Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified {alpha}-mannosidase from Candida albicans

Héctor M. Mora-Montes2, Everardo López-Romero2, Samuel Zinker3, Patricia Ponce-Noyola2 and Arturo Flores-Carreón1,2

2 Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Guanajuato, Guanajuato 36000, Mexico, and the 3 Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14-740, México, D.F. 07000, Mexico

Received on October 14, 2003; revised on January 8, 2004; accepted on January 9, 2004


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A soluble {alpha}-mannosidase from Candida albicans was purified to homogeneity by sequential size exclusion, ion exchange, and affinity chromatographies in columns of Sepharose CL6B, DEAE Bio-Gel A, and Concanavalin A Sepharose 4B, respectively. Analytical electrophoresis of the purified preparation in 10% SDS–polyacrylamide gels stained with Coomassie blue revealed a single polypeptide of 43 kDa that was responsible for enzyme activity. The purified enzyme primarily trimmed Man9GlcNAc2 to produce Man8GlcNAc2 isomer B and mannose as a function of time of incubation up to 12 h at 37°C. Prolonged incubation with the enzyme resulted in the accumulation after 24 h of other oligosaccharides corresponding to Man7GlcNAc2 and probably Man6GlcNAc2. These two products were also observed when Man8GlcNAc2 isomer B instead of Man9GlcNAc2 was used as substrate. Other oligosaccharides, such as Man6GlcNAc2-Asn, Man5GlcNAc2-Asn, and the {alpha}1,3- and {alpha}1,6-linked mannobiosides, were not hydrolyzed at all. These properties are consistent with an {alpha}1,2-mannosidase that may represent a new member of the glycosylhydrolase family 47.

Key words: {alpha}1,2-mannosidase / Candida albicans / protein N-glycosylation


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Synthesis of glycoprotein N-glycans is a complex process that involves the transfer of the Glc3Man9GlcNAc2 oligosaccharide from Glc3Man9GlcNAc2-PP-dolichol to asparagine residues of nascent proteins in the endoplasmic reticulum (ER) exhibiting the consensus sequence Asn-X-Ser or Thr where X can be any amino acid except proline. Shortly after transfer, the Glc3Man9GlcNAc2 oligosaccharide is processed by {alpha}-glucosidases I and II, which sequentially trim the three glucose units, and by {alpha}-mannosidases, which remove at least one mannose residue depending on the organism (Abeijon and Hirschberg, 1992Go; Herscovics and Orlean, 1993Go; Herscovics, 1999aGo,bGo). Hydrolysis of {alpha}1,2-linked mannose residues gives rise to hybrid and high-mannose N-glycan, whereas removal of {alpha}1,3- and {alpha}1,6-linked mannoses leads to formation of complex N-glycans (Herscovics, 1999bGo; Kornfeld and Kornfeld 1985Go).

Based on amino acid sequence analysis and some biochemical properties, {alpha}-mannosidases are grouped into glycosylhydrolase families 47 and 38, which include class I {alpha}1,2-mannosidases and class II {alpha}1,2-, {alpha}1,3-, and {alpha}1,6-mannosidases, respectively (Bourne and Henrissat, 2001Go; Henrissat and Davies, 1997Go; Herscovics, 1999bGo). Members of family 47 exhibit highly conserved catalytic domains but differ in N-terminal sequences, including the cytoplasmic and transmembrane domains. Depending on substrate specificity, two types of {alpha}1,2-mannosidases can be recognized in this family: those that reside in the ER of yeast and mammalian cells and eliminate a mannose unit from Man9GlcNAc2 (M9) to form the Man8GlcNAc2 isomer B (M8B) (Jelinek-Kelly et al., 1985Go; Tremblay and Herscovics, 1999Go) and the Golgi-resident {alpha}-mannosidases IA, IB, and IC, which release the four {alpha}1,2-linked mannoses from M9 to produce Man5GlcNAc2 (M5). In this case, the removal of mannose residues yields a mixed population of oligosaccharide isomers (Lal et al., 1998Go; Tremblay and Herscovics, 2000Go).

A good bit of evidence supports the notion that demannosylation of intermediate oligosaccharides is related to glycoprotein quality control in the ER, that is, in a function comparable to that established for {alpha}-glucosidase II (Cabral et al., 2001Go; Dennis et al., 2001Go; Helenius and Aebi, 2001Go). For instance, a misfolded carboxypeptidase Y is rapidly degraded in yeast cells harboring the {alpha}1,2-mannosidase-encoding MNS 1 gene but not in mutants lacking this function (Knop et al., 1996Go).

A major interest of this laboratory has focused on the study of {alpha}-glycosidases involved in glycoprotein processing in pathogenic fungi such as Candida albicans. This has led us to the purification and characterization of soluble {alpha}-mannosidases (Vázquez-Reyna et al., 1993Go, 1999Go, 2000Go) presumably involved in N-glycan processing in this organism. Here we report the purification of a soluble {alpha}-mannosidase from C. albicans with the ability to trim the natural substrates M9 and M8B and propose that it corresponds to an {alpha}1,2-mannosidase of the glycosylhydrolase family 47.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Enzyme purification and identification of the active polypeptide by zymogram analysis
Results of purification of {alpha}-mannosidase by gel filtration chromatography of the high-speed supernatant in Sepharose CL6B were essentially similar to those previously described (Vázquez-Reyna et al., 1999Go, 2000Go). Further purification in DEAE resolved {alpha}-mannosidase into a small and a large peak of activity eluting at 0.1 and 0.125 M NaCl, respectively, well separated from contaminating proteins, which emerged mostly in the salt-free buffer washing (Figure 1). Most active fractions associated to the large peak, routinely 77–87, were used for further purification by affinity chromatography to Concanavalin A (Con A) as described later. Enzyme failed to bind the lectin as all activity and about half of the applied protein emerged in the {alpha}-methyl D-mannoside ({alpha}MM)-free buffer washing (data not shown). After this step, enzyme was purified 402-fold with a recovery of 47% over the starting material (Table I). Analytical electrophoresis revealed the presence in the Con A–purified sample of a single polypeptide of 43 kDa with the ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl {alpha}-D-mannopyranoside (MU{alpha}Man) (Figure 2). Hydrolysis of this substrate occurred optimally at pH 6.0 with 50 mM citrate-phosphate buffer (data not shown).



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Fig. 1. Separation of {alpha}-mannosidase by ion exchange chromatography. The enzyme fraction obtained after gel filtration in Sepharose CL6B was applied on a column (2.8 x 7.3 cm) of DEAE Bio-Gel A equilibrated with buffer A. After washing with the same buffer, the sample was eluted with a discontinuous gradient of 0–4.0 M NaCl in the same buffer (see Materials and methods). Two-milliliter fractions were collected and used to monitor elution of protein (closed symbols) and enzyme activity using MU{alpha}Man as substrate (open symbols). Peaks corresponding to {alpha}-mannosidase I (E-I) and II (E-II) are indicated.

 

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Table I. Purification of {alpha}-mannosidase E-II from C. albicans.

 


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Fig. 2. Analytical gel electrophoresis and zymogram analysis of purified {alpha}-mannosidase. Samples of 10 µg purified enzyme were applied in sodium dodecyl sulfate–polyacrylamide (10%) gels either after heat denaturation (A) or without heating (B) and separated by electrophoresis in the conditions described in the text. Lane A was stained with Coomassie blue, and lane B was incubated for 1 h at 37°C with MU{alpha}Man to reveal the enzyme band. Molecular weight markers of 93, 66, 45, and 29 kDa correspond to phosphorylase B, bovine serum albumin, ovalbumin, and soybean trypsin, respectively.

 
Activity of purified {alpha}-mannosidase on natural substrates and {alpha}-linked mannobiosides
To get an insight into the role of purified {alpha}-mannosidase in N-glycan processing, the ability of the enzyme to hydrolyze M9 was examined in a time course experiment. After 2 h of incubation, a product with the same retention time of M8B started to accumulate at the expense of a decrease in the amount of M9, reaching a maximum after 12 h. After longer times of incubation, two other products with lower retention times corresponding to Man7GlcNAc2 (M7) and probably Man6GlcNAc2 (M6) were observed (Figure 3, left). As expected, increasing amounts of mannose accumulated as a function of oligosaccharide hydrolysis (Figure 3, right). Results obtained after 24 h of incubation suggested that {alpha}-mannosidase was also active on products resulting from M9 trimming. This was tested in a similar time course experiment using M8B as substrate. After 6 h of incubation, it was possible to observe the progressive accumulation of oligosaccharides with lower retention times than M8B (Figure 4, left) and corresponding amounts of mannose (Figure 4, right). Under the same experimental conditions, the enzyme failed to hydrolyze the Man6GlcNAc2-Asn (M6Asn) and Man5GlcNAc2-Asn (M5Asn) oligosaccharides after 6 h of incubation (Figure 5) as well as the Man-{alpha}1,3-Man and Man-{alpha}1,6-Man disaccharides after 24 h of incubation (data not shown).



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Fig. 3. Hydrolysis of M9 by purified {alpha}-mannosidase. Reaction mixtures containing the purified enzyme (1.5 µg protein), M9 (2 µg), and buffer A in a total volume of 30 µl were incubated at 37°C. After the indicated times, samples were heated in boiling water and cooled, and the volume was adjusted to 60 µl with deionized water. Hydrolysates were subjected to gel filtration in a column (0.3 x 105 cm) of Bio-Gel P-6 eluted with deionized water, and fractions were analyzed by HPAEC as described in the text. Elution profiles of oligosaccharides and mannose are indicated in left and right panels, respectively. M9 and M8B correspond to Man9GlcNAC2 and Man8GlcNAc2 isomer B, respectively. M7 corresponds to Man7GlcNAc2, and M6 is probably Man6GlcNAc2.

 


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Fig. 4. Hydrolysis of M8B by purified {alpha}-mannosidase. Same legend as in Figure 3 except that M8B was used as substrate. Elution profiles of oligosaccharides and mannose are indicated in left and right panels, respectively. M8B corresponds to Man8GlcNAc2 isomer B, M7 to Man7GlcNAc2, and M6 is probably Man6GlcNAc2.

 


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Fig. 5. Hydrolysis of M6Asn and M5Asn by purified {alpha}-mannosidase. Same legend as in Figure 3 except that M5Asn (left) and M6Asn (right) were used as substrates.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Early studies of {alpha}-mannosidase from C. albicans showed that about 80% of total cellular activity was present in a soluble form (Vázquez-Reyna et al., 1993Go). Later, purification of this fraction by size exclusion and ion exchange chromatographies resolved the enzyme into two peaks of activity that were named as E-I and E-II. These were further separated by preparative nondenaturing electrophoresis and shown to exhibit molecular masses of 54.3 and 93.3 kDa, respectively. Analysis of enzyme activity on different substrates revealed that E-I and E-II released mannose preferentially from an {alpha}1,6-linked mannan and an {alpha}1,3-linked mannobioside, respectively (Vázquez-Reyna et al., 1999Go), and also converted the processing-inert Man10GlcNAc oligosaccharide into a mannose-accepting substrate (Vázquez-Reyna et al., 2000Go). The very low recovery of E-I and E-II activities (0.5% and 1.5%, respectively, over the starting material) obtained in early studies precluded the identification of the active polypeptide. Also the enzyme ability to process the physiological substrates M9 and M8B remained undetermined.

In this study, several modifications were made to the initial protocol of purification: (1) the protease inhibitor phenylmethylsulfonyl fluoride was not added to the working buffer; (2) the Mono Q column was substituted by DEAE, thus allowing the run of this step at 4°C; and (3) affinity to Con A replaced nondenaturing electrophoresis as the final step. As previously observed, sample fractionation in DEAE resolved activity into a small (E-I) and a large (E-II) peak, the latter representing over 70% of recovered activity. Further separation of E-II by affinity chromatography in Con A resulted in enzyme purification and recovery of 402-fold and 47%, respectively, over the starting material. These values are much higher than those of 8.2-fold and 1.5% previously reported (Vázquez-Reyna et al., 1999Go).

Analytical electrophoresis of the Con A–purified fraction revealed a single polypeptide of 43 kDa that, as judged from zymogram analysis, was responsible for {alpha}-mannosidase activity. This molecular mass is very similar to that of 45 kDa reported for a soluble {alpha}-mannosidase purified from Saccharomyces cerevisiae (Jelinek-Kelly and Herscovics, 1988Go) and about half of that previously estimated for E-II from C. albicans (Vázquez-Reyna et al., 1999Go). Other molecular weights reported for {alpha}1,2-mannosidases from different sources range from 56.6 kDa for the enzyme from Penicillium citrinum (Yoshida and Ichishima, 1995Go) to 91.5 kDa for that from Aspergillus nidulans (Eades and Hintz, 2000Go). The enzyme purified in this study acted on M9 as an ER-resident {alpha}1,2-mannosidase (Herscovics, 1999bGo), producing M8B and mannose as the sole products of hydrolysis after 12 h of incubation. As incubation continued, oligosaccharides of lower retention times than M8B, such as M7 and probably M6, were detected after 24 h. These results are similar to those by Herscovics et al. (2002)Go, who observed also that incubation of M9 with purified {alpha}-mannosidase from human and yeast cells for no longer than 24 h produced M8B only. Longer times of incubation or increased amounts of enzyme generated manno-oligosaccharides shorter than the primary product of hydrolysis. Our results thus indicate that {alpha}1,2-mannosidase is active not only on M9 but also on M8B and M7, providing that these accumulate in sufficient amounts. There are presently no evidences supporting the trimming of M8 in vivo because oligosaccharides shorter than this have not been isolated. Furthermore, reaching of a critical concentration of M8 in the ER seems unlikely because this is rapidly transported into the Golgi apparatus for further processing.

{alpha}-Mannosidase, on the other hand, failed to recognize M6Asn as substrate despite the presence of a terminal {alpha}-1,2-linked mannose, a result probably due to stereospecific restraints imposed by the oligosaccharide structure. Inability to trim M5Asn was expected because this oligosaccharide lacks an {alpha}-1,2-bonded mannose. Lack of activity on {alpha}1,3- and {alpha}1,6-linked mannobiosides further confirmed the substrate specificity of the purified {alpha}-mannosidase.

Presently, we cannot fully explain the discrepancy between these findings and those obtained earlier in this laboratory (Vázquez-Reyna et al., 1999Go) in terms of the enzyme molecular weight and substrate specificity. However, it is clear that the modified isolation procedure used in this study resulted in the purification of a 43-kDa enzyme whose catalytic properties strongly resemble an {alpha}1,2-mannosidase of the ER.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Man9GlcNAc2, Man8GlcNAc2 isomer B, Man7GlcNAc2, Man5NAc2-Asn, Man-{alpha}1,3-Man, Man-{alpha}1,6-Man, MU{alpha}Man, {alpha}MM, and Con A Sepharose 4B were obtained from Sigma Chemical Company (St. Louis, MO). Man6GlcNAc2-Asn was prepared according to Huang et al. (1970)Go. Sepharose CL6B was from Pharmacia LKB Biotechnology (Uppsala, Sweden). Bio-Gel P-6, DEAE Bio-Gel A, and all electrophoresis reagents were purchased from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of reagent grade.

Organism and culture conditions
C. albicans strain ATCC 26555 was used in this study. It was maintained on slants of solid YPG medium (0.3% yeast extract, 1% peptone, 2% glucose, and 2% agar, pH 6.3) described by Bartnicki-García and Nickerson (1962)Go. For propagation of yeast cells, 2-L Erlenmeyer flasks containing 600 ml liquid YPG medium were inoculated with an overnight culture to a final density of 7 x 105 cells/ml and shaken at 80 rpm for 12 h at 28°C.

Preparation of cell-free extracts and enzyme purification
The following procedure was carried out at 4°C. Yeast cells were collected by low-speed centrifugation, washed twice with 50 mM MES-Tris buffer pH 6.0 (buffer A), resuspended in 10–15 ml of the same buffer, and broken with glass beads (0.45 mm diameter) in a Braun MSK cell homogenizer (Braun, Melsungen, Germany) by alternate periods of breakage (20 s) and cooling until 2 min of breakage were completed. The homogenate was centrifuged at 1000 x g for 10 min, and the resulting supernatant was further centrifuged at 105,000 x g for 1 h. The high-speed supernatant was collected, freeze-dried, and kept at –20°C until use.

For enzyme purification, the freeze-dried high-speed supernatant was resuspended in 6.0 ml buffer A and subjected to gel filtration in a column (2.7 x 57.8 cm) of Sepharose CL6B equilibrated and eluted with the same buffer. Fractions of 4 ml were collected and used to measure protein by absorbance at 280 nm and enzyme activity with a fluorogenic substrate as described shortly. Most active fractions were pooled and subjected to ion exchange chromatography in a column (2.8 x 7.3 cm) of DEAE Bio-Gel A equilibrated with buffer A. The column was washed with buffer A, and the sample was eluted with a discontinuous gradient of 0 to 4 M NaCl in the same buffer. Two-milliliter fractions were collected and used to monitor elution of protein and enzyme activity as described. Most active fractions eluting at 0.125 M NaCl, routinely 77 to 87, were pooled and freeze-dried. The freeze-dried sample was resuspended in 1–1.5 ml buffer A containing 1 mM MgCl2, 1 mM CaCl2, and 1.5 M NaCl (buffer B). After centrifugation at low speed to remove undissolved NaCl, the supernatant was applied on a column (0.8 x 6 cm) of Con A Sepharose 4B equilibrated with buffer B. The column was successively washed with buffer B and buffer B supplemented with 0.2 {alpha}MM. Fractions of 1 ml were collected and used to measure protein and enzyme activity.

{alpha}-Mannosidase assay
Two methods were used to determine enzyme activity. In a fluorometric method, the procedure described by Yoshihisa et al. (1988)Go was followed. Briefly, the enzyme fraction (10–100 µg protein) was incubated at 37°C with 40 µM MU{alpha}Man and buffer A in a final volume of 200 µl. After 30 min, the reaction was stopped by adding 3.3 ml 50 mM glycine-NaOH buffer, pH 11, and the fluorescence of released MU was read in a Perkin-Elmer LS-5B luminiscence spectrofluorometer with excitation and emision set at 350 nm and 440 nm, respectively. Activity was expressed as nmoles of MU released in 1 min. Specific activity was referred to 1 mg protein. Alternatively, activity was determined in reaction mixtures containing 1.5 µg purified {alpha}-mannosidase and 2 µg M9, M8B, M6Asn, or M5Asn and buffer A in a total volume of 30 µl. Control mixtures in which the enzyme or the oligosaccharides were omitted were run in parallel. After different periods of incubation at 37°C, the reaction was stopped by heating the mixtures in boiling water for 10 min; after cooling, the volume was adjusted to 60 µl with deionized water. To remove salt and protein and also to separate mannose from manno-oligosaccharides, hydrolyzates were passed through a column (0.3 x 105 cm) of Bio-Gel P-6 precalibrated with bovine serum albumin, M9, M8B, M7, M6Asn, M5Asn, and mannose standards. Products were eluted with deionized water containing 0.02% sodium azide, and fractions of 120 µl were collected. Fractions enriched with oligosaccharides or mannose were pooled, freeze-dried, resuspended in 50 µl deionized water and analyzed by high-performance anion exchange chromatography (HPAEC) as follows.

For oligosaccharide analysis, 25-µl aliquots of samples were injected in a Dionex carbohydrate analyzer equipped with a CarboPac PA-100 column (4.6 x 250 mm), a guard column, and a PED-2 pulse electrochemical detector. The sample was eluted with a linear gradient of 10–100 mM sodium acetate in 100 mM NaOH at a flow rate of 0.8 ml/min for 30 min. After this time, the column was washed with 250 mM sodium acetate in 100 mM NaOH for 10 min and equilibrated with 10 mM sodium acetate in 100 mM NaOH for 10 min prior to the next run. Standards of M9 (1 µg) and M8B, M7, M6Asn, M5Asn (0.5 µg) were run in the same conditions. For mannose analysis, aliquots of 25 µl were processed in the same way except that the sample was eluted with an isocratic gradient of 8 mM NaOH. The column was washed with 200 mM NaOH for 10 min and equilibrated with 8 mM NaOH for 10 min prior to the next run. Oligosaccharide and mannose peaks were plotted and integrated using a Varian 4400 integrator.

Protein determination
Protein was measured by absorbance at 280 nm and by the method of Bradford (1976)Go using bovine serum albumin as standard.

Electrophoresis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was carried out in 10% gels following standard protocols (Laemmli, 1970Go). Proteins were revealed with Coomassie blue according to Merril (1990)Go. For enzyme detection, proteins were separated in gels run in the same conditions except that the enzyme sample was not heated. Gels were washed three times with 1% Triton X-100 in buffer A for 10–15 min at room temperature, immersed in a revealing solution containing 40 µM MU{alpha}Man in the same buffer, and incubated at 37°C. After 60 min, the gel was inspected under UV illumination, and the image was captured in a Stratagene Model Eagle II gel analyzer.


    Acknowledgements
 
We thank Ana Gómez-Villanueva for technical asistance. This work was supported by grant no. CONACyT-2002-CO1–39528/A-1 from Consejo Nacional de Ciencia y Tecnología, Mexico.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: floresca{at}quijote.ugto.mx


    Abbreviations
 
{alpha}MM, {alpha}-methyl D-mannoside; Con A, Concanavalin A; ER, endoplasmic reticulum; HPAEC, high-performance anion exchange chromatography; MU, 4-methylumbelliferone


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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