was applied to determination of endo-b-galactosidase activity with b- D -glucosidase as a coupled enzyme (Fig. 4C). When 4 was used as a substrate, 100 ng of the enzyme was required for determination of the activity by this assay method. The addition of 50 mU of coupled enzyme to the assay system was sufficient to obtain the maximal activity of endo-b-galactosidase (Fig. 4D). Kinetics of endo-b-galactosidase In order to elucidate in more detail the substrate specificity of endo-b-galactosidase, parameters of Michaelis–Menten- type kinetics for 1–5 were evaluated by a 1/v–1/[S] plot (Fig. 5). Compound 2 was assayed with A. orientalis b-NAHase as a coupled enzyme and 3–5 with almond b- D -glucosidase using the newly developed colorimetric assay described above. Compound 1 was assayed by HPLC. The kinetic parameters are summarized in Table 3. Com- pound 2 was the best among the synthetic substrates with a k cat /K m value of 911Æm M )1 Æs )1 for E. freundii endo-b-galac- tosidase. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group at the terminal position, because the k cat /K m value of 1 was 31% of that of 2. However, the k cat /K m value of 4, in which Glc had been replaced with GlcNAc, was only 0.9% of that of 2.The same tendency was seen in a comparison of 1 and 3.This was also the case for B. fragilis endo-b-galactosidase as showninTable3. Transglycosylation reaction of endo-b-galactosidase from E. freundii In order to examine the transglycosylation activity of endo-b-galactosidase, E. freundii endo-b-galactosidase was Fig. 3. Principle of the colorimetric assay method for endo-b-galac- tosidase using compounds 2 and 4 through a coupled reaction involving b-NAHase or b- D -glucosidase. Fig. 4. Effects of coupled enzymes on the release of p-nitrophenol from synthetic substrates. (A) Effect of b-NAHase on the production of p-nitrophenol from 2. Compound 2 (125 nmol) was incubated with E. freundii endo-b-galactosidase (9.3 ng) in 0.5 mL 10 m M sodium acetate buffer, pH 5.8, in the presence or absence of b-NAHase (25 mU). The reaction was stopped by the addition of 1.0 M Na 2 CO 3 , and then liberated p-nitrophenol was determined spectrophotometri- cally at 405 nm. d, Endo-b-galactosidase and b-NAHase; s, endo- b-galactosidase; m, b-NAHase. (B) Time course of the production of p-nitrophenol formed from 2 through a coupled reaction involving b-NAHase. (C) Effect of b- D -glucosidase on the production of p-nitrophenol from 4. Compound 4 (250 nmol) was incubated with E. freundii endo-b-galactosidase (93 ng) in the presence or absence of b- D -glucosidase (50 mU) as a coupled enzyme. d, Endo-b-galactosi- dase and b- D -glucosidase; s, endo-b-galactosidase; m, b- D -glucosi- dase. (D) Time course of the production of p-nitrophenol from 4 through a coupled reaction involving b- D -glucosidase. 3714 T. Murata et al. (Eur. J. Biochem. 270) Ó FEBS 2003 incubated with a high substrate concentration of 1 (8.4%). After the incubation, the reaction mixture was analysed by HPLC. As shown in Fig. 1A, two peaks, A and B, which were presumed to be transglycosylation products, appeared at retention times of 20 and 25 min, respectively. Based on the results of ESI-MS analysis, the molecular mass of peak B was estimated to be 1072, which coincides with the value of GlcNAc-Gal-GlcNAc-Gal-GlcNAc-pNP. Peak B was collected and lyophilized to produce 9 (3.3% actual yield base on the donor substrate 1). The 1 H- and 13 C-NMR signals of 9 were easily assigned by correlation with the spectra of 1 and 2 (Table 1). The most direct evidence that the GlcNAcb1-3Gal unit is bound to the C-4¢ position of GlcNAc was obtained from an 8-p.p.m. downfield shift of C-4¢ and the HMBC spectrum (Fig. 6). The results of NMR and ESI-MS analyses revealed that 9 is a pentasaccharide b-glycoside, GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4Glc- NAcb-pNP. Peak A was also subjected to ESI-MS analysis. The molecular mass of peak A was estimated to be 1437.5, which coincides with the theoretical value of GlcNAc-Gal- GlcNAc-Gal-GlcNAc-Gal-GlcNAc-pNP. This result shows that the GlcNAcb1-3Gal residue from 1 was also trans- ferred to 9 by the enzyme. Taking into account the regioselective synthesis of 9 from 1, peak A was estimated to be a heptasaccharide b-glycoside, GlcNAcb1-3Galb1- 4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (10). The time course of the production of transfer products and the degradation of 1 was observed by HPLC analysis as shown in Fig. 1B. The maximum production of 9 and 10 was reached at 4 h, and their amounts were gradually decreased during the subsequent reaction, finally causing complete hydrolysis. Discussion Endo-b-galactosidases have been used widely as analytical tools in the field of glycobiology and glycotechnology. However, there have been few studies on kinetics of the enzyme because a simple and sensitive assay method has not been developed. The conventional assay for endo-b-galac- tosidase activity is based on the method of Park and Johnson using keratan sulfate as a substrate. However, this method is not always reproducible because of lack of uniformity of the polysaccharide. Therefore, we synthesized p-nitrophenyl di, tri, and tetrasaccharide b-glycosides 1–8 as substrates for endo-b-galactosidase by combining a glycos- idase-catalysed transglycosylation and a glycosyltrans- ferase-catalysed reaction. Endo-b-galactosidases from E. freundii and B. fragilis hydrolysed p-nitrophenyl oligo- saccharide b-glycosides 1–6 as shown in Table 2. Based on the hydrolytic action toward the enzyme, 2 wasfoundtobe the best substrate among the synthetic substances. Com- pound 1, in which terminal Gal was trimmed from 2, still acts as a fairly good substrate. The present assay system has better reproducibility and is simpler than the method of Park and Johnson. From a practical viewpoint, 2, a well- defined substrate, was shown to be very useful for a routine submicrogram assay of endo-b-galactosidase in biological materials. Further kinetic studies on endo-b-galactosidases were carried out using a series of modified substrates mentioned above. Kinetic parameters of activities of E. freundii and B. fragilis endo-b-galactosidases toward 1–5 are summar- ized in Table 3. The catalytic efficiencies of 2 for both enzymes were the highest among the synthetic substrates. The value of V max /K m of 2 for the E. freundii enzyme was 1.7-times higher than that for keratan sulfate as shown in Table 3, indicating that it is very useful as a substrate instead of keratan sulfate for analytical use in the endo- b-galactosidase assay. In addition, this similarity in the values of V max /K m suggests that the sulfate group on the 6-position on GlcNAc of keratan sulfate is not always essential for the hydrolytic action of the enzyme. Replace- ment of Glc by internal GlcNAc of 2 resulted in a remarkable reduction in the catalytic efficiency on 4.This was also true for a comparison of 1 and 3. The increase in Fig. 5. Effects of concentrations of substrate on endo-b-galactosidase activity using compounds 2 (A) and 4 (B). The results shown in (A) were obtained from an experiment in which the substrate concentration was varied over a range of 0.02–0.75 m M , and the results shown in (B) were obtained from an experiment in which the substrate concentration was varied over a range of 0.25–2.0 m M . Insets show the Linweaver–Burk plots. Ó FEBS 2003 Kinetic studies on endo-b-galactosidase (Eur. J. Biochem. 270) 3715 catalytic efficiency was clearly due to the N-acetyl group on C-2 of GlcNAc linked to the aglycon moiety. However, compound 4 still acts as a fairly good substrate: the V max /K m value of 4 is14.1%ofthatofGalb1-4GlcNAcb1- 3Galb1-4Glcb-Cer. It may be used as a substitute for the detection of glycosphingolipid-degrading endo-b-galactosi- dase. This concept for the enzyme assay could be applied to other types of endo-b-galactosidases from Diplococcus pneumonia [25], Clostridium perfringens [26,27], and mollusk Painopecten sp. [28], which hydrolyse internal b-galactosidic linkages of blood group A and B antigens, Gala1-4Galb1- 4GlcNAc and GlcNAca1-4Galb1-4GalNAc, and GlcAb1- 3Galb1-3Gal structures, respectively. The structure of the site of cleavage by the enzyme, which was deduced from results of kinetic studies using well- defined synthetic oligosaccharides, is shown in Fig. 7A. We propose a binding structure so that Galb1-4GlcNAcb1- 3Galb1-4GlcNAcb-pNP has a matching shape, which can accommodate a chain of five residues (A, B, C, D, and E) in the active site. Synthetic tri- and tetrasaccharide glycosides had only one cleavage site for the endo-b-galactosidase, which splits the glycoside bond between C and D. On the other hand, Fukuda and Matsumura reported that endo- b-galactosidases from E. freundii hydrolysed corneal Fig. 6. HMBC spectrum of compound 9 with 1 Hand 13 C spectra on the sides of the two-dimensional spectrum. Only the expanded region of the HMBC spectrum showing connectivity of GlcNAc C-1¢¢ with Gal C-1¢, newly formed by endo-b-galactosidase-mediated transglycosylation, is presented. Fig. 7. Proposed structure of the cleavage site of endo-b-galactosidase. TheenzymehydrolysesbothN-acetyllactosamine-repeating tetrasac- charide b-glycoside (A) and keratan sulfate (B). Arrows show the cleavage site of the glycosidic linkages of each substrate. Table 3. Kinetic parameters of endo-b-galactosidases from Escherichia freundii and Bacteroides fragilis. The parameters of Michaelis–Menten-type kinetics were evaluated by 1/v-1/[S] plots and the least-squares method. This summary was compiled from results reported here and from data in the literature. The substrate concentration ranges used for compounds 1, 2, 3, 4 and 5 were 0.05–0.4, 0.02–0.75, 0.1–0.8, 0.25–2.0 and 0.25–1.5 mM, respectively. K m , Mean ± SEM (mM); V max , Mean ± SEM (lmolÆmin )1 Æmg )1 ); k cat , Mean ± SEM (sec )1 ); k cat /K m , sec )1 ÆmM )1 .–,not determined. 3716 T. Murata et al. (Eur. J. Biochem. 270) Ó FEBS 2003 keratan sulfate, releasing GlcNAc6SO 3 – b1-3Gal and GlcNAc6SO 3 – b1-3Gal6SO 3 – b1-4GlcNAc6SO 3 – b1-3Gal as major products [8]. This finding suggests that the enzyme tolerates C-6 sulfation of the sugar residues A and B, which have to be partially O-sulfated in keratan sulfate, but not C-6 sulfation of the sugar residue C. The hydrolysates GlcNAc6SO 3 – b1-3Gal and GlcNAc6SO 3 – b1-3Gal6- SO 3 – b1-4GlcNAc6SO 3 – b1-3Gal may occupy corresponding sites B-C and -A-B-C, respectively, as shown in Fig. 7B. The sugar residue D at the cleavage site influences the sensitivity of oligosaccharides to the enzyme, because hydrolytic action was promoted by the presence of an N-acetyl group on C-2 of GlcNAc corresponding to sugar residue D (Table 3). Disaccharide b-glycoside 7 did not act as a substrate. Reduction of the reducing-end residue of Galb1-4Glc- NAcb1-3Galb1-4Glc inhibited the action of endo-b-gal- actosidases from E. freundii [7,29]. These results indicate that a sugar pyranose structure such as GlcpNAc or Glcp on leaving site D is required for the hydrolytic action of endo- b-galactosidases. Furthermore, the mode of linkage of sugar residues A to B was strict for the binding locus in the active site, because conversion of the (1–4) into (1–3)-linkages of terminus Gal to GlcNAc residues remarkably decreased the enzyme action (Table 2). These observations indicate that a tetrasaccharide sequence consisting of two LacNAc repeat- ing units such as 2 is preferable to the binding locus in the active site. A series of chromogenic substrates were shown to be advantageous as probes for substrate recognition at theactivesiteintheenzyme. Generally, glycosidase has transglycosylation activity as a reverse reaction of hydrolysis. Taking into account the hydrolytic action of endo-b-galactosidase, 1 wasusedasa substrate for transglycosylation reaction. The enzyme produced 9 and 10 as major products through consecutive transfer of the GlcNAcb1-3Gal unit (Fig. 8). Thus, the enzyme catalysed regioselective transfer to the OH-4¢ position of 1 of the GlcNAcb1-3Gal unit from the same substrate to produce 9.Inthiscase,1 serves as both donor and acceptor substrates in the transglycosylation. Furthermore, the disaccharide elongation reaction pro- gresses in order and produces heptasaccharide glycoside 10 from 9. However, once 9 and 10 had reached maximum concentrations, they gradually decreased in a subsequent reaction and finally caused complete degrada- tion (Fig. 1B,C) indicating that 9 and 10 are fairly good substrates for endo-b-galactosidase and reflect an N- acetyllactosamine-repeating structure. The analytical yield of 9 was estimated by HPLC analysis (Fig. 1) to be 7.0% based on the donor substrate. The large difference between yields in actual and analytical data is though to be caused by a loss of 9 through the process of chromatographic separation procedures. Poly (N-acetyllactosamine) has been shown to be present on membrane glycoconjugates [30,31] and has been identi- fied as a precursor of Lewis X, sialyl Lewis X, and blood group antigens. The amounts of poly (N-acetyllactosamine) chains have been shown to be changed during cellular differentiation [32] and malignant transformation of cells [33,34]. Furthermore, poly (N-acetyllactosamine)s are recognized with high affinity by galectins [35] and are involved in apoptosis [36]. Recently, Ando et al. reported that sialylated poly (N-acetyllactosamine) on the cell surface facilitated apoptotic cell uptake by macrophages [37]. On the other hand, it has been reported that Helicobacter pylori selectively interacted with poly (N-acetyllactosamine) of human erythrocytes [38,39] and with Neu5Aca2-3Galb1- 4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4Glcb-Cer and NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1- 3Galb1-4GlcNAcb1-3Galb1-4Glcb-Cer isolated from human gastric adenocarcinoma [40]. These findings suggest that poly (N-acetyllactosamine) chains involved in glyco- conjugates play important roles in biological events such as metastasis, apoptosis, phagocytosis, and infection. Accord- ingly, compounds 9 and 10 should be useful as model compounds for studying biological functions of N-acetyl- lactosamine repeating domains involved in glycoproteins and glycolipids. In this study, the first enzymatic synthesis of poly (N-acetyllactosamine) has been achieved by endo-b-galac- tosidase-mediated transglycosylation. This enzyme would be an excellent tool for producing a poly (N-acetyllactosa- mine) chain as well as for the detection of poly (N-acetyl- lactosamine)s involved in glycoconjugates. Acknowledgements We thank Yamasa Corporation and Meiji Milk Products for the gift of UDP-GlcNAc and UDP-Gal and the gift of b- D -galactosidase from B. circulans ATCC31382, respectively. We also thank JEOL Hightech Ltd. (Akishima, Japan) for ESI-MS analysis of the transfer products. This work was supported by a Grant-in-Aid for Scientific Research (No. 14760047) from the Ministry of Education, Science, Sports, Culture, and Technology of Japan. References 1. 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Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-b-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material Glycobiology 10, 1291–1309 40 Roche, N. , Larsson, T., Angstrom, J & Teneberg, S (2001) Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma...Ó FEBS 2003 Kinetic studies on endo-b-galactosidase (Eur J Biochem 270) 3719 38 Miller-Podraza, H., Milh, M .A. , Bergstrom, J & Karlsson, K .A (1996) Recognition of glycoconjugates by Helicobacter pylori: an apparently high -a nity binding of human polyglycosylceramides, a second sialic acid-based specificity Glycoconj J 13, 453–460 39 Karlsson, H., Larsson, T., Karlsson, K & Miller-Podraza, H (2000) Polyglycosylceramides... spectrometry of permethylated undegraded material Glycobiology 10, 1291–1309 40 Roche, N. , Larsson, T., Angstrom, J & Teneberg, S (2001) Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma Glycobiology 11, 935–944 . Kinetic studies on endo-b-galactosidase by a novel colorimetric assay and synthesis of N -acetyllactosamine-repeating oligosaccharide b-glycosides using its transglycosylation activity Takeomi. of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAc b1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (9 )and GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1- 4GlcNAcb-pNP. Consecutive additions of Glc- NAc and Gal to Galb1-4GlcNAcb-pNP by b3GnT and b4GalT. (B) Consecutive additions of GlcNAc and Gal to Galb1-4Glcb-pNP by b3GnT and b- D -galactosidase or b4GalT. (C) N- acetylglucosaminy- lation