Key words: Candida albicans/glycosylation/[alpha]-mannosidase
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. 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
Table I. 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
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.
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. Introduction
Results
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
Discussion
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
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.
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.
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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