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Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure, activity and stability
Biochimica et Biophysica Acta 1750 (2005) 61 – 68 http://www.elsevier.com/locate/bba
Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure, activity and stability Javad Jafari-Aghdama, Khosro Khajehb, Bijan Ranjbarb, Mohsen Nemat-Gorgania,c,* b
a Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran Department of Biochemistry, Faculty of Basic Science, Tarbiat Modarres University, P.O. Box 14115-175, Tehran, Iran c Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA
Received 28 December 2004; received in revised form 17 March 2005; accepted 22 March 2005 Available online 15 April 2005
Abstract A comparative structure – function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures. D 2005 Elsevier B.V. All rights reserved. Keywords: Glucoamylase; Irreversible thermoinactivation; Aggregation; Refolding; Deglycosylation
1. Introduction Glycosylation is one of the major naturally occurring modifications of the covalent structure of proteins. Most secretary proteins become glycosylated as soon as the growing polypeptide chains enter the endoplasmic reticulum, before the final native-like folded state is attained. Accordingly, the reasons for the occurrence of such events and their consequences in relation to structure and function of proteins have been investigated extensively. There are two different types of protein glycosylation: O-glycosylation at hydroxyl groups of serine and threonine residues and N-glycosylation at asparagine residues in the consensus sequence of AsnX-Ser/Thr. The biological functions of glycoproteins are well established but the role that carbohydrates play in these * Corresponding author. Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran. Tel.: +1 650 812 1961; fax: +1 650 812 1975. E-mail address: [email protected] (M. Nemat-Gorgani). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.03.011
functions is, for the most part, unclear with contradictory reports frequently observed in the literature [1– 6]. Glucoamylase, also known as amyloglucosidase-EC 3. 2. 1. 3, is an exo-amylase which removes glucose from the non-reducing ends of starch and a variety of other carbohydrate polymers and oligomers [7]. The enzyme extracted from the fungus Aspergillus niger has various industrial applications and is extensively employed in hydrolysis of starch, production of glucose, high fructose syrups and in alcohol fermentation [8,9]. It is believed to break down a (1, 4) bonds more rapidly than a (1, 6). Although in the case of a (1, 6) bonds, the rate of activity is only 0.2% of a (1, 4), even this amount of activity is considered sufficient for the purpose of industrial applications [10 –13]. The enzyme is produced by a variety of microorganisms, with the ones extracted from Aspergillus niger, Aspergillus awamori and Rhizopus oryzae being considered as most important [14]. All enzymes within glucoamylase family consist of a catalytic domain and a starch-binding domain which are attached to each other by means of an O-glycosylation linker [10]. The enzyme
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contains a very specific carbohydrate region consisting of 30 chains in the form of di- or trisaccharides (mainly mannose) [15]. In the present study, GA was chosen as a model glycoprotein and a comparative study was performed using the native protein structure and a deglycosylated preparation obtained by the use of a-mannosidase. Results indicate that upon deglycosylation, flexibility of the enzyme is enhanced and its thermostability diminished. Studies on irreversible thermoinactivation combined with elucidation of the structural properties of the two forms suggest that loss of sugar molecules result in exposure of hydrophobic residues in the protein molecule, thereby facilitating aggregation of the more flexible structure at high temperatures.
2. Materials and methods 2.1. Materials Glucoamylase from Aspergillus niger, a-mannosidase, m-aminophenylboronic acid, concanavalinA-S4B and subtilisin were obtained from Sigma (St. Louis, MO, USA). All other chemicals were obtained from Merck (Darmstadt, Germany) and were reagent grade. 2.2. Determination of enzymatic activity and protein concentration Glucoamylase was assayed colorimetrically at room temperature, using soluble starch as substrate in 16 mM sodium acetate, pH 4.8. Concentration of reducing sugars obtained from the catalyzed reaction was measured by the dinitrosalicylic acid method according to Bernfeld [16]. Protein concentration was determined by the Lowry et al. [17] and Bradford methods [18].
from a m-aminophenylboronic acid affinity column [23] provided a sharp band corresponding to the deglycosylated form (Fig. 1). This preparation was subsequently used as the deglycosylated form in the present work. 2.4. Fluorescence measurements Fluorescence studies was carried out on a Perkin-Elmer luminescence spectrometer LS 50 B. Intrinsic fluorescence was determined using 50 Ag/ml protein and an excitation wavelength of 280 nm. Emission spectra were recorded between 300 and 400 nm. Extrinsic fluorescence studies were carried out as outlined earlier [24 – 26], using ANS (8anilino-1-naphthalene-sulfonate) as a fluorescence probe. All experiments were carried out at 25-C with ANS and protein concentrations of 50 AM and 50 Ag/ml, respectively, in 16 mM sodium acetate buffer. An excitation wavelength of 350 nm was used. 2.5. Fluorescence quenching Fluorescence quenching was carried out by the addition of 2 M acrylamide to protein solutions (50 Ag/ml). Quenching data were analyzed in terms of the Stern-Volmer constant, K sv, which was calculated from the ratio of the unquenched and the quenched fluorescence intensities, F o/F, using the relationship F o/F = 1+K sv [Q]. Here Q is the molar concentration of the quencher [27]. The intrinsic protein fluorescence F was corrected for the acrylamide inner filter effect, f, the latter being defined as f = 10q[Q]/2, using an extinction coefficient q of 4.3 M1 cm1 for acrylamide at 280 nm. 2.6. Proteolytic cleavage
2.3. Deglycosylation of glucoamylase
Proteolytic cleavage of native and deglycosylated forms of GA was performed by treating the enzyme (0.1 mg/ml)
Glucoamylase was treated with a-mannosidase using a 0.1-M sodium acetate buffer (pH 4.5), containing 0.1 mM ZnCl2 and 0.1% (V/V) h-mercaptoethanol, at 37-C for 24 h. Three units of a-mannosidase were used for each 1 mg of GA. The extent of carbohydrate removal was tested by the use of SDS-PAGE as described earlier [15,19,20,21]. The deglycosylated form showed enhanced mobility, presumably due to higher extents of SDS-binding [22]. This was in accord with earlier reports demonstrating a preponderance of carbohydrate moieties in hydrophobic regions of glycoproteins [15]. A greater affinity of the deglycosylated form for interaction with hydrophobic adsorbents further supported this conclusion (Jafari-Aghdam, J. and NematGorgani, M., unpublished data). All attempts involving use of size-exclusion chromatography failed in separating the two forms. Also the fraction of the mannosidase treated preparation which did not bind to ConA Sepharose 4B was not homogeneous on the gel. However, the flow through
Fig. 1. SDS-PAGE of native and deglycosylated GA. Lanes: 1, native; 2, amannosidase; 3, GA digest consisting of native, deglycosylated forms and a-mannosidase; 4, unbound fraction of GA digest on ConA-S4B; 5, unbound fraction of GA digest on m-aminophenylboronic acid affinity matrix. Further details are provided in Materials and methods.
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with various concentrations of subtilisin, using 50 mM borate, 10 mM CaCl2, pH 10. Digestion was carried out at 45-C for 6 h and required volumes were removed from the reaction mixtures for SDS-PAGE. 2.7. Circular dichroism (CD) measurements CD measurements were conducted using a Jasco (Tokyo Japan) J-715 spectropolarimeter equipped with a thermostatically-controlled cell holder. Tm measurement was done using a CD spectropolarimeter (Jasco J-715) employing a protein concentration of 0.1 mg/ml. Thermal unfolding was monitored by recording the change of the CD signal at 222 nm as a function of sample temperature. The rate of increase in temperature was adjusted at 1-C/min. 2.8. Heat stability Native or deglycosylated enzyme preparations (0.1 mg/ ml) in sodium acetate buffer were incubated at different temperatures. At regular intervals, samples were removed cooled on ice and the remaining activity determined. Activity of the same enzyme solution kept on ice throughout the procedure was considered as control (100%). 2.9. pH stability determination A mixed buffer containing acetate, Caps and Tris, each at 40 mM concentration and adjusted to the indicated pH was used. The procedure involved use of 100 Al of free and deglycosylated forms (0.1 mg/ml) which were added to 400 Al of the buffer and incubated for 2 h at 25-C. This was followed by addition of 50 Al of each of the samples to 450 Al of 16 mM acetate buffer, pH 4.8 which were then left for 30 min at room temperature. Activity determination was subsequently carried out in the usual manner. 2.10. Aggregation measurements The extent of aggregation was determined by measuring turbidity at 415 nm as reported earlier [28 –30]. The protein solution (0.1 mg/ml in 16 mM sodium acetate) was incubated at the desired temperature. The increase in absorbance (apparent) against time was plotted using a Shimadzu (UV-160) spectrophotometer. 2.11. Determination of deamidation
3. Results and discussion It is an accepted fact that protein sequence ultimately determines its assigned function. To this end, information on the three-dimensional structure of the macromolecule is necessary in order to unequivocally determine the specific contribution of each of its constituent components to its functional property as a whole. Several roles have been suggested for the carbohydrate moieties of glycoproteins among which stabilization of protein conformation [15], protection from proteolysis [3,5] participation in cell –cell interactions and protein folding [32] are a few examples. However, none of these roles was consistently demonstrated for all glycoproteins. Despite many efforts directed toward elucidation of the role of carbohydrates in glycoproteins, the mechanism of stabilization by glycosylation is not entirely clear. In the present study, GA was treated with alphamannosidase to provide a deglycosylated preparation which was compared with the native form in relation to a number of structure – function properties. 3.1. Structure and function of native and deglycosylated GA No significant changes were observed in the V max, K m, activation energy (E a) and pH optima of native and deglycosylated GA (Table 1). These results indicate that deglycosylation does not affect the active site nor the mechanism of catalysis. In addition, no detectable differences in the far-UV CD spectra of the two forms were found (Fig. 2), thus suggesting that the secondary structure of the native protein is maintained upon loss of sugar molecules. This has also been observed for a GA from a different source than the one used in the present investigation [19]. Circular dichroism spectra (far-UV) of the native and carbohydrate-depleted GA with endo-B-N-acetylglucosaminidase were found to be identical [19]. Another approach taken in this investigation involved intrinsic fluorescence studies. As indicated in Fig. 3, enhancement of fluorescence was observed upon deglycosylation. This would result in a different pattern of conformational changes in the protein structure with respect to deglycosylation, followed by alteration of the microenvironment of excitable tryptophan residues, as supported by an obvious change in the fluorescence intensity (Fig. 3). The Table 1 Kinetic parameters, Stern – Volmer constant and ammonia production for native and deglycosylated GA pH(optim.) K m
Production of ammonia during thermoinactivation of GA was determined by incubating samples of the enzyme in 16 mM sodium acetate buffer and different pH in sealed tubes, at 70-C for 15 min. The tubes were then cooled, opened and the amount of dissolved ammonia was determined enzymatically using glutamate dehydrogenase [27,31].
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GA 4.8 Deglycosylated 4.8 GA
V max
Ea
Ksv
Ammoniaa
1.2 T 0.1 10 T 0.2 0.0414 0.0235 20 T 1.5 1.1 T 0.2 9.7 T 0.3 0.0397 0.0248 26 T 2.5
K m (mg/ml) and V max (Amol/min) were determined at 25-C using different concentrations of starch in 16 mM acetate sodium, pH 4.8, by performing the assays at least in triplicates. K sv and E a are in mol1 and kcal mol1, respectively. For further details, please see Materials and methods. a Amol/L in 70-C (10 min)
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application is dependent on its thermostability. Thermal stabilities of native and deglycosylated forms of the enzyme determined at 70-C and three different pH values are depicted in Fig. 4. As indicated, the native structure is clearly more stable than the deglycosylated form confirming the role of carbohydrate moieties in thermostabilization of the protein structure. A reduction of thermal stability upon removal of carbohydrate was also evident at other temperatures (60 – 65– 75 –80-C, results not shown). Proteins unfold both reversibly and irreversibly, as represented by: N$U!I
Fig. 2. Far-UV spectra of native and deglycosylated forms of glucoamylase. For details, please see Materials and methods.
results of fluorescence quenching experiments allow us to assess the relative solvent exposure of different types of fluorophores. The more exposed a fluorophore is, the more effective a collisional quencher will be in reducing the fluorescence intensity displayed by that molecule [27,33,34]. The Stern –Volmer plot for quenching of intrinsic protein fluorescence by acrylamide at pH 4.5 was prepared and the K sv values were determined (Table 1). The data suggest that the aromatic amino acids are exposed to similar extents in native and deglycosylated forms. 3.2. Effect of deglycosylation on the irreversible thermoinactivation A clear understanding of operational stability constitutes an important goal in enzyme technology today. Since temperature is an important physical variable in enzymecatalyzed reactions, the efforts aimed at elucidation of structure –stability relationships in enzymes on their stabilization have been focused mostly on thermostability. This is especially true for an enzyme such as GA whose potential
Fig. 3. Intrinsic fluorescence of native (N) and deglycosylated (D) glucoamylase. Details are described in Materials and methods.
where N is the native form of the protein, U and I are the reversibly- and irreversibly-unfolded forms, respectively [31,42,46]. I arises from modification of U through events such as aggregation, covalent modification, peptide bond hydrolysis and autooxidation [31,34 –37]. There are many examples in the literature where deglycosylation has clearly altered the UYI reaction [38 – 40]. A GA from Rhizopus niveus was found to be thermally less stable upon deglycosylation [19], as also observed for the GA used in the present study. This was shown by both of these proteins in spite of the fact that the carbohydrate moieties are Olinked in GA from Aspergillus niger and N-linked in the Rhizopus niveus enzyme, and that the two proteins are significantly different in relation to their amino acid and carbohydrate composition [41]. Removal of carbohydrates from human liver a-l-fucosidase did not affect its catalytic activity, or its gross conformation [20]. 3.2.1. Aggregation A substantially higher degree of aggregation was observed at pH 3.0 and 4.8, upon deglycosylation (Fig. 5). This is in line with improvement of surface hydrophobicity of the protein structure as suggested by enhancement of ANS fluorescence (Fig. 6) and increase in affinity for
Fig. 4. Irreversible thermoinactivation of native and deglycosylated forms of glucoamylase at 70-C: Native (?), deglycosylated (>) at pH 3; native (h), deglycosylated (g) at pH 4.8; native (r), deglycosylated (‚) at pH 8. Further details are described under Materials and methods.
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Fig. 5. Aggregation of native and deglycosylated forms of glucoamylase at 70-C: Native (?), deglycosylated (>) at pH 3; native (h), deglycosylated (g) at pH 4.8; native (r), deglycosylated (‚) at pH 8. For further details, please see Materials and methods.
interaction of deglycosylated form with hydrophobic matrices. ANS is essentially non-fluorescent in aqueous solution, whereas its fluorescence increases in a hydrophobic environment. A lower degree of aggregation for the native enzyme would be expected from the presence of hydrophilic sugar components providing repulsive interactions, loss of which could cause aggregation (Fig. 5). Other reasons for enhancement of aggregation are related to a higher proportion of beta-sheet structure of the starch binding site [10]. It has been suggested that the beta structures in glycoproteins show a high tendency toward aggregation and that upon deglycosylation they will be more exposed to the surface [42]. These observations, combined with the fact that sugar molecules are abundant at the hydrophobic regions of the protein molecule, would provide the reasons for the higher aggregation observed upon deglycosylation (Fig. 5). Lack of any detectable aggregation at alkaline pH may be explained in terms of
Fig. 6. ANS Fluorescence of native (N) and deglycosylated (D) forms of glucoamylase. Details are described in Materials and methods.
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repulsive interactions due to a significant difference between the pH at which the experiment was carried out and the pI of the protein (3.5 –3.7), including those contributed by negatively-charged aspartic acid residues [43,44]. Accordingly, inclusion of MgCl2 or CaCl2 at different concentrations was found to enhance this process (data not shown). These observations clearly establish that aggregation, an important mechanism of irreversible thermoinactivation, may indeed be expected to occur more extensively for the deglycosylated enzyme in the course of thermoinactivation. There are a number of reports in the literature describing enhancement of aggregation upon deglycosylation. For example, glucose oxidase from Aspergillus niger and yeast invertase are less soluble and more prone to aggregation after deglycosylation [39]. Also, glycosylation has been shown to improve the solubility of unfolded or partially folded invertase molecules from yeast, leading to suppression of aggregation [45]. Many recombinant versions of human proteins including a-antitrypsin (aAT) undergo aggregation upon storage. This is attributed to lack of carbohydrates in the recombinant protein structures [46,47]. 3.2.2. Deamidation Deamidation of Gln and Asn is an important event which may be involved in thermoinactivation of a protein [48], and which is dependent on pH [49]. Results presented in Table 1 indicate no significant differences in the two forms. This was to be expected since it has been reported that deamidation of Asn and Gln is enhanced if these residues are placed before or after a Ser or a Thr residue in the protein structure [50]. In the process of deglycosylation, sugar molecules are released from Ser or Thr of GA.
Fig. 7. Cleavage of Asp-X bonds in native (1Y6) and deglycosylated (7Y12) forms of glucoamylase (0.1 mg/ml) at pH 3, 4.8 and 8 (as indicated in the Figure), before (B) and after (A) heating for 10 min at 70-C. A mixed buffer containing acetate, Caps and Tris, each at 40 mM concentration and adjusted at the three pH values was used. SDS-PAGE of samples was performed in the usual manner.
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Fig. 8. pH stability of native (h) and deglycosylated (g) forms of glucoamylase. For details, please see Materials and methods.
Fig. 10. Effect of polyols (20%) on aggregation of glucoamylase at 70-C: native (h) and deglycosylated (g) forms in the absence of polyols; native (r) and deglycosylated (‚) forms in the presence of mannose; native (?) and deglycosylated (>) forms in the presence of trehalose.
3.3. pH stability of native and deglycosylated forms of GA However, none of the Asn or Gln residues in this enzyme is found adjacent to a Thr or a Ser. 3.2.3. Cleavage of Asp-X bonds Asp-X bonds are known to be very labile at elevated temperatures [51]. Cleavage of these bonds may therefore be another cause of thermoinactivation in a protein molecule [49,51]. As indicated (Fig. 7), treatment of the two forms of GA result in breaking up of the structure, significantly more for the deglycosylated enzyme than for the native form, in line with previous reports [e.g., 37]. These results were not unexpected since Asp is abundantly found in GA and predominantly at the protein surface [10,37]. Furthermore, loss of sugar molecules may occur with a concomitant loss of a number of structurally-important hydrogen bonds between the sugar molecules themselves or the sugar molecules and protein backbone structure, thereby leading to enhanced flexibility.
Fig. 9. Effect of polyols (20%) on irreversible thermoinactivation of glucoamylase at 70-C: native (h) and deglycosylated (g) forms in the absence of polyols; native (r) and deglycosylated (‚) forms in the presence of mannose; native (?) and deglycosylated (>) forms in the presence of trehalose.
The deglycosylated form clearly showed higher stabilities in alkaline conditions (Fig. 8). Accordingly, while the native form lost almost all activity at pH 12, about half of the activity was recovered for the deglycosylated protein. 3.4. Irreversible thermoinactivation and the effect of polyols Irreversible thermoinactivation and aggregation of native and deglycosylated forms of GA in the presence of 20% (W/V) of mannose and trehalose are depicted in Figs. 9 and 10. As shown, these polyols afford protection for both forms, presumably by being preferentially excluded from the protein surface [52]. Sorbitol behaved similarly. It has been suggested that these additives may strengthen hydrophobic interactions among non-polar amino acid residues, thereby making them more resistant to unfolding and thermal denaturation. The cohesive force of sugars responsible for the increase in the surface tension of water is also suggested to be an important factor. Accordingly, prefer-
Fig. 11. Proteolytic cleavage of native (1Y4) and deglycosylated (5Y8) forms of glucoamylase (0.5 mg/ml) by subtilisin, used at zero (1 and 5), 0.3 (2 and 6), 0.6 (3 and 7) and 1 (4 and 8) mg/ml. Further details are described in Materials and methods.
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ential interaction of proteins with solvent components, in aqueous sugar systems, may take place concomitant with stabilization [53]. 3.5. Proteolytic cleavage by subtilisin As indicated in Fig. 11, the deglycosylated form was more sensitive to proteolysis by subtilisin than the native enzyme. This may be due to a direct hindrance of approach provided by the sugar molecules and/or enhanced flexibility as a result of deglycosylation. While deglycosylation did not affect the stability of saposin B [54] toward proteolysis, hen ovomucoid and low-density-lipoprotein receptor were found more sensitive toward proteolytic digestion [55] upon deglycosylation. In conclusion, results presented in this communication provide some mechanistic features related to the role of carbohydrate moieties in stabilization of a glycoprotein. They also suggest that this property cannot be generalized and each observation should be analyzed in terms of specific structural property in relation to the function exhibited by the protein molecule.
Acknowledgement This work was supported by a grant from the Research Council, University of Tehran.
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[30] A. Guagliardi, L. Cerchia, M. Rossi, Prevention of in vitro protein thermal aggregation by the sulfolobus solfataricus chaperonin. Evidence for nonequivalent binding surfaces on the chaperonin molecule, J. Biol. Chem. 270 (1995) 28126 – 28132. [31] S. Zale, A.M. Klibanov, Why does ribonuclease irreversibly inactive at high temperature? Biochemistry 25 (1986) 5432 – 5444. [32] N. Kojima, M. Shiota, Y. Sadahira, K. Handa, S. Hakamori, Cell adhesion in a dynamic flow system as compared to static system. Glycosphingolipid – glycosphingolipid interaction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B16 melanoma cells to non-activated endothelial cells, J. Biol. Chem. 267 (1992) 17264 – 17270. [33] P.G. Varley, R.H. Pain, Relation between stability, dynamics and enzyme activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus, J. Mol. Biol. 220 (1991) 531 – 538. [34] S. d Amico, J.C. Marx, C. Gerday, G. Feller, Activity – stability relationships in extremophilic enzymes, J. Biol. Chem. 278 (2003) 7891 – 7896. [35] T. Ahern, A.M. Klibanov, The mechanisms of irreversible enzyme inactivation at 100-C, Science 228 (1985) 1280 – 1284. [36] K. Khajeh, H. Naderi-Manesh, B. Ranjbar, A. Moosavi-Movahedi, M. Nemat-Gorgani, Chemical modification of lysine residues in Bacillus a-amylases: effect on activity and stability, Enzyme Microb. Technol. 28 (2001) 543 – 549. [37] O. Munch, D. Tritsch, Irreversible thermoinactivation of glucoamylase from Aspergillus niger and thermoinactivation by chemical modification of carboxyl groups, Biochim. Biophys. Acta 1041 (1990) 111 – 116. [38] G. Williamson, N.J. Belshaw, T.R. Noel, S.G. Ring, M.P. Williamson, O-glycosylation and stability, unfolding of glucoamylase induced by heat and guanidine hydrochloride, Eur. J. Biochem. 207 (1992) 661 – 670. [39] K. Takegawa, K. Fujiwara, S. Iwahara, K. Yamamoto, T. Tochikura, Effect of deglycosylation Of N-linked sugar chains on glucose oxidase from Aspergillus niger, Biochem. Cell. Biol. 67 (1989) 460 – 464. [40] J.X. Gu, T. Matsuda, R. Nakamura, H. Ishiguro, M. Sasaki, N. Takahashi, Chemical deglycosylation of hen ovomucoid: protective effect of carbohydrate moiety on tryptic hydrolysis and heat denaturation, J. Biochem. (Tokyo) 106 (1989) 66 – 70. [41] J.H. Pazur, B.L. Liu, F.J. Miskiel, Comparison of the properties of glucoamylases from Rhizopus niveus and Aspergillus niger, Biotechnol. Appl. Biochem. 12 (1990) 63 – 78.
[42] A.L. Fink, Protein aggregation: folding aggregates, inclusion bodies and amyloid, Fold 3 (1998) R9 – R23. [43] A.S. Vandersall, R.G. Cameron, C.J. Nairn, G. Yelenosky, R.J. Wodzinski, Identification, characterization and partial purification of glucoamylase from Aspergillus niger (syn A. ficuum) NRRL 3135, Prep. Biochem. 25 (1995) 29 – 55. [44] E. Wenisch, P. Schneider, S.A. Hansen, R. Rezzonico, P.G. Righetti, Isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes, exemplified by purification of glucoamylase, J. Biochem. Biophys. Methods 27 (1993) 199 – 213. [45] N. Schulke, F.X. Schmid, Effect of glycosylation on the mechanism of renaturation of invertase from yeast, J. Biol. Chem. 263 (1988) 8832 – 8837. [46] K. Kwon, M. Hee Yu, Effect of glycosylation on the stability of aantitrypsin toward urea denaturation and thermal deactivation, Biochem. Biophys. Acta 335 (1996) 265 – 272. [47] S. Vemuri, C.T. Yu, N. Roosdorp, Formulation and stability of recombinant alpha 1-antitrypsin, Pharm. Biotechnol. 5 (1993) 263 – 286. [48] S.J. Tomazic, A.M. Klibanov, Mechanisms of irreversible thermal inactivation of Bacillus a-amylases, J. Biol. Chem. 263 (1988) 3086 – 3091. [49] A.M. Klibanov, Stabilization of enzymes against thermal inactivation, Adv. Appl. Microb. 29 (1983) 1 – 28. [50] H.T. Wright, Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins, Crit. Rev. Biochem. Mol. Biol. 26 (1991) 1 – 52. [51] A.S. Inglis, Cleavage at aspartic acid, Methods Enzymol. 91 (1984) 332 – 342. [52] T. Arakawa, S.N. Timasheff, Stabilization of protein structure by sugars, Biochemistry 21 (1982) 6536 – 6544. [53] J.C. Lee, K. Gekko, S.N. Timasheff, Measurements of preferential solvent interactions by densimetric techniques, Methods Enzymol. 61 (1976) 26 – 49. [54] M. Hiraiwa, S. Soeda, B.M. Martin, A. Fluharty, Y. Hirabayashi, J. Brien, Y. Kishimoto, The effect of carbohydrate removal on stability and activity saposin B, Arch. Biochem. Biophys. 303 (1993) 326 – 331. [55] K. Kozarsky, D. Kingsley, M. Krieger, Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4335 – 4339.
Deglycosylation of glucoamylase from Aspergillus niger: Effects on structure, activity and stability Javad Jafari-Aghdama, Khosro Khajehb, Bijan Ranjbarb, Mohsen Nemat-Gorgania,c,* b
a Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran Department of Biochemistry, Faculty of Basic Science, Tarbiat Modarres University, P.O. Box 14115-175, Tehran, Iran c Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA
Received 28 December 2004; received in revised form 17 March 2005; accepted 22 March 2005 Available online 15 April 2005
Abstract A comparative structure – function study was performed to establish possible roles of carbohydrates in stabilization of glycoproteins, using glucoamylase (GA) as a model system. In addition to kinetic properties, stability toward elevated temperatures, extremes of pH, high salt concentrations together with circular dichroism, intrinsic/extrinsic fluorescence studies, proteolysis and affinity for interaction with hydrophobic ligands were investigated. Related to all the main properties examined, with one exception, glycosylation provided improvement in functional characteristics of the enzyme, especially in relation to its thermostability. Results are explained in terms of provision of stabilizing intermolecular interactions by the sugar molecules. The improvement in protein rigidity together with reduction of surface hydrophobicity appear to be especially important in relation to prevention of aggregation, an important mechanism of irreversible thermoinactivation, occurring at elevated temperatures. D 2005 Elsevier B.V. All rights reserved. Keywords: Glucoamylase; Irreversible thermoinactivation; Aggregation; Refolding; Deglycosylation
1. Introduction Glycosylation is one of the major naturally occurring modifications of the covalent structure of proteins. Most secretary proteins become glycosylated as soon as the growing polypeptide chains enter the endoplasmic reticulum, before the final native-like folded state is attained. Accordingly, the reasons for the occurrence of such events and their consequences in relation to structure and function of proteins have been investigated extensively. There are two different types of protein glycosylation: O-glycosylation at hydroxyl groups of serine and threonine residues and N-glycosylation at asparagine residues in the consensus sequence of AsnX-Ser/Thr. The biological functions of glycoproteins are well established but the role that carbohydrates play in these * Corresponding author. Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran. Tel.: +1 650 812 1961; fax: +1 650 812 1975. E-mail address: [email protected] (M. Nemat-Gorgani). 1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.03.011
functions is, for the most part, unclear with contradictory reports frequently observed in the literature [1– 6]. Glucoamylase, also known as amyloglucosidase-EC 3. 2. 1. 3, is an exo-amylase which removes glucose from the non-reducing ends of starch and a variety of other carbohydrate polymers and oligomers [7]. The enzyme extracted from the fungus Aspergillus niger has various industrial applications and is extensively employed in hydrolysis of starch, production of glucose, high fructose syrups and in alcohol fermentation [8,9]. It is believed to break down a (1, 4) bonds more rapidly than a (1, 6). Although in the case of a (1, 6) bonds, the rate of activity is only 0.2% of a (1, 4), even this amount of activity is considered sufficient for the purpose of industrial applications [10 –13]. The enzyme is produced by a variety of microorganisms, with the ones extracted from Aspergillus niger, Aspergillus awamori and Rhizopus oryzae being considered as most important [14]. All enzymes within glucoamylase family consist of a catalytic domain and a starch-binding domain which are attached to each other by means of an O-glycosylation linker [10]. The enzyme
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contains a very specific carbohydrate region consisting of 30 chains in the form of di- or trisaccharides (mainly mannose) [15]. In the present study, GA was chosen as a model glycoprotein and a comparative study was performed using the native protein structure and a deglycosylated preparation obtained by the use of a-mannosidase. Results indicate that upon deglycosylation, flexibility of the enzyme is enhanced and its thermostability diminished. Studies on irreversible thermoinactivation combined with elucidation of the structural properties of the two forms suggest that loss of sugar molecules result in exposure of hydrophobic residues in the protein molecule, thereby facilitating aggregation of the more flexible structure at high temperatures.
2. Materials and methods 2.1. Materials Glucoamylase from Aspergillus niger, a-mannosidase, m-aminophenylboronic acid, concanavalinA-S4B and subtilisin were obtained from Sigma (St. Louis, MO, USA). All other chemicals were obtained from Merck (Darmstadt, Germany) and were reagent grade. 2.2. Determination of enzymatic activity and protein concentration Glucoamylase was assayed colorimetrically at room temperature, using soluble starch as substrate in 16 mM sodium acetate, pH 4.8. Concentration of reducing sugars obtained from the catalyzed reaction was measured by the dinitrosalicylic acid method according to Bernfeld [16]. Protein concentration was determined by the Lowry et al. [17] and Bradford methods [18].
from a m-aminophenylboronic acid affinity column [23] provided a sharp band corresponding to the deglycosylated form (Fig. 1). This preparation was subsequently used as the deglycosylated form in the present work. 2.4. Fluorescence measurements Fluorescence studies was carried out on a Perkin-Elmer luminescence spectrometer LS 50 B. Intrinsic fluorescence was determined using 50 Ag/ml protein and an excitation wavelength of 280 nm. Emission spectra were recorded between 300 and 400 nm. Extrinsic fluorescence studies were carried out as outlined earlier [24 – 26], using ANS (8anilino-1-naphthalene-sulfonate) as a fluorescence probe. All experiments were carried out at 25-C with ANS and protein concentrations of 50 AM and 50 Ag/ml, respectively, in 16 mM sodium acetate buffer. An excitation wavelength of 350 nm was used. 2.5. Fluorescence quenching Fluorescence quenching was carried out by the addition of 2 M acrylamide to protein solutions (50 Ag/ml). Quenching data were analyzed in terms of the Stern-Volmer constant, K sv, which was calculated from the ratio of the unquenched and the quenched fluorescence intensities, F o/F, using the relationship F o/F = 1+K sv [Q]. Here Q is the molar concentration of the quencher [27]. The intrinsic protein fluorescence F was corrected for the acrylamide inner filter effect, f, the latter being defined as f = 10q[Q]/2, using an extinction coefficient q of 4.3 M1 cm1 for acrylamide at 280 nm. 2.6. Proteolytic cleavage
2.3. Deglycosylation of glucoamylase
Proteolytic cleavage of native and deglycosylated forms of GA was performed by treating the enzyme (0.1 mg/ml)
Glucoamylase was treated with a-mannosidase using a 0.1-M sodium acetate buffer (pH 4.5), containing 0.1 mM ZnCl2 and 0.1% (V/V) h-mercaptoethanol, at 37-C for 24 h. Three units of a-mannosidase were used for each 1 mg of GA. The extent of carbohydrate removal was tested by the use of SDS-PAGE as described earlier [15,19,20,21]. The deglycosylated form showed enhanced mobility, presumably due to higher extents of SDS-binding [22]. This was in accord with earlier reports demonstrating a preponderance of carbohydrate moieties in hydrophobic regions of glycoproteins [15]. A greater affinity of the deglycosylated form for interaction with hydrophobic adsorbents further supported this conclusion (Jafari-Aghdam, J. and NematGorgani, M., unpublished data). All attempts involving use of size-exclusion chromatography failed in separating the two forms. Also the fraction of the mannosidase treated preparation which did not bind to ConA Sepharose 4B was not homogeneous on the gel. However, the flow through
Fig. 1. SDS-PAGE of native and deglycosylated GA. Lanes: 1, native; 2, amannosidase; 3, GA digest consisting of native, deglycosylated forms and a-mannosidase; 4, unbound fraction of GA digest on ConA-S4B; 5, unbound fraction of GA digest on m-aminophenylboronic acid affinity matrix. Further details are provided in Materials and methods.
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with various concentrations of subtilisin, using 50 mM borate, 10 mM CaCl2, pH 10. Digestion was carried out at 45-C for 6 h and required volumes were removed from the reaction mixtures for SDS-PAGE. 2.7. Circular dichroism (CD) measurements CD measurements were conducted using a Jasco (Tokyo Japan) J-715 spectropolarimeter equipped with a thermostatically-controlled cell holder. Tm measurement was done using a CD spectropolarimeter (Jasco J-715) employing a protein concentration of 0.1 mg/ml. Thermal unfolding was monitored by recording the change of the CD signal at 222 nm as a function of sample temperature. The rate of increase in temperature was adjusted at 1-C/min. 2.8. Heat stability Native or deglycosylated enzyme preparations (0.1 mg/ ml) in sodium acetate buffer were incubated at different temperatures. At regular intervals, samples were removed cooled on ice and the remaining activity determined. Activity of the same enzyme solution kept on ice throughout the procedure was considered as control (100%). 2.9. pH stability determination A mixed buffer containing acetate, Caps and Tris, each at 40 mM concentration and adjusted to the indicated pH was used. The procedure involved use of 100 Al of free and deglycosylated forms (0.1 mg/ml) which were added to 400 Al of the buffer and incubated for 2 h at 25-C. This was followed by addition of 50 Al of each of the samples to 450 Al of 16 mM acetate buffer, pH 4.8 which were then left for 30 min at room temperature. Activity determination was subsequently carried out in the usual manner. 2.10. Aggregation measurements The extent of aggregation was determined by measuring turbidity at 415 nm as reported earlier [28 –30]. The protein solution (0.1 mg/ml in 16 mM sodium acetate) was incubated at the desired temperature. The increase in absorbance (apparent) against time was plotted using a Shimadzu (UV-160) spectrophotometer. 2.11. Determination of deamidation
3. Results and discussion It is an accepted fact that protein sequence ultimately determines its assigned function. To this end, information on the three-dimensional structure of the macromolecule is necessary in order to unequivocally determine the specific contribution of each of its constituent components to its functional property as a whole. Several roles have been suggested for the carbohydrate moieties of glycoproteins among which stabilization of protein conformation [15], protection from proteolysis [3,5] participation in cell –cell interactions and protein folding [32] are a few examples. However, none of these roles was consistently demonstrated for all glycoproteins. Despite many efforts directed toward elucidation of the role of carbohydrates in glycoproteins, the mechanism of stabilization by glycosylation is not entirely clear. In the present study, GA was treated with alphamannosidase to provide a deglycosylated preparation which was compared with the native form in relation to a number of structure – function properties. 3.1. Structure and function of native and deglycosylated GA No significant changes were observed in the V max, K m, activation energy (E a) and pH optima of native and deglycosylated GA (Table 1). These results indicate that deglycosylation does not affect the active site nor the mechanism of catalysis. In addition, no detectable differences in the far-UV CD spectra of the two forms were found (Fig. 2), thus suggesting that the secondary structure of the native protein is maintained upon loss of sugar molecules. This has also been observed for a GA from a different source than the one used in the present investigation [19]. Circular dichroism spectra (far-UV) of the native and carbohydrate-depleted GA with endo-B-N-acetylglucosaminidase were found to be identical [19]. Another approach taken in this investigation involved intrinsic fluorescence studies. As indicated in Fig. 3, enhancement of fluorescence was observed upon deglycosylation. This would result in a different pattern of conformational changes in the protein structure with respect to deglycosylation, followed by alteration of the microenvironment of excitable tryptophan residues, as supported by an obvious change in the fluorescence intensity (Fig. 3). The Table 1 Kinetic parameters, Stern – Volmer constant and ammonia production for native and deglycosylated GA pH(optim.) K m
Production of ammonia during thermoinactivation of GA was determined by incubating samples of the enzyme in 16 mM sodium acetate buffer and different pH in sealed tubes, at 70-C for 15 min. The tubes were then cooled, opened and the amount of dissolved ammonia was determined enzymatically using glutamate dehydrogenase [27,31].
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GA 4.8 Deglycosylated 4.8 GA
V max
Ea
Ksv
Ammoniaa
1.2 T 0.1 10 T 0.2 0.0414 0.0235 20 T 1.5 1.1 T 0.2 9.7 T 0.3 0.0397 0.0248 26 T 2.5
K m (mg/ml) and V max (Amol/min) were determined at 25-C using different concentrations of starch in 16 mM acetate sodium, pH 4.8, by performing the assays at least in triplicates. K sv and E a are in mol1 and kcal mol1, respectively. For further details, please see Materials and methods. a Amol/L in 70-C (10 min)
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application is dependent on its thermostability. Thermal stabilities of native and deglycosylated forms of the enzyme determined at 70-C and three different pH values are depicted in Fig. 4. As indicated, the native structure is clearly more stable than the deglycosylated form confirming the role of carbohydrate moieties in thermostabilization of the protein structure. A reduction of thermal stability upon removal of carbohydrate was also evident at other temperatures (60 – 65– 75 –80-C, results not shown). Proteins unfold both reversibly and irreversibly, as represented by: N$U!I
Fig. 2. Far-UV spectra of native and deglycosylated forms of glucoamylase. For details, please see Materials and methods.
results of fluorescence quenching experiments allow us to assess the relative solvent exposure of different types of fluorophores. The more exposed a fluorophore is, the more effective a collisional quencher will be in reducing the fluorescence intensity displayed by that molecule [27,33,34]. The Stern –Volmer plot for quenching of intrinsic protein fluorescence by acrylamide at pH 4.5 was prepared and the K sv values were determined (Table 1). The data suggest that the aromatic amino acids are exposed to similar extents in native and deglycosylated forms. 3.2. Effect of deglycosylation on the irreversible thermoinactivation A clear understanding of operational stability constitutes an important goal in enzyme technology today. Since temperature is an important physical variable in enzymecatalyzed reactions, the efforts aimed at elucidation of structure –stability relationships in enzymes on their stabilization have been focused mostly on thermostability. This is especially true for an enzyme such as GA whose potential
Fig. 3. Intrinsic fluorescence of native (N) and deglycosylated (D) glucoamylase. Details are described in Materials and methods.
where N is the native form of the protein, U and I are the reversibly- and irreversibly-unfolded forms, respectively [31,42,46]. I arises from modification of U through events such as aggregation, covalent modification, peptide bond hydrolysis and autooxidation [31,34 –37]. There are many examples in the literature where deglycosylation has clearly altered the UYI reaction [38 – 40]. A GA from Rhizopus niveus was found to be thermally less stable upon deglycosylation [19], as also observed for the GA used in the present study. This was shown by both of these proteins in spite of the fact that the carbohydrate moieties are Olinked in GA from Aspergillus niger and N-linked in the Rhizopus niveus enzyme, and that the two proteins are significantly different in relation to their amino acid and carbohydrate composition [41]. Removal of carbohydrates from human liver a-l-fucosidase did not affect its catalytic activity, or its gross conformation [20]. 3.2.1. Aggregation A substantially higher degree of aggregation was observed at pH 3.0 and 4.8, upon deglycosylation (Fig. 5). This is in line with improvement of surface hydrophobicity of the protein structure as suggested by enhancement of ANS fluorescence (Fig. 6) and increase in affinity for
Fig. 4. Irreversible thermoinactivation of native and deglycosylated forms of glucoamylase at 70-C: Native (?), deglycosylated (>) at pH 3; native (h), deglycosylated (g) at pH 4.8; native (r), deglycosylated (‚) at pH 8. Further details are described under Materials and methods.
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Fig. 5. Aggregation of native and deglycosylated forms of glucoamylase at 70-C: Native (?), deglycosylated (>) at pH 3; native (h), deglycosylated (g) at pH 4.8; native (r), deglycosylated (‚) at pH 8. For further details, please see Materials and methods.
interaction of deglycosylated form with hydrophobic matrices. ANS is essentially non-fluorescent in aqueous solution, whereas its fluorescence increases in a hydrophobic environment. A lower degree of aggregation for the native enzyme would be expected from the presence of hydrophilic sugar components providing repulsive interactions, loss of which could cause aggregation (Fig. 5). Other reasons for enhancement of aggregation are related to a higher proportion of beta-sheet structure of the starch binding site [10]. It has been suggested that the beta structures in glycoproteins show a high tendency toward aggregation and that upon deglycosylation they will be more exposed to the surface [42]. These observations, combined with the fact that sugar molecules are abundant at the hydrophobic regions of the protein molecule, would provide the reasons for the higher aggregation observed upon deglycosylation (Fig. 5). Lack of any detectable aggregation at alkaline pH may be explained in terms of
Fig. 6. ANS Fluorescence of native (N) and deglycosylated (D) forms of glucoamylase. Details are described in Materials and methods.
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repulsive interactions due to a significant difference between the pH at which the experiment was carried out and the pI of the protein (3.5 –3.7), including those contributed by negatively-charged aspartic acid residues [43,44]. Accordingly, inclusion of MgCl2 or CaCl2 at different concentrations was found to enhance this process (data not shown). These observations clearly establish that aggregation, an important mechanism of irreversible thermoinactivation, may indeed be expected to occur more extensively for the deglycosylated enzyme in the course of thermoinactivation. There are a number of reports in the literature describing enhancement of aggregation upon deglycosylation. For example, glucose oxidase from Aspergillus niger and yeast invertase are less soluble and more prone to aggregation after deglycosylation [39]. Also, glycosylation has been shown to improve the solubility of unfolded or partially folded invertase molecules from yeast, leading to suppression of aggregation [45]. Many recombinant versions of human proteins including a-antitrypsin (aAT) undergo aggregation upon storage. This is attributed to lack of carbohydrates in the recombinant protein structures [46,47]. 3.2.2. Deamidation Deamidation of Gln and Asn is an important event which may be involved in thermoinactivation of a protein [48], and which is dependent on pH [49]. Results presented in Table 1 indicate no significant differences in the two forms. This was to be expected since it has been reported that deamidation of Asn and Gln is enhanced if these residues are placed before or after a Ser or a Thr residue in the protein structure [50]. In the process of deglycosylation, sugar molecules are released from Ser or Thr of GA.
Fig. 7. Cleavage of Asp-X bonds in native (1Y6) and deglycosylated (7Y12) forms of glucoamylase (0.1 mg/ml) at pH 3, 4.8 and 8 (as indicated in the Figure), before (B) and after (A) heating for 10 min at 70-C. A mixed buffer containing acetate, Caps and Tris, each at 40 mM concentration and adjusted at the three pH values was used. SDS-PAGE of samples was performed in the usual manner.
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Fig. 8. pH stability of native (h) and deglycosylated (g) forms of glucoamylase. For details, please see Materials and methods.
Fig. 10. Effect of polyols (20%) on aggregation of glucoamylase at 70-C: native (h) and deglycosylated (g) forms in the absence of polyols; native (r) and deglycosylated (‚) forms in the presence of mannose; native (?) and deglycosylated (>) forms in the presence of trehalose.
3.3. pH stability of native and deglycosylated forms of GA However, none of the Asn or Gln residues in this enzyme is found adjacent to a Thr or a Ser. 3.2.3. Cleavage of Asp-X bonds Asp-X bonds are known to be very labile at elevated temperatures [51]. Cleavage of these bonds may therefore be another cause of thermoinactivation in a protein molecule [49,51]. As indicated (Fig. 7), treatment of the two forms of GA result in breaking up of the structure, significantly more for the deglycosylated enzyme than for the native form, in line with previous reports [e.g., 37]. These results were not unexpected since Asp is abundantly found in GA and predominantly at the protein surface [10,37]. Furthermore, loss of sugar molecules may occur with a concomitant loss of a number of structurally-important hydrogen bonds between the sugar molecules themselves or the sugar molecules and protein backbone structure, thereby leading to enhanced flexibility.
Fig. 9. Effect of polyols (20%) on irreversible thermoinactivation of glucoamylase at 70-C: native (h) and deglycosylated (g) forms in the absence of polyols; native (r) and deglycosylated (‚) forms in the presence of mannose; native (?) and deglycosylated (>) forms in the presence of trehalose.
The deglycosylated form clearly showed higher stabilities in alkaline conditions (Fig. 8). Accordingly, while the native form lost almost all activity at pH 12, about half of the activity was recovered for the deglycosylated protein. 3.4. Irreversible thermoinactivation and the effect of polyols Irreversible thermoinactivation and aggregation of native and deglycosylated forms of GA in the presence of 20% (W/V) of mannose and trehalose are depicted in Figs. 9 and 10. As shown, these polyols afford protection for both forms, presumably by being preferentially excluded from the protein surface [52]. Sorbitol behaved similarly. It has been suggested that these additives may strengthen hydrophobic interactions among non-polar amino acid residues, thereby making them more resistant to unfolding and thermal denaturation. The cohesive force of sugars responsible for the increase in the surface tension of water is also suggested to be an important factor. Accordingly, prefer-
Fig. 11. Proteolytic cleavage of native (1Y4) and deglycosylated (5Y8) forms of glucoamylase (0.5 mg/ml) by subtilisin, used at zero (1 and 5), 0.3 (2 and 6), 0.6 (3 and 7) and 1 (4 and 8) mg/ml. Further details are described in Materials and methods.
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ential interaction of proteins with solvent components, in aqueous sugar systems, may take place concomitant with stabilization [53]. 3.5. Proteolytic cleavage by subtilisin As indicated in Fig. 11, the deglycosylated form was more sensitive to proteolysis by subtilisin than the native enzyme. This may be due to a direct hindrance of approach provided by the sugar molecules and/or enhanced flexibility as a result of deglycosylation. While deglycosylation did not affect the stability of saposin B [54] toward proteolysis, hen ovomucoid and low-density-lipoprotein receptor were found more sensitive toward proteolytic digestion [55] upon deglycosylation. In conclusion, results presented in this communication provide some mechanistic features related to the role of carbohydrate moieties in stabilization of a glycoprotein. They also suggest that this property cannot be generalized and each observation should be analyzed in terms of specific structural property in relation to the function exhibited by the protein molecule.
Acknowledgement This work was supported by a grant from the Research Council, University of Tehran.
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