Purification and biochemical characterization of two soluble [alpha]-mannosidases from Candida albicans

Ana Bertha Vázquez-Reyna1, Patricia Ponce-Noyola1, Carlos Calvo-Méndez1,2, Everardo López-Romero1,2 and Arturo Flores-Carreón1,2,3

1Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad Autónoma de Guanajuato, Apartado Postal 187, Guanajuato, Gto. CP 36000, México and 2Departamento de Genética y Biología Molecular, CINVESTAV, IPN Apartado Postal México D.F. CP 07000, México

Received on July 31, 1998; revised on December 3, 1998; accepted on December 7, 1998

Two soluble [alpha]-mannosidases, E-I and E-II, were purified from C.albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-[alpha]-D-mannopyranoside and p-nitrophenyl-[alpha]-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42°C. Apparent Km values for hydrolysis of 4-methylumbelliferyl-[alpha]-D-mannopyranoside and p-nitrophenyl-[alpha]-D-mannopyranoside by E-I were 0.83 µM and 2.4 mM, respectively. Corresponding values for E-II were 0.25 µM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-[alpha]-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-[alpha]-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+ and Ca2+ in the range of 0.5-2 mM. At the same concentrations, Mg2+ was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved [alpha]-1,6 and [alpha]-1,3 linkages, respectively.

Key words: Candida albicans/glycosylation/[alpha]-mannosidase

Introduction

Newly synthesized glycoproteins are remodeled in the endoplasmic reticulum (ER) and in the Golgi complex by specific glycosidases and glycosyltransferases to generate a number of different mature structures from a common N-linked oligosaccharide precursor (Moremen et al., 1994). Early steps of processing are similar in yeast and mammalian cells. However, later in the pathway several mannose residues are removed by specific [alpha]-mannosidases in mammalian glycoproteins whereas in Saccharomyces cerevisiae a single mannose moiety is cleaved from Man9GlcNAc2 to form the essential intermediate Man8GlcNAc2 (Byrd et al., 1982). A soluble and an ER membrane-bound form of the yeast specific [alpha]-mannosidase have been purified and shown to migrate as 60 kDa (Jelinek-Kelly and Herscovics, 1988) and 67 kDa (Ziegler and Trimble, 1991) polypeptides by SDS-PAGE, respectively. The MNS1 gene encoding for the enzyme has been isolated (Camirand et al., 1991) and shown to be not essential for viability or outer chain synthesis in S.cerevisiae (Puccia et al., 1993). No such studies exist in Candida albicans, a human pathogen with the ability to undergo morphogenesis (Odds, 1985). Previous results from this laboratory have demonstrated that about 80% of [alpha]-mannosidase activity in this organism is in a soluble form and that it may be involved in the maturation of N-linked glycans (Vázquez-Reyna et al., 1993). To gain further insight into the nature of this activity and its potential role in glycosylation, here we describe the purification to homogeneity and characterization of two soluble [alpha]-mannosidases.

Results

Purification of [alpha]-mannosidase

Results of purification of [alpha]-mannosidase activity by gel chromatography in Sepharose CL6B were very similar to those previously described (Vázquez-Reyna et al., 1993). Accordingly, enzyme activity as measured with 4-methylumbelliferyl-[alpha]-D-mannopyranoside (4-MU-Man) eluted with a Ve/Vo ratio in the range of 1.4-2.2 with an optimum at 1.8 (not shown). Most active fractions were pooled, freeze-dried, and further purified by FPLC in a Mono Q column as described in Materials and methods. Results are shown in Figure 1. Two major peaks of protein were eluted, one with the salt-free buffer followed by another that emerged with the 0.1 M NaCl-containing buffer. Following elution with the 0.1-0.3 M NaCl gradient, two peaks of activity were resolved in a low UV-absorbance eluate with maxima at 0.21 and 0.24 M NaCl, the latter usually representing about 70% of total activity. It should be mentioned, however, that in some experiments the 0.21 M-peak contained more activity than the second one. Fractions 80-86 (E-I) and 92-102 (E-II) were pooled separately, freeze-dried, and resuspended in 1 ml of buffer A and dialyzed against the same buffer. Enzyme samples were stored frozen. The results of purification up to this step are illustrated in Table I. Enrichments of 6.5- and 8.0-fold over the starting material were obtained for E-I and E-II, respectively, with corresponding recovery values of 0.5% and 1.5%. Analysis of E-I and E-II in native gels revealed the presence of three and two major bands, respectively, in addition to other minor bands in both preparations (not shown). To purify enzymes to homogeneity, E-I and E-II were subjected to preparative native-PAGE at 10 mA for 180 min to better resolve the bands. Following enzyme visualization with 4-MU-Man as described above, fluorescent bands were cut off, electroeluted, and analyzed by SDS-PAGE at 10%. As depicted in Figure 2, silver staining revealed single polypeptides of 54.3 kDa and 93.3 kDa for E-I and E-II, respectively.

Table I. Purification of soluble [alpha]-mannosidases I and II from C.albicans
Fraction Total protein (mg) Activitya Recovery (%) Purification (n-fold)
Specific Total
Supernatant 517.5 10.6 5485 100 1
Sepharose CL6B 105.6 24.8 2618 48 2.3
MONO Q:
Enzyme 1 0.42 68.5 28.8 0.5 6.5
Enzyme II 0.95 86.8 82.5 1.5 8.2
aAs measured with 4-MU-Man as substrate.

Biochemical characterization of E-I and E-II


Figure 1. Elution profile of [alpha]-mannosidase activity from a Mono Q column. The enzyme sample (80 mg of protein in 10 ml of buffer A) previously separated by size exclusion chromatography in a Sepharose CL6B column, was subjected to anion exchange chromatography in a Mono Q HR 5/5 column (0.5 cm × 5 cm; 1 ml) equilibrated with buffer A, in a FPLC equipment. After washing with the same buffer, the sample was eluted with a linear 0.1-0.3 M NaCl gradient as described in the text. Open circles, absorbency at 280 nm; solid circles, fluorescence.


Figure 2. SDS-PAGE of purified [alpha]-mannosidases. Samples of 1 µg protein each of purified E-I (lane 1) and E-II (lane 2) were applied to a 10% SDS-polyacrylamide gel and run in the conditions described in the text. Polypeptide bands were stained with silver nitrate.

The low activity recovered after purification by preparative electrophoresis precluded the biochemical characterization of [alpha]-mannosidase in these preparations. Therefore, this was carried out in enzyme fractions purified by ion exchange chromatography in the Mono Q matrix and routinely with 4-MU-Man as substrate.

For both enzymes, optimum activity occurred at pH 6.0 in 50 mM Mes-Tris buffer and at 42°C. At 1 mM, Mn2+ and Ca2+ had no effect on E-I and were inhibitory at higher concentrations (5 mM). On the other hand, Mg2+ and Ca2+ slightly stimulated (40-50%) E-II activity in the range of 0.5 to 2.5 mM and Mn2+ was slightly inhibitory. When measured as a function of increasing concentrations of 4-MU-Man or p-nitrophenyl-[alpha]-D-mannopyranoside (p-NP-Man), enzyme velocity exhibited typical Michaelian kinetics. Lineweaver-Burk plots revealed Km values of 0.83 µM and 0.25 µM 4-MU-Man for E-I and E-II, respectively. Corresponding values for p-NP-Man were 2.04 mM and 1.86 mM (not shown).

We then considered it important to investigate the effect of [alpha]-mannosidase inhibitors such as 1-deoxymannojirimicin (DMJ) and swansonine (SW) on enzyme activity. Hydrolysis of p-NP-Man by E-I was slightly stimulated by both inhibitors (60% at 20 µM SW and 40% at 25 µM DMJ) whereas that by E-II was not affected (Figure 3A,B). On the contrary, hydrolysis of 4-MU-Man by either enzyme was strongly inhibited by SW and DMJ. IC50 values were 0.096 mM and 0.18 mM SW for E-I and E-II, respectively. Corresponding values for DMJ were 0.24 mM and 0.68 mM (Figure 4A,B).


Figure 3. Effect of swainsonine (A) and deoximannojirimicin (B) on the activity of E-I and E-II using p-NP-Man as substrate. Reaction mixtures containing 2.6 µg of E-I or 5 µg of E-II, 2.5 mM p-NP-Man and the indicated concentrations of inhibitors in a final volume of 1 ml with buffer A were incubated for 60 min at 37°C. For E-I and E-II, 100% activity values correspond to specific activities of 8.5 and 9.6 µmol/min/mg of protein, respectively. Open circles, E-I; solid circles, E-II.


Figure 4. Same as in Figure 3 caption except that 20 µM 4-MU-Man was used as substrate and the final volume was reduced to 200 µl. For E-I and E-II, 100% activity values correspond to specific activities of 110 and 108 nmol/min/mg of protein, respectively. Open circles, E-I; solid circles, E-II.

Linkage specificity of E-I and E-II was investigated with various natural substrates such as cell wall mannans obtained from the wild type strain and the mnn1 and mnn2 mutants of S.cerevisiae, the M6 oligosaccharide (AsnGlcNAc2Man6) obtained from ovoalbumin as described in Materials and methods and the disaccharide 3-O-[alpha]-D-mannopyranosyl-D-mannopyranoside (Man-[alpha]-1,3-Man). Following incubation with E-I and E-II, released mannose was quantified with the Dionex analyzer. Results are shown in Table II. All substrates were hydrolyzed by E-I and E-II although at different extents. Thus, whereas wild type mannan was digested with comparable efficiencies by E-I and E-II, hydrolysis of mnn1 mannan and Man-[alpha]-1,3-Man by E-I was 2.2- and 2.4-fold higher, respectively, than that obtained with E-II. More striking differences were observed, however, when lytic activities on mnn2 mannan and M6 were compared. Specific activity of E-I on mnn2 mannan was 11.6-fold higher than that of E-II whereas the latter digested M6 with a 13.8-fold higher efficiency than E-I.

Discussion

Purification of soluble [alpha]-mannosidase activity from C.albicans yeast cells by a three-step procedure involving size exclusion and anion exchange chromatographies and preparative gel electrophoresis led to the separation of two enzyme fractions named here as [alpha]-mannosidase I (E-I) and [alpha]-mannosidase II (E-II). In terms of total activity, the relative proportion of E-I and E-II varied in the different experiments, a result probably due to the drastic procedure used for cell disruption. Recently, we observed that purification of enzyme activity from protoplast homogenates resulted in isolation of mannosidase E-I only. These results suggest that E-I and E-II might be forming a complex which dissociates upon cell disruption with each enzyme retaining its catalytic activity. Alternatively, E-II might be a loosely ER-bound mannosidase which is released into the soluble fraction during cell breakage. In line with this possibility, it has been described that the ER-bound [alpha]-glucosidase II from mammalian cells is made up of two subunits, namely, the 104 kDa [alpha]-subunit containing the catalytic domain and the 58 kDa [beta]-subunit probably responsible for the localization of the enzyme to the ER (Trombetta et al., 1996).

Table II. Substrate specificity of [alpha]-mannosidases I and II from C.albicans
Substrate Linkage Specific activity
Enzyme I Enzyme II
Mannan (WT) [alpha]-1,2;1,3;1,6 0.983 0.676
Mannan (mnn 1) [alpha]-1,2;1,6 1.042 2.319
Mannan (mnn 2) [alpha]-1,6 9.062 0.779
M6 (ovalbumin) [alpha]-1,2;1,3;1,6 0.810 10.380
Man-[alpha]-1,3-Man [alpha]-1,3 17.8 42.4
Reaction mixtures containing: 5-10 µg of protein (E-I or E-II), 0.6 mM of Man-[alpha]-d-Man, 0.25 µM M6 or 90 µg of mannan (WT or mutants) and 50 mM Mes-Tris, pH 6.0 in a final volume of 100 µl, were incubated at 37°C for 4 h (Man-[alpha]-d-Man) or 24 h (all other substrates). Enzyme and substrate controls were run in parallel. After incubation, samples were heated in boiling water for 10 min, cooled, and centrifuged in a Microfuge at 14,000 rpm for 10 min. Supernatants were saved and used to determine the amount of mannose by anion exchange chromatography.

Except for the rat liver [alpha]-1,2 mannosidase which is a tetramer of 57-58 kDa subunits (Tulsiani et al., 1982), most [alpha]-1,2 mannosidases ([alpha]-mannosidase I) are monomeric polypeptides with molecular masses in the range of 51-73 kDa whereas [alpha]-1,3 mannosidases ([alpha]-mannosidase II) are commonly dimers or tetramers of subunits over 100 kDa (Bischoff and Kornfeld, 1986; Moremen et al., 1991). Molecular masses calculated here for E-I and E-II, which appear to be monomeric polypeptides, were 54.3 and 93.3 kDa, respectively.

Several reports indicate that [alpha]-aryl mannosides are cleaved by [alpha]-mannosidase II and ER/cytosolic [alpha]-mannosidase (Dewald and Touster, 1973; Haeuw et al., 1991) but not by [alpha]1,2-mannosidase (Jelinek-Kelly et al., 1985; Schweden et al., 1986; Jelinek-Kelly and Herscovics, 1988; Schweden and Bause, 1989; Yoshihisa and Anraku, 1989; Bischoff et al., 1990; Bonay and Hughes, 1991; Moremen and Robbins, 1991; Schatzle et al., 1992; Merkle and Moremen, 1993). Here, both E-I and E-II hydrolyzed 4-MU-Man, p-NP-Man and Man-[alpha]-1,3-Man (see below). Whereas E-I and E-II hydrolyzed the fluorogenic substrate with comparable efficiencies, E-II was more active than E-I on p-NP-Man and Man-[alpha]-1,3-Man. The use of some natural substrates revealed that although they were unespecifically digested by the two enzymes, E-I and E-II preferentially cleaved [alpha]-1,6- and [alpha]-1,3-linkages, respectively. Recent results from this laboratory indicate that E-I and E-II show also the ability to hydrolyze a glycan (GlcNAc-Man10) obtained from the mnn1, mnn9, ldb2 a triple mutant of S.cerevisiae (Mañas et al., 1997) releasing 1 and 2 mol of mannose per mole of substrate, respectively (Vázquez-Reyna et al., unpublished observations).

It has been described that the [alpha]-mannosidase inhibitors DMJ and SW preferentially inhibit [alpha]-mannosidases I and II, respectively (Moremen et al., 1994). Here, hydrolysis of p-NP-Man by either E-I or E-II was not blocked by the inhibitors; instead, they brought about a slight activation of both enzymes. On the contrary, when the substrate was 4-MU-Man, E-I and E-II were strongly inhibited by both SW and DMJ. It is worth noting that E-I was nearly 2- and 3-fold more sensitive to inhibition by SW and DMJ, respectively, than E-II, as judged from the IC50 values. Presently, we have no explanation for the observed influence of the substrate on the inhibitory effect of DMJ and SW. Some reports suggest that inhibition of [alpha]-mannosidases by DMJ depends not only on the enzyme affinity for a given substrate but also from the nature of the substrate itself (Moremen et al., 1994).

Further experiments will be necessary to establish the physiological relatedness between E-I and E-II and their specific role in glycoprotein processing.

Materials and methods

Organism and culture conditions

Candida albicans, strain ATCC 26555, was maintained and propagated in the yeast-like form in the media and in the conditions described previously (Vázquez-Reyna et al., 1993).

Preparation of the enzymatic fraction

Yeast cells (14-15 g, wet weight) were resuspended in 15-20 ml of 50 mM Mes-Tris buffer, pH 6.0 (buffer A), supplemented with 1 mM PMSF to minimize proteolytic activity and broken ballistically as described before (Vázquez-Reyna et al., 1993). The cell homogenate was centrifuged at 1500 × g for 10 min to remove unbroken cells and heavy fragments, and the resulting supernatant was further centrifuged at 105,000 × g for 60 min. The membrane pellet was discarded and the high-speed supernatant was saved, freeze-dried, and used as the starting material for the purification of [alpha]-mannosidase activity as described below.

Assay of [alpha]-mannosidase activity

Enzyme activity was assayed with the fluorogenic substrate 4-MU-Man as described previously (Vázquez-Reyna et al., 1993) or with p-NP-Man according to Jelinek-Kelly et al. (1985). Activity was expressed as nanomoles of either 4-methylumbelliferone or p-nitrophenolate liberated in 1 min. Alternatively, mannose released from natural substrates was quantified by ion-exchange chromatography (see below) and activity was expressed as nanomoles of mannose liberated in 1 min. With either method, specific activity was referred to 1 mg of protein.

Polyacrylamide electrophoresis (PAGE)

Denaturing and native PAGE was carried out in 10% and 6% gels, respectively, according to Laemmli (1970) and protein bands were revealed with silver nitrate (Heukeshoven and Dernick, 1985).

nzyme purification

(1) Size exclusion chromatography in Sepharose CL6B. The freeze-dried, high-speed supernatant obtained as described above was resuspended in 4 ml of buffer A and subjected to gel filtration in a Sepharose CL6B column (2.6 × 83 cm) essentially as described previously (Vázquez-Reyna et al., 1993). (2) Fast performance liquid chromatography (FPLC). The enzyme sample obtained in the preceding step, routinely 80 mg of protein in 4 ml of buffer A, was dialyzed overnight against 10 mM Mes-Tris buffer, pH 6.0 (buffer B) and subjected to anionic exchange chromatography in a HR 5/5 Mono Q column (0.5 mm × 5 cm; 1 ml) equilibrated with the same buffer. The sample was successively eluted with 20 ml of buffer B and buffer B plus 0.1 M NaCl followed by a 0.1-0.3 M lineal gradient of NaCl in the same buffer. Fractions of 1 ml were collected, absorbance was measured at 280 nm, and aliquots were removed to determine activity with the fluorogenic substrate. Most active fractions were pooled, dialyzed against buffer B aliquoted, and stored at -70°C. (3) Preparative native-PAGE. E-I and E-II were subjected to preparative native-PAGE in 6% gels as described above. To visualize the enzyme, gels were incubated with 0.4 mM 4-Mu-Man in buffer A. After 30 min at 37°C, active polypeptides appeared as fluorescent bands under ultraviolet illumination at 254 nm. Bands were cut off, electroeluted overnight at 4°C at 2-4 mA with a Bio-Rad model 422 Electro-Eluter, and freeze-dried.

Hydrolysis of natural substrates by [alpha]-mannosidases and analysis of released mannose by ion-exchange chromatography

Reaction mixtures containing 5-10 µg of enzyme protein (E-I or E-II), the substrate (either O.6 mM Man-[alpha]-1,3-Man, 90 µg of mannan obtained from the wild-type strain or the mnn1 or mnn2 mutants of S.cerevisiae or 0.25 µM Man6) and buffer A in a final volume of 100 µl were incubated at 37°C. After 4 h (Man-[alpha]-1,3-Man) or 24 h (all other substrates), samples were heated in boiling water for 10 min, cooled, and centrifuged in a Microfuge at 14,000 r.p.m. for 10 min. Mock mixtures containing either enzyme or substrate alone were run in parallel. Supernatants were saved and used to quantify mannose by high-performance anion-exchange chromatography (HPAE). To this purpose, 25 µl aliquots were injected in a Dionex carbohydrate analyzer equipped with a CarboPac PA-1 column (4.6 × 250 mm), a guard column, and a PED-2 pulse electrochemical detector. The sample was eluted with 4 mM NaOH at a flow rate of 0.8 ml/min during 30 min. After this time, the column was washed with 200 mM NaOH for 10 min and equilibrated with 4 mM NaOH for 10 min prior to the next run. A standard containing 5.5 nmol of mannose was run in the same conditions. Peaks were plotted and integrated using a Varian 4400 integrator.

Determination of protein

Protein content was measured by either the Lowry's (1951) or the Bradford's (1976) method using bovine serum albumin as standard.

Chemicals

The substrates 4-MU-Man, p-NP-Man, and 3-O-[alpha]-D-mannopyranosyl-D-mannopyranoside and the inhibitors 1-deoxymannojirimicin and swansonine were purchased from Sigma Chemical Co. (St. Louis, MO). Mannans from the wild-type strain and the mnn1 and mnn2 mutants of S.cerevisiae were a gift of Dr. L.M.Hernández (University of Extremadura, Spain). The Man6 oligosaccharide was obtained from ovalbumin according to the procedure of Huang et al. (1970). All other chemicals were from reliable commercial sources.

Acknowledgments

We thank Dr. L.M.Hernández (University of Extremadura, Spain) for providing us with mannans from the wild type strain and the mnn1 and mnn2 mutants of Saccharomyces cerevisiae and Dr. A.Flores-Martínez for his help in editing the figures. This work was partially supported by grants from CONACyT, CONCyTEG, and Universidad Autónoma de Guanajuato.

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3To whom correspondence should be addressed at: Dr. Arturo Flores-Carreón, IIBE, Facultad de Química, Universidad Autónoma de Guanajuato, Apartado Postal 187, Guanajuato, Gto. CP 36000, México


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