A spectrophotometric assay for a-mannosidase activity

Glycobiology vol. 6 no. 3 pp. 265-270, 1996 A spectrophotometric assay for a-mannosidase activity C.H.Scaman 2, F.Lipari 1 and A.Herscovics 1 Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 and 'McGill Cancer Centre, McGill University. Montreal, Quebec, Canada H3G 1Y6 ^ o whom correspondence should be addressed: Department of Food Science, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada A simple and versatile spectrophotometric assay for a- mannosidase activity, which can be used with unlabelled natural substrate or synthetic substrates, was developed. The reducing mannose released from the substrate by the enzyme is quantitated using glucose oxidase, peroxidase and o-dianisidine. Using recombinant al,2-mannosidase obtained from Saccharomyces cerevisiae and Man, GlcNAc, the spectrophotometric assay yielded values of 03 mm for K^ and 15 mu//tg for V,,,, which are comparable to those obtained using the traditional radiochemical assay. The assay was also used to evaluate some alternative oligosaccharides as substrates for the enzyme. Mans-CMCr^VCOOCH, shows potential as an alternative synthetic substrate as the enzyme retained its specificity for a single al,2-mannose residue. Kinetic results suggest that the lower 13 linked arm of Man 9 Glc- NAc is more critically involved in substrate recognition than the upper 1,6 linked arm. Key words: a-mannosidase/saccharomyces cerevisiae/ spectrophotometric assay/substrate recognition Introduction All glycoproteins with asparagine-linked oligosaccharides arise from the transfer of an oligosaccharide, Glc 3 Man 9 Glc- NAc 2, from dolichol pyrophosphate to the growing polypeptide chain (Kornfeld and Kornfeld, 1985. Subsequent enzymatic modifications are initiated by the removal of the terminal glucose residues, with further processing of mannose residues by a-mannosidases (for review, see Moremen et ai, 1994. al,2-mannosidases are essential for the maturation of asparagine-linked carbohydrates in mammalian cells. Several al,2-mannosidases in the ER and Golgi of mammalian cells are involved in the processing of Man 9 GlcNAc 2 to Man 5 GlcNAc 2, the substrate leading to the formation of hybrid and complex oligosaccharide structures. In the yeast Saccharomyces cerevisiae, there is only one al,2-mannosidase responsible for the specific hydrolysis of a single al,2-linked mannose residue from the Man 9 GlcNAc 2 precursor (Byrd et ai, 1982; Jelinek-Kelly etal, 1985. This enzyme has been purified (Jelinek-Kelly and Herscovics, 1988; Ziegler and Trimble, 1991 and cloned (Camirand etal., 1991. The yeast enzyme shows good homology to the catalytic domain of several al,2-mannosidases cloned from mammalian tissues and from other sources (Bause et al, 1993; Herscovics et ai, 1994; Lai et ai, 1994; Kerscher et ai, 1995. These enzymes have been termed Class 1 al,2-mannosidases (Moremen et ai, 1994. They are all type II membrane proteins with a large C-terminal catalytic region and an N-terminal transmembrane domain. In addition to their sequence homology, they have similar properties in that they do not hydrolyze p-nitrophenyl-a-d-mannopyranoside, require calcium for activity, and are inhibited by 1-deoxymannojirimycin but not by swainsonine (Moremen et ai, 1994. It has recently been possible to produce mg quantities of the recombinant yeast al,2-mannosidase which can be used as a model for determining the mechanism of action and for identifying the active site topography of this family of al,2-mannosidases (Lipari and Herscovics, 1994; Lipari et ai, 1995. Since the yeast al,2-mannosidase, as other Class 1 al,2- mannosidases, does not act on simple chromogenic substrates such as p-nitrophenyl-a-d-mannopyranoside, the enzyme assay has required the preparation and isolation of metabolically labelled 3 H-Man 9 GlcNAc. Quantitation of the 3 H-mannose released by the a-mannosidase is achieved after separation of the oligosaccharide substrate and products, using a variety of techniques, with the most facile being Concanavalin A and polyethylene glycol precipitation of the oligosaccharide (Herscovics and Jelinek-Kelly, 1987. Alternatively, 3 H-oligosaccharide alditols have been used as substrates following reduction with NaB[ 3 H] 4 and the substrate and products were separated by HPLC (Ziegler and Trimble, 1991. These assays have been useful, but they are time consuming in the preparation of the radiolabelled substrate whose specific activity varies with each preparation. We report on a simple and versatile spectrophotometric assay for a-mannosidases which can be used with the unlabelled natural substrate or with synthetic substrates. Reducing mannose released from the substrate is detected using glucose oxidase, peroxidase and the chromogenic agent, o-dianisidine (Scheme I. The utility of the assay is demonstrated by evaluating the Michaelis parameters of the yeast al,2-mannosidase with Man 9 GlcNAc and with some truncated synthetic oligosaccharides as substrates. Kinetic results obtained with truncated substrates suggest that for the yeast enzyme, the lower 1,3 linked arm of Man 9 GlcNAc is more critically involved in substrate recognition than the upper 1,6 linked arm. Results and Discussion Assay evaluation and mannose detection Use of the coupled enzymes, glucose oxidase and peroxidase followed by o-dianisidine as a chromogenic agent, is an effective method for detection of mannose in solution, with no interference from residual substrate. Previously, Oxford University Press 265

CH-Scaman, F.Lipari and A.Herscovics a-d-man( 1 -»2a-D-Man( 1 -»6\ a-d-man(l a-d-man( 1 ->2a-D-Man( 1-43 a-d-man( 1 ->2a-D-Man( 1 -»2a-D-Man( 1 - fi-d-man( 1 -+4P-D-GlcN Ac a 1,2 -Mannosldase D-Mannose a- D-Man( 1 ->2a-D-Man( 1 -»6 2(H 2 O Glucose Oxidase oc-d-man(l-»6 a-d-man(l->3 P-D-Man(l->4P-D-GlcNAc a-d-man( 1 ->2a-D-Man( l->2a-d-man(l Mannonic acid + H 2 O 2 o-dianisidine (colorless Peroxidase Oxidized o-dianisidine (Brown Color a similar enzymatic assay was developed to detect glucose released from a trisaccharide substrate by glucosidase I (Neverova et al, 1994. Mannose, however, is a relatively poor substrate for glucose oxidase, with a turnover rate of 1% or less that of glucose (Pazur, 1966. Therefore, 10 times more glucose oxidase was included in the developing solution used for mannose detection. This did increase background absorbance readings slightly, to approximately 0.065 AU. However, detection of mannose was linear over an assay range of 1-28 nmol, with a detection limit of approximately 1 nmol of substrate (Figure 1. There are limitations to using the spectrophotometric assay. It is approximately 25 times less sensitive than the corresponding radiolabelled assay, as determined by a comparison of the amount of enzyme and the volume of assay used to calculate the K ra value using 3 H-Man,GlcNAc (Lipari and Herscovics, 1994. As well, the spectrophotometric assay did require an extended incubation period, for conversion of reducing mannose to an acid by glucose oxidase. However, there are advantages to using the spectrophotometric assay over the radiochemical assay. The assay presented here does not require the preparation, isolation, and calibration of the 3 H-labelled oligosaccharide substrate. These steps may be important sources of variability when assaying for enzyme activity with labelled substrate, as the incorporation of label into the substrate occurs randomly. Also, the spectrophotometric assay does not require separation of the products of the reaction from the remaining substrate, and with the common use of microplate readers in laboratories, multiple samples may be evaluated simultaneously. The assay is also useful for assaying alternative substrates or inhibitors which are not amenable to the Concanavalin Scheme I A/polyethylene glycol precipitation method or are difficult to prepare with a radioisotope label. Kinetic evaluation of substrates The reliability of the spectrophotometric assay was investigated by evaluating the kinetic parameters of Man 9 Glc- NAc, the substrate commonly used to determine activity of the yeast a-mannosidase. Using the spectrophotometic assay, values for K m and V^ of 0.3 mm (± 0.07 and 15 mu//ig (± 1.3 were obtained for the recombinant yeast al,2-mannosidase (Table I. This compares well with values of 0.3 mm and 6 mu//xg for K m and V,,,, reported using the traditional radiochemical assay with the same recombinant enzyme (Lipari and Herscovics, 1994, and indicates that the spectrophotometric detection of mannose is a reliable alternative for detection of a-mannosidase activity and evaluating kinetic parameters. Kinetic parameters have been published for the same enzyme, purified by conventional methods from S.cerevisiae, and using Man 9 GlcNAc[ 3 H]ol as a substrate (Ziegler and Trimble, 1991. The K m value reported was 0.2 mm which compares well to those reported above. However, the V, value was reported as 220 mu/fig (Ziegler and Trimble, 1991, which is considerably higher than that found for the recombinant enzyme as assayed using either tritiated substrate or the spectrophotometric assay. The reason for this is not clear, but different substrates were used in these studies, either Man 9 GlcNAc or Man 9 Glc- NAcol. It seems possible that this difference in the reducing end of the oligosaccharide may affect catalysis by the enzyme. It has been shown previously (Bause et al, 1992 266

A spectrophotometric assay for a-mannosidase activity E c o e c o 0.1-0.0 10 20 Mannose (nmols 30 Fig. 1. Mannose standard curve ranging from 1.39 to 27.8 nmol/assay. Various concentrations of D-mannose in 50 /xl of buffer were reacted with 250 /xl of developing solution directly in microplate wells Linear regression analysis was used to fit the data. that differences at the reducing end of Man 9 -containing oligosaccharides can affect the specificity of a mammalian al,2-mannosidase. In addition, some intrinsic differences between the recombinant enzyme used in this work and the native enzyme may be responsible for the observed differences in catalytic activity. The Man 5 -O(CH 2 8 COOCH 3 substrate was evaluated as a substrate for the yeast al,2-mannosidase. This compound retains the critical middle arm and the upper arm of Man 9 GlcNAc, but is missing the three mannose residues present on the lower branch and the /31,4-mannose attached to GlcNAc of the core (Table I. There was an approximately 30-fold increase in the Km/V^ ratio, as the K ra value increased to 4 mm and the V,,^ decreased to 6 mu/pig. Although Man5-O(CH 2 8 COOCH3 was a poorer substrate than Man 9 GlcNAc, it has potential to be used as a synthetic substrate with the enzyme. The activity of the yeast al,2-mannosidase has been suggested to be relatively independent of the two 1,2 linked mannoses on the lower 1,3 linked arm of Man 9 GlcNAc (Ziegler and Trimble, 1991. The evaluation of Man 5 - O(CH 2 gcooch 3 as a substrate indicates that removal of the entire lower arm affects binding affinity more than turnover, as the K,,, value increased approximately 13-fold, while V mu values decreased only about 2.5-fold. It is possible that the octyl ester linking arm, to which the five mannose residues are attached, affected binding of the substrate in the active site by interfering with the alignment of the substrate through hydrophobic interactions with the enzyme. There was some concern as to whether the specificity of the enzyme would be retained with the truncated Man 5 - O(CH 2 8 COOCH 3 substrate. Therefore, a preparative scale incubation with the Man5-O(CH 2 8 COOCH3 was carried out. Product from the reaction was isolated using a C-18 Sep Pak cartridge, and characterized by 'H-NMR. The product showed four distinct a-anomeric signals, of approximately equal intensity, indicating that the enzyme had selectively removed a single mannose residue from the substrate (Figure 2. An additional signal was observed at 5.095 ppm, with a coupling constant inconsistent with that for an anomeric signal, and was attributed to a hydrophobic contaminant in the sample. The signal is not consistent with any of the anomeric signals of the Man 5 substrate and was not present in the 'H-NMR spectrum of the Man 5 substrate. It was not possible to assign each signal to an individual mannose residue due to the limited amount of product available. However, based on evidence previously presented for the specificity of the enzyme, it seems likely that the expected al,2-mannose residue on the 1,3 linked branch of the Man 5 -O(CH 2 8 COOCH 3 oligosaccharide was removed (Jelinek-Kelly and Herscovics, 1988; Lipari and Herscovics, 1994. As well, a comparison of the chemical shifts of the anomeric signals for the three trimannosyl compounds described below (data not shown, with those of the Man 5 substrate and Man 4 product support the loss of the expected residue. The specificity of the enzyme was further examined using three Man3-O(CH 2 8 COOCH 3 compounds, each representing a single branch of the Man 9 GlcNAc substrate (Table I. The Michaelis parameters for a-d-manl» 2a-D-Manl -> 3a-D-Man-O(CH 2 8 COOCH 3, corresponding to the middle arm which normally contains the hydrolyzed residue, were evaluated. As expected, the trisaccharide was a much poorer substrate for the yeast a-mannosidase, with a K,, and V,^ of 9 (± 1.3 mm and 0.7 (± 0.04 mu//ig, respectively. There is approximately a 30-fold increase in K m and a 21-fold decrease in V^ for this compound, com- 267

C.H.Seaman, F.Lipari and A.Herscovics Table I. Oligosaccharides evaluated as substrates for al,2-mannosidase. Substrate Man, (mm V max (mu/lg a-d-man(l»2a-d-man(la-d-man(l->2a-d-man(l->3' a-d-man(l a-d-man( 1 >2a-D-Man( I >2a-D-Man(l- Man 5 a-d-man(l-»4/9-d-glcnac 0.3 (0.07 15(1.3 a-d-man( l-«2a-d-man(l 6 a-d-man(l >2a-D-Man(l >3 a-d-man-o(ch 2 8 COOCH 3 4(0 2 6(0.1 Man 3 Upper arm a-d-man(1 2a-D-Man(l-»6a-D-Man-O(CH 2 8 COOCH 3 Middle arm a-d-man(l->2a-d-man(l->3a-d-man-o(ch 2 8 COOCH 3 n.d. 9(1.3 n.d. 0 7 (0.04 Lower arm a-d-man( l->2a-d-man( l->2a-d-man-o(ch 2 8 COOCH 3 Values of K m and V,^ determined using the spectrophotometric assay, with standard error given in brackets n.d. Not determined. pared with Man 9 GlcNAc as substrate. This indicates that the Man 3 compound binds poorly, with almost equally poor turnover. However, there is only about a two fold increase in K m compared to the Man 5 -octyl ester, while the V,, value decreased almost ninefold. This would suggest that the upper arm of the Man 9 GlcNAc substrate is not as critically involved in substrate recognition as the lower arm. Limited quantities of the other two Man 3 substrates, a-d-manl -» 2a-D-Manl -+ 6a- D -Man-O(CH 2 8 COOCH 3 (upper branch anda-d-manl >2a-D-Manl >2a-D-Man- O(CH 2 8 COOCH 3 (lower branch, precluded a full kinetic analysis. However, the relative rate of hydrolysis of the three trisaccharides at 15 mm is shown in Table II. Despite its strict specificity with the larger substrates, the mannosidase does hydrolyze a mannose residue from both of the alternate trisaccharide compounds, albeit at a much reduced rate. It was interesting that the enzyme shows strict specificity for a single mannose residue in the natural substrate and Man 5 -O(CH 2 8 COOCH 3, yet catalyzes the hydrolysis of a residue from the two Man 3 substrates with the 'incorrect' linkages. Although the active site configuration is not optimally configured for hydrolysis of these alternatively linked trisaccharides, there may also be little steric hinderance for these truncated substrates, allowing them to act as substrates, albeit poor ones. The assay for a-mannosidase activity presented here is a reliable and versatile alternative to using radiolabelled Man 9 GlcNAc as a substrate. The utility of the assay to evaluate a number of alternative substrates for the yeast enzyme has been demonstrated. A comparison of the kinetic parameters of Man 9 GlcNAc, Man 5 -O(CH 2 S - COOCH 3, and Man 3 -O(CH 2 8 COOCH 3 substrates suggests that the lower arm of Man 9 GlcNAc is more cntically n.d. involved in substrate binding than the upper arm. Although the assay was set up using the recombinant yeast a-mannosidase, it should be a useful method for the assay of other mannosidases with different specificities. Methods and materials Enzyme and substrates Kinetic studies were carried out using a recombinant, secreted form of a\,2-mannosidase obtained from Saccharomyces cerevisiae as previously described (Lipari and Herscovics, 1994. Unlabelled Man 9 GlcNAc was obtained from soy bean agglutinin as described previously (Bhattacharyya etai. 1988. Potential alternative substrates, Man5-O(CH 2 8 COOCH 3 and three Man r O(CH 2 8 COOCH3 were synthesized as previously described (Distler el ai, 1991. Compounds, synthesized as mannose-6-phosphate derivatives, were dephosphorylated by incubation with alkaline phosphatase (Boehringer-Mannheim, grade I, calf intestine and recovered by adsorption onto a preequilibrated C-18 Sep Pak cartridge (Waters followed by elution with methanol. Assay procedures The kinetic parameters of the hydrolysis of Man 9 GlcNAc were determined with the following protocol. Reactions were initiated by the addition of 100 ng of enzyme to 0.05 to 1.0 mm substrate, in 500 y\ of 100 mm PIPES buffer, ph 6.5, containing 1 mg/ml BSA and 1 mm sodium azide. These were incubated at 37 C for 0.5 h and quenched by placing in boiling water for 1 min. The reaction solution was then microfuged to pellet precipitated protein, and the supernatant transferred to a new microfuge tube. The protein pellet and reaction tube were washed with 4 X 100 fj.\ of methanol, and washes were combined with the supernatant. These were lyophilized, and transferred to a well on a microassay plate using 3 X 100 ^il aliquots of developing solution, containing glucose oxidase (55 U/ml, horseradish peroxidase (1 purpurogallin unit/ml and o-dianisidine dihydrochloride (40 mg/ml in 1 M Tris-HCI, ph 7.2. Microassay plates used were the flat-bottomed, low protein binding type from Corning. The solutions were protected from light by covering with aluminum foil and left to develop for 3.5-4 h at 37 C or until there was n.d 268

A spectrophotometric assay for a-mannosidase activity ppm 5.10 i ' ' ' ' r 5.05 5.00 4.95 4.90 4.85 ppm Fig. 2. The partial 'H-NMR spectrum of the product of the preparative scale incubation of Man r O(CH 2 8COOCH 3 with a-mannosidase. The presence of four distinct alpha anomeric signals of approximately equal intensity indicates that a single a\2 mannose residue was removed from the substrate. no further increase in absorbance. Absorbances were read at 450 nm to 650 nm in a Molecular Devices microplate reader. A typical background absorbance reading for the blank containing all reaction components except substrate was 0.065 AU. Substrate itself, did not contribute to background when incubated with the developing solution, in the absence of enzyme. Absorbance values were converted to nmol of mannose using a mannose standard curve, obtained as described below. K^ values were obtained byfittingthe rate data to the Michaelis-Menten equation, using nonlinear regression analysis employing the Marquardt-Levenberg algorithm (SigmaPlot, Jandel Scientific, 1991. The protocol above was modified to evaluate Manj-O(CH 2,COOCH 3 and Man 3 -O(CH 2 8 COOCH 3 as substrates. The higher K,, values for these compounds allowed higher concentrations of substrate to be used in a smaller volume of 10 15 /xl. For these compounds, substrate was pipetted 269

C.H.Seaman, F.Lipari and A.Herscovics Table II. A comparison of the enzyme activity of al,2-mannosidase on a series of Man3-O(CH 2 8 COOCH 3 oligosaccharides at 15 mm Oligosaccharide a-d-manl->2a-d-manl->3a-r>man-o(ch 2 g COOCH 3 a-d-manl->2a-d-manl->2a-d-man-o(ch 2 8 COOCH 3 a-d-manl->2a-d-manl-»6a-r>man-o(ch 2 8 COOCH 3 Velocity at 15 mm (mu/lg 0.44 0.11 0.05 into 500 fi.\ microfuge tubes and lyophilized. Substrate concentrations ranged from 1 to 10 mm for Man 5 -O(CH 2 8 COOCH 3 and 5 to 50 mm for Man3-O(CH 2 S COOCH3. Buffer and enzyme were added to initiate the reaction. For Man3-O(CH 2 8 COOCH 3, 200 ng of enzyme was incubated for 3 h, and for Man5-O(CH 2 8 COOCH 3, 80 ng of enzyme was incubated for 1.5 h. Incubations were quenched using Tris, by addition of 1.25 M Tris-HCl, ph 7.6 to bring the total volume to 50 fi\ (Jelinek- Kelly and Herscovics, 1988. The quenched reaction mixture was transferred to a microplate well, 250 jtl of developing solution was added to each well, and the plate was left to develop and was read as described above. A standard curve was obtained by reacting 1.39-27.8 nmol (27.8-556 fim of D-mannose in 50 /xl of 100 mm PIPES buffer containing 1 mg/ ml BSA and 1 mm sodium azide, with 250 /il of developing solution directly in mictoplate wells. Triplicates of each mannose level were developed and read as described above. Blanks containing only buffer and developing solution were subtracted from the absorbance readings, and the data was analysed by linear regression analysis. Preparative scale incubation A preparative scale incubation of Man5-O(CH 2 8 COOCH 3 with yeast a- mannosidase was carried out. Approximately 0.3 mg of substrate and 400 ng of enzyme were incubated in 10 ^1 of buffer overnight at 37 C. The reaction was loaded onto a Sep-Pak C-18 cartridge, which had been preequilibrated with 20 ml HPLC grade methanol, followed by 20 ml Milli-Q water. Buffer salts and D-mannose were washed from the cartridge using 20 ml of water, and material retained on the Sep-Pak was eluted with 20 ml of methanol. The methanol eluant, containing the product of the reaction, was lyophilized, and characterized by 'H-NMR spectroscopy using a Varian Unity 500 instrument and D 2 O as the solvent. Acknowledgements We thank Dr. Albin Otter for his enthusiastic assistance in obtaining the 'H-NMR spectra of the compounds. We also thank Dr. O.Hindsgaul and Dr. O.P.Srivastava for making the Manj-O(CH 2 8 COOCH 3 and Man 3 - O(CH 2 8 COOCH3 compounds available. C.S. would like to thank Dr. M.Palcic for financial support to carry out this work. Funding for this study was provided by the Natural Sciences and Engineering Research Council of Canada to Dr. M.M.Palcic and by National Institute of Health grant GM31265 (to A.H.. F. L. would like to thank the Natural Sciences and Engineering Research Council of Canada for a scholarship for graduate studies. References Bause.E., Breuer,W., Schweden J., Roeser,R. and Geyer,R. (1992 Effect of substrate structure on the activity of Maivmannosidase from pig liver involved in N-linked oligosaccharide processing. Eur. J. Biochem., 208,451-457. Bause.E., Bieberich,E., Rolfs,A., V61ker,C. and Schmidt,B. (1993 Molecular cloning and primary structure of Maivmannosidase from human kidney. Eur. J. Biochem., 217, 535-540. Bhattacharyya.L., Haraldssonjvl. and Brewer,C.F. 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