Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris

Mark A. Yoshimasu2, Takuji Tanaka3, Jong-Kun Ahn4 and Rickey Y. Yada1,2

2 Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1; 3 Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8; 4 Department of Agricultural Science, Korea National Open University,169, Dongsung-Dong, Chongro-Ku, Seoul, 110-791, Korea

Received on August 1, 2003; revised on October 16, 2003; accepted on October 21, 2003


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar Km values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.

Key words: conformational stability / glycosylation / pepsin / structure–function relationship


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycosylation, a posttranslational process in which carbohydrate residues are covalently attached as the protein is being processed, has been found to facilitate proper folding, prevent protein aggregation, protect against proteolytic attack, and act as surface cell receptors and targeting signals (Imperiali and Rickert, 1995Go; Matthews, 1993Go; Paulson, 1990Go; Rademacher et al., 1988Go), however, specific roles appear to be protein-dependent. For some glycoproteins, N-linked oligosaccharides are necessary for overall stability (Hasagawa et al., 1999Go; Wang et al., 1996Go), whereas for others the presence of the carbohydrate chains is only required during the folding process (Letourneur et al., 2001Go). Some proteins fold more efficiently without carbohydrates, whereas partial removal of the oligosaccharide chains is essential for others (Helenius, 1994Go). For glycoproteins containing multiple N-linked motifs, some sites have more impact on the stability and function than others (Haraguchi et al., 1995Go; McGinnes and Morrison, 1995Go; Newrzella and Stoffel, 1996Go). On the other hand, removal of the N-linked sites has occasionally had little effect on structure and function (Levy et al., 1998Go). For example, staphylokinase, an enzyme that contains the N-linked motif but is not found in a naturally glycosylated state, was expressed from a eukaryotic system in a glycosylated form, and in this case, glycosylation was shown to have no effect on the structure, stability, or activity (Miele et al., 1999Go).

Much of the research on the role of glycosylation has been devoted to naturally glycosylated proteins. Using site-directed mutagenesis and various expression systems, more systematic analysis of individual N-linked carbohydrate chains on glycoproteins has been achieved. It has allowed researchers to study multiple N-linked sites to determine the impact each oligosaccharide has on the structure and function of the glycoprotein (Haraguchi et al., 1995Go; McGinnes and Morrison, 1995Go; Newrzella and Stoffel, 1996Go). Conversely, little research has been conducted on the effect of glycosylation on nonglycosylated proteins. Kato and co-workers (Nakamura et al., 1993Go; Shu et al., 1998Go) engineered N-linked sites into lysozyme with an intent to improve its emulsifying properties.

Porcine pepsin A (EC 3.4.23.1), a nonglycosylated protein, belongs to the aspartic protease family. Aspartic proteinases are bilobal proteins with each lobe contributing a catalytic aspartic acid residue located at the center of the binding cleft between the domains (Andreeva et al., 1984Go; Cooper et al., 1990Go; Sielecki et al., 1990Go). Due to the catalytic aspartate residues, the active pH ranges between 1.0 and 5.0. The structure of porcine pepsin and its zymogen pepsinogen have been determined and are highly homogenous to other aspartic proteinases (Andreeva et al., 1984Go; Baldwin et al., 1993Go; Blundell et al., 1990Go; Pedersen and Foltmann, 1973Go; Sepulveda et al., 1975Go; Wlodawer and Erickson, 1993Go). Enzyme specificity and kinetics, activation mechanism of pepsinogen, catalysis, and inhibition of pepsin have been well documented (Dunn et al., 1988Go; Kageyama and Takahashi, 1983Go; Kay, 1985Go; McPhie, 1975Go; Tang, 1971Go; Tang and Wong, 1987Go). Because pepsin and pepsinogen have been well characterized, they represent an ideal model to study the effects of glycosylation on structure, function, and stability.

Recently, a eukaryotic system capable of posttranslational modification was developed for the expression of porcine pepsinogen (Yoshimasu et al., 2002Go). This system provides an excellent means to study the effect of N-linked glycosylation on the structure and function of a protein. Through site-directed mutagenesis, the asparagine-x-serine/threonine motif was engineered into four sites (two on the N-terminal domain and two on the C-terminal domain) and the various glycosylated pepsins were analyzed for changes in structure (secondary and tertiary), enzyme activity, substrate specificity, and stability (pH, temperature).


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Expression and purification of glycosylated recombinants
Through site-directed mutagenesis, the N-linked motif was successfully incorporated into four specific sites on the pepsinogen gene (Table I). Transformation of each plasmid led to successful expression of four different mutants. Each mutant migrated slightly higher in molecular weight compared to commercial pepsinogen indicating possible glycosylation. Two bands were detected for each mutant that could have resulted from (1) possible N- and O-linked glycosylation because O-linked carbohydrates were detected in the wild type (Yoshimasu et al., 2002Go), or (2) heterogeneous N-linked glycosylation because heterogeneous glycosylation (i.e., oligosaccharides varying in the number of sugar residues) has been observed with P. pastoris (Kang et al., 1998Go; Montesino et al., 1998Go, 1999Go). To confirm the presence of N-linked oligosaccharides, each mutant was treated with N-glycosidase F, an enzyme that cleaves the bond between the GlcNAc and Asn, liberating the entire sugar chain. After treatment with N-glycosidase F, a single band corresponding to the molecular weight of commercial pepsinogen was detected for each mutant (Figure 1). Because N-glycosidase F is specific for only N-linked glycosylation, this would suggest that two N-linked glycoforms were present for each mutant. Heterogeneous glycosylation through the P. pastoris expression system has been reported by several researchers (Kang et al., 1998Go; Montesino et al., 1998Go, 1999Go) with experiments being conducted on the mixture of glycosylated variants or glycoforms (Nakakubo et al., 2000Go; Pratap et al., 2000Go). A number of chromatographic techniques were attempted to separate the two forms but were unsuccessful. This would indicate that the two isoforms for each mutant share similar structural and biochemical characteristics. Thus all experiments were conducted on a mixture of the two glycoforms forms for each mutant.


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Table I. Incorporation of N-linked motif into primary structure of pepsinogen

 


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Fig. 1. Western blot of recombinant pepsins treated with N-glycosidase F. Lanes (1) standard, (2) commercial pepsinogen, (3) mutant 77, (4) deglycosylated mutant 77, (5) mutant 110, (6) deglycosylated mutant 110, (7) mutant 244, (8) deglycosylated mutant 244, (9) mutant 281, (10) deglycosylated mutant 281. Approximately 0.1 mg protein per well was applied for each sample.

 
Structural characterization of mutants
Far- and near-UV scans using CD spectroscopy were conducted on each mutant to analyze its secondary and tertiary structure, respectively. The far-UV spectra of the active form for each mutant are presented in Figure 2. All mutants exhibited similar spectra with a strong negative ellipticity around the 218 nm range. The spectra generally overlapped with the wild type for all mutants, which suggests that the secondary structure was not altered. Tertiary structure was analyzed in the near-UV region (Figure 3). The spectra for all mutants appeared to overlap with the wild type, which was not unexpected because it was thought that these mutations would induce a more local change. These findings are in agreement with other studies that found that glycosylation did not alter the secondary and tertiary structure of the staphylokinase, porcine pepsin, RNAase, and invertase (Acosta et al., 2000Go; Joao et al., 1992Go; Miele et al., 1999Go; Tanaka et al., 1998Go).



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Fig. 2. Far-UV CD spectra of wild-type and glycosylated pepsins. (A) represents mutants 77 and 110 and (B) represents mutants 244 and 281. Samples were scanned four times using the JASCO 600 spectropolarimeter at 25°C in 20 mM sodium acetate buffer, pH 5.2. Final spectra obtained were the average of the four scans minus the buffer. Analysis was performed in triplicate using 0.2 mg/mL.

 


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Fig. 3. Near-UV CD spectra of wild-type and glycosylated pepsins. (A) represents mutants 77 and 110 and (B) represents mutants 244 and 281. Samples were scanned four times using the JASCO 600 spectropolarimeter at 25°C in 20 mM sodium acetate buffer, pH 5.2. Final spectra obtained were the average of the four scans minus the buffer. Analysis was performed in triplicate using 1.0 mg/mL.

 
Conformational stability of mutants
Conformational stability of each mutant was determined by monitoring the unfolding equilibrium using the denaturant guanidinium chloride and analyzed based on the transitional phase in the denaturation curve. For wild-type pepsin, the sigmoidal change observed was steep and narrow and occurred at Gdn-HCl concentrations of 1.75 and 2.7 M (Figure 4). A similar denaturation range was observed with mutant 110. This narrow, steep transition phase translated into apparent {Delta}G(H2O) values of 5.42 ± 0.36 and 5.59 ± 0.49 kcal/mol for wild type and mutant 110, respectively. Mutants 77, 244, and 288 also exhibited the typical sigmoidal change, however, the denaturation range using Gdn-HCl was from 2.0 to 3.6 M, which led to slightly larger apparent {Delta}G(H2O) values of 6.79 ± 0.81, 6.39 ± 0.33, and 6.85 ± 0.20, respectively (Figures 4 and 5).



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Fig. 4. Conformational stability of wild type and mutants. Gdn-HCl induced unfolding transitions (A) of mutant 77 (dots), mutant 110 (diamonds), and wild type (line). Denaturation was followed using enzyme activity and absorbance (300 nm). The graph is modeled based on a two-state derivation of {Delta}G assuming a linear dependence of {Delta}G on Gdn-HCl concentration. (B) shows the influence of denaturant concentration on the change in free energy of denaturation in the transition state.

 


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Fig. 5. Conformational stability of wild type and mutants. Gdn-HCl induced unfolding transitions (A) mutant 244 (triangles), mutant 281 (boxes), and wild type (line). Denaturation was followed using enzyme activity and absorbance (300 nm). The graph is modeled based on a two-state derivation of {Delta}G assuming a linear dependence of {Delta}G on Gdn-HCl concentration. (B) shows the influence of denaturant concentration on the change in free energy of denaturation in the transition state.

 
Kinetic characterization of glycosylated and nonglycosylated mutants
Table II summarizes the kinetic analysis of each mutant at pH 2.0. The Km values for the mutants were not statistically significant (p >= 0.05) from the wild type, however, kcat values for each mutant were approximately one-third those of the wild type.


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Table II. Kinetic analysis of glycosylated and nonglycosylated pepsins at pH 2.0

 
To promote N-linked glycosylation in pepsinogen, a change in primary structure was required. To ensure that any differences observed between glycosylated mutants and wild type were due to N-linked glycosylation and not from changes in the primary structure, a nonglycosylated form of each mutant was produced through the addition of the antibiotic tunicamycin during expression. Tunicamycin blocks the enzyme required to initiate N-linked glycosylation in the Golgi network. Nonglycosylated forms of each mutant were expressed, purified and analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blot (data not shown). The relative mobility and molecular mass of each nonglycosylated mutant corresponded with the wild type, which would indicate that the mutants expressed were not glycosylated. Kinetic constants were measured at pH 2.0 for each nonglycosylated mutant (Table II). Both Km and kcat values were similar (p > 0.05) to those observed with the wild type; thus any differences detected between the wild type and glycosylated mutants were attributed to the presence of the carbohydrate chain and not to changes in the primary structure.

Effect of pH on activation of mutants
Figure 6 represents the effect of pH on activation of the wild type and glycosylated mutants. The wild type exhibited almost 100% relative activity between pH 1.0 to 3.0 with loss of activity above pH 4.0, indicating that the majority of pepsinogen was activated at pH values equal to or less than 3.5. For all mutants except 77, activation occurred up to pH 5.5 with 60–80% relative activity observed at pH 4.0. For mutant 77, an activation profile was similar to other mutants, however, very little activation occurred between pH 5.0 and 6.0 (less than 5% relative activity).



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Fig. 6. Effect of pH on activation of glycosylated pepsins. Samples were incubated at various pH values for 30 min, quenched at pH 8.0 (overnight, 4°C) and remaining pepsinogen activated at pH 1.0 for 30 min. The sample was buffered to pH 5.3 and hydrolysis of the synthetic substrate (Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu) measured at 300 nm. The relative activity was expressed as a percentage of the highest activity over the pH range examined. Each data point represents the mean of three determinations. (A) represents mutant 77 (dots), mutant 110 (diamonds), and wild type (line), and (B) represents mutant 244 (triangles), mutant 281 (squares), and wild type (line).

 
Effect of pH on activity and stability of mutants
Enzyme activity for each mutant was investigated over a broad pH range (Figure 7). The wild type exhibited between 70% and 100% relative activity up to pH 5.0, followed by a sharp decrease with no activity at pH 6.0. Mutants 244 and 281, for which glycosylation was located on the C-domain, behaved in a similar manner to the wild type, however, some activity was observed between pH 6.0 and 6.5 (<5% relative activity). Mutant 77 exhibited between 90 and 100% relative activity from pH 1.0 to pH 3.0, which slowly declined to pH 5.0 and showed no activity beyond pH 5.5. Approximately 40–50% relative activity was initially observed with mutant 110, however, activity peaked at pH 3.5 and then quickly declined with no activity observed at 5.5.



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Fig. 7. Effect of pH on activity of glycosylated pepsins. Hydrolysis of the synthetic substrate was measured at 300 nm over the various pH values. The relative activity was expressed as a percentage of the highest activity over the pH range examined. Each data point represents the mean of three determinations. (A) represents mutant 77 (dots), mutant 110 (diamonds), and wild type (line), and (B) represents mutant 244 (triangles), mutant 281 (boxes), and wild type (line).

 
The effect of pH on enzyme stability was also examined on the active form. The wild type was stable up to pH 6.5; however, when incubated at pH 7.0, activity was not observed (data not shown). A small amount of activity was detected at pH 7.0 for mutants 77 and 110 (~5%), however, activity was not detected beyond pH 7.5. Mutants 244 and 281 displayed a slightly higher relative activity at pH 7.5 (~15%) with a small amount of activity observed at pH 8.0 (<5% relative activity).

Thermal stability of mutants
Differential scanning calorimetry (DSC) of wild-type pepsin resulted in thermal profiles containing two peaks. Melting points (Tm) of 38.8 ± 1.2°C and 66.2 ± 0.7°C were detected for the wild type (Yoshimasu et al., 2002Go). Similar two-peak thermal profiles were obtained with each mutant, however, the melting point(s) for some of the peaks had increased in temperature (Table III).


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Table III. Thermodynamic characteristics of mutant pepsins

 
The effect of temperature on enzyme stability based on activity was also examined for each mutant (Figure 8). The wild type, including all mutants, exhibited 100% enzyme activity at 25°C, however, as temperature increased, a steady decline was observed. At 60°C, less than 5% activity was observed with the wild type, however, all mutants exhibited activity ranging from 20% to 60%. At 65°C, a small amount of activity was detected with each mutant (5–10%).



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Fig. 8. Effect of temperature on stability of glycosylated pepsins. Samples were incubated for 10 min at each temperature and cooled to 4°C; hydrolysis of the synthetic substrate was measured at 300 nm. The relative activity was expressed as a percentage of the highest activity over the pH range examined. Each data point represents the mean of three determinations. (A) represents mutant 77 (dots), mutant 110 (diamonds), and wild type (line), and (B) represents mutant 244 (triangles), mutant 281 (boxes), and wild type (line).

 
Cleavage profile of mutants
Hydrolytic specificity of each glycosylated mutant was investigated using oxidized insulin B-chain. Pepsin has broad cleavage specificity; however, the catalytic aspartates have demonstrated a preference for hydrophobic residues (Powers et al., 1977Go). Analysis of high-performance liquid chromatography (HPLC) chromatograms for each mutant over a 24-h period indicated similar digestion profiles compared with the wild type (data not shown); thus substrate specificity was not altered.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
P. pastoris was an adequate expression system to study the effects of N-linked glycosylation on the aspartic proteinase pepsin. Four recombinant pepsinogen proteins glycosylated at different sites were expressed and purified, however, heterogeneous glycosylation occurred during expression.

Conformational stability of mutants
Enzyme activity can be considered the most sensitive probe to study changes in conformational structure because subtle changes in the active site due to small conformational variations can be detected. The sigmoidal change observed for both wild type and mutant 110 were similar. This would indicate that glycosylation at position 110 did not influence the conformational stability of the structure. In contrast, mutants 77, 244, and 288 exhibited the same sigmoidal change, however, the denaturation range was from 2.0 to 3.6 M Gdn-HCl. This would indicate that glycosylation on the C-domain lead to a slight increase in conformational stability of the enzyme. We propose that glycosylation has decreased flexibility or added rigidity to the structure, which in turn has added conformational stability. Several research groups have concluded that glycosylation added stability to the structure via decreased flexibility (Live et al., 1996Go; Mer et al., 1996Go; Rudd et al., 1994Go; Yanez et al., 1998Go). More specifically, the two GlcNAc and first mannose residue on the carbohydrate chain have been found to interact with nearby amino acid side chains, either through direct steric interactions or possible local ordering of solvents around the hydroxyl groups on the oligosaccharide core (Wormald et al., 1991Go; Wyss et al., 1995Go; Yanez et al., 1998Go). Furthermore, Wyss et al. (1995)Go found that a single GlcNAc residue was sufficient to add stability to the structure. Similar findings were observed with the GalNAc residue in O-glycosylated proteins (Davis et al., 1994Go; Gerken et al., 1989Go).

Pepsin is composed of two similar domains contributing a catalytic aspartate located at the bottom of the cleft between the domains (Andreeva et al., 1984Go; Sielecki et al., 1990Go). A flexible "flap" loop located on the N-domain spans across the active site to the C-domain (Abad-Zapatero et al., 1990Go). For mutant 77, which is glycosylated on the flexible loop, it is postulated that the carbohydrate chain is interacting with the C-domain and that this interaction has a stabilizing effect on the overall conformation of the protein through steric interactions. Steric interactions between the sugar residues and protein structure have been reported to have stabilizing effects in other glycosylated proteins (Gerken et al., 1989Go; Rudd et al., 1994Go).

Kinetic characterization of glycosylated and nonglycosylated mutants
Kinetic analysis of each mutant (Table II) revealed a decrease in kcat values compared with the wild type. This decrease in kcat has been observed by other researchers who analyzed the effects of glycosylation on enzyme activity (Haraguchi et al., 1995Go; Newrzella and Stoffel, 1996Go; Yanez et al., 1998Go). In pepsin, the cleft is large enough to accommodate seven to eight amino acids (Andreeva et al., 1984Go). Within the binding site are a number of subsites that align the substrate into position for cleavage that are thought to maximize binding between enzyme and substrate, thus increasing cleavage efficiency (Dunn et al., 1995Go). These subsites have been created through the interaction of amino acid side chains from both the N- and C-terminal domain as well as the flap loop. We have proposed that the presence of the N-linked oligosaccharide chain has made the protein structure more rigid, which in turn has influenced the position and role of the subsites. Adjustments in the subsites would affect the cleavage and formation of new bonds, which in turn would affect the free energy of the enzyme (Nelson and Cox, 2000Go). Thus once the substrate is in position, the affinity would not be altered between the substrate and enzyme (Km) but a change would occur in the conversion rate from enzyme–substrate to enzyme plus product (kcat). This was observed in mutants 110, 244, and 288.

The flap loop covering the active site is flexible (Abad-Zapatero et al., 1990Go). Residues Tyr75, Thr77, and Ser79, located at the tip of the flap, interact with the substrate forming subsites S1 and S2 (Hartsuck et al., 1992Go; Sielecki et al., 1990Go; Tanaka et al., 1998Go). Gly76 has also been shown to participate in hydrogen bonding to the substrate and has been suggested to be involved in its stabilization (James and Sielecki, 1985Go; Okoniewska et al., 2000Go; Pearl, 1987Go). For mutant 77, where glycosylation was located at the tip of this loop, it is postulated that the presence of the carbohydrate chain has altered the mobility of the loop. Thus once the substrate is in position, subtle changes in the position of residues 75, 76, 77, and 79 (subsites S1 and S2) would lead to changes in kcat.

Effect of pH on activation of mutants
Under acidic conditions, activation of pepsinogen occurs through destabilization of electrostatic interactions between basic residues in the prosegment and acidic residues in the active enzyme portion (James and Sielecki, 1986Go). As the pH shifts from a neutral to acidic environment, the acidic residues become protonated, however, the protein structure is able to compensate for these internal interactions. As the pH drops below 4.0, the protein is unable to maintain its zymogenic form. The N- and C-terminal domains of pepsinogen undergo structural rearrangement; the prosegment unfolds from the cleft and is cleaved forming pepsin (Abad-Zapatero et al., 1990Go; Sali et al., 1992Go; Sielecki et al., 1990Go). It is proposed that increased rigidity due to carbohydrate residues on the C-domain may hinder the ability of the structure to adjust to changes in pH. As pH changed from an alkaline to acidic environment, the rigidified structure could not compensate as readily to fluctuations in electrostatic interactions between the prosegment and domains. This loss in flexibility led to activation at a higher pH (below 6.0), which was observed with mutants 110, 244, and 281. For mutant 77, activation at a higher pH may have been due to a shift in position of the flexible loop over the active site. This disruption in position could affect the accessibility of the active site to environmental conditions. Because the prosegment rests in the active site, disruption of the loop could disturb the interaction between the prosegment and the N- and C-domain, leading to premature activation of the enzyme.

Effect of pH on activity and stability of mutants
As discussed, a rigidified structure might prolong the unfolding process, thus a glycosylated protein would be more resistant to pH denaturation compared to a nonglycosylated form. Glycosylation on the C-domain (mutants 244 and 281) displayed a similar pH activity profile to the wild type; however, low levels of relative enzyme activity were detected beyond pH 6.0 (Figure 7). This residual amount of activity past pH 6.0 could be attributed to increased stability of the structure, which was confirmed by higher {Delta}G(H2O) values compared with the wild type and an increase in melting point of both domains in the thermal stability studies. Mutants 77 and 110 displayed much different pH profiles compared with the wild type. Mutant 77 exhibited between 80% and 100% relative activity up to pH 3.0; however, it declined steadily with loss of activity after pH 5.5, unlike the wild type, which had ~20% relative activity at pH 5.5 (Figure 7).

As discussed earlier, it was proposed that glycosylation on the loop led to disruption in its position over the active site. This disturbance in position may have led to partial destabilization of the N-domain and loss of enzyme activity as pH approached 5.0. For mutant 110, less then 50% relative activity was observed between pH 1.0 and 2.5 and activity rapidly increased to ~100% at pH 3.5 and steadily declined as pH increased to 5.5. We propose that as the pH approached 3.5, flexibility was added to the structure through the initial stages of destabilization of the N-domain, which would account for the increase in activity. However, as pH continued to increase, the N-domain quickly began to denature similar to mutant 77 with loss of activity at pH 5.0. Conformational studies indicated that glycosylation did not affect stability of this mutant. Based on these results, it would appear that the N-terminal domain was more easily disrupted compared to the C-terminal domain.

At pH values below 6.0, pepsin is largely stabilized by hydrophobic interactions (Fruton, 1960Go). If glycosylation has rigidified the structure, it would help maintain the conformation of the protein, which in turn would help sustain the hydrophobic interactions between the N- and C-domain (Privalov et al., 1981Go). At pH 7.0, ~15–20% activity was observed with mutants 244 and 281 (glycosylation on C-domain), and trace amounts of activity was observed up to pH 8.0. Because activity was demonstrated with these mutants, it would suggest that the N-domain was still intact because the active site is composed of both the N- and C-domain. Conversely, only a slight amount of activity was detected with mutant 110 (~8% relative activity) at pH 7.0, which would indicate that the rigidity imposed by the oligosaccharides on the N-domain was not enough to maintain its structure for activity compared to mutant 244 and 281. The {Delta}G(H2O) value of mutant 110 was similar to the wild type, which would imply no increase in conformational stability; however, thermal stability studies did indicate an increase in structural stability of only the N-domain. This increase in stability of the N-domain could account for the slight activity observed with mutant 110 at pH 7.0. Although the {Delta}G(H2O) value for mutant 77 was higher than that for the wild type, thereby indicating greater conformational stability, results from enzyme activation and enzyme activity did not reflect this increased stability. It would, however, imply that pH did have an influence on the position of the glycosylated loop. Thus mutant 77 did not show any pH stability at neutral or alkaline conditions. Results from this portion of the study would indicate that glycosylation on the C-domain had an influence on the structural stability of the N-domain which would support the results obtained from both conformational and thermal stability studies for mutants 244 and 281.

Thermal stability of mutants
Differential scanning calorimetry of wild-type pepsin resulted in thermal profiles containing two peaks. Melting points (Tm) of 38.8 ± 1.2°C and 66.2 ± 0.7°C were observed (Yoshimasu et al., 2002Go). Two distinct denaturation peaks for pepsin were also observed by Privalov and co-workers (1981)Go, who concluded that each lobe acted more or less as an independent substructure that behaved as individual cooperative units in a melting process. The authors also concluded that the N-terminal domain denatured at lower temperatures due to its flexibility. Similar thermal profiles were obtained with each mutant (data not shown); however, different melting points for each peak were obtained (Table III). The increase in thermal stability observed with the glycosylated mutants could be attributed to the increased rigidity imposed by the N-linked oligosaccharide. This rigidified structure would restrict the mobility of the N- and C-domain as it begins to unfold, thus stabilizing the structure. This was observed with mutants 244 and 281, where peaks representing the C-terminal domain increased ~3°C compared with the wild type. The increase in rigidity of the C-domain may have helped maintain the conformation of both N- and C-domains, which in turn helped maintain the hydrophobic interactions between the domains (Privalov et al., 1981Go). This would account for the increase in thermal stability observed in the N-domain (5–15°C) for mutants 244 and 281. Stabilization of the C-domain was not as prominent with mutant 110, however, a 10°C increase in Tm of the N-terminal domain was observed, which would indicate an increase in structural stability. An increase in melting point of both domains was observed with mutant 77. As proposed earlier, the oligosaccharide chain on the loop may be interacting with the lobe of the C-domain through steric interactions. This interaction with the C-domain may have helped maintain the overall structure and stability. Thus these results would support the findings from the conformation study and provide more detail on the increased structural stability of only the N-domain for mutant 110.

Results from the study on temperature and enzyme stability based on activity would support the hypothesis that glycosylation was able to increase thermal stability for each mutant. However, for mutant 110, which showed no conformational stability based on its {Delta}G(H2O) value compared to the wild type, DSC results demonstrated an increase in structural stability of only the N-domain. This could account for the slight increase in thermal stability based on enzyme activity. Yasuda et al. (1999)Go and Pratap et al. (2000)Go observed an increase in thermal stability in a glycosylated form of cathepsin E and streptokinase, respectively. They concluded that the N-linked glycosylation added rigidity to the structure, which led to an increase in thermal stability.

Several studies have been conducted on the role of glycosylation and its impact on increased thermal stability of naturally glycosylated proteins (Tang et al., 2001Go; Wang et al., 1996Go). Through nuclear magnetic resonance, Rudd et al. (1994)Go discovered that glycosylation caused a decrease in dynamic fluctuations throughout the entire molecule, which led to an increase in thermal stability. Even in the presence of heterogeneous carbohydrates, the cooperativity of the unfolding transition was not altered (DeKoster and Robertson, 1997Go; Wang et al., 1996Go).

Conclusion
The present study indicated that N-linked glycosylation had no effect on the secondary and tertiary structures of the various pepsinogen mutants but did influence their activity and stability. It is believed that the presence of the carbohydrate residues added rigidity to the structure, which had different effects on the function and stability but was site-dependent. Glycosylation affected activation of pepsinogen and its activity and stability under different pH conditions. Glycosylation on the C-domain led to a more global stabilization of the structure at different pH and temperature conditions, whereas glycosylation on the N-domain had more of a stabilizing affect on that portion of the protein. However, glycosylation on the flexible loop led to an increase in temperature stability and structural stability of both domains.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
The Pichia expression kit containing the host strain P. pastoris KM71 (arg4 his4 aox1: ARG4), Escherichia coli TOP10F' and plasmid vector pHIL-S1 was purchased from Invitrogen (San Diego, CA). QIAquick Gel Extraction and polymerase chain reaction (PCR) purification kits were purchased from Qiagen (Mississauga, ON). N-glycosidase F, Pwo polymerase, DNA ligase, and restriction enzymes were obtained from Roche Biochemical (Laval, QC). Goat anti-rabbit IgG-alkaline phosphatase was purchased from BioRad (Hercules, CA). DEAE Sepharose Fast Flow and Concanavalin A Sepharose 4B were obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). Schiff's reagent, porcine pepsinogen, methyl-{alpha}-D-mannopyranoside, and oxidized insulin B-chain were purchased from Sigma (St. Louis, MO). Oligonucleotide primers and the synthetic octapeptide substrate Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu [Phe(NO2) is p-nitrophenylalanine] were synthesized at the Institute of Molecular Biotechnology at McMaster University (Hamilton, ON) and the Core Facility for Protein/DNA Chemistry at Queen's University (Kingston, ON), respectively. All other reagents were of the highest grade commercially available for biochemical use.

Cloning and mutagenesis
The pepsinogen gene in plasmid pPSP2000 (Yoshimasu et al., 2002Go) was subcloned into the EcoR I site of vector pHIL-S1. The following oligonucleotide primers were synthesized for site-directed mutagenesis. The engineered sites for the N-linked motif are underscored with the base changes from the original sequence highlighted in bold.

Oligonucleotide primers used for amplification of the pepsinogen gene were as follows:

XhoI-F: 5'-CGC TCG AGT ATT CAT GC-3'
EcoR-F: 5'-CAG GAA GAA TTC CTT GAG G-3'
R-primer: 5'-TCA GAA TCC TCT ACA TGG AGG-3'

One PCR reaction was carried out using primers EcoR-F and R-primer and a second reaction using XhoI-F and one of the primers incorporating the N-linked sequence. Each PCR reaction was carried out for 25 cycles. The former reaction produced a complete fragment and the latter produced a partial fragment containing the mutation. Fragments were isolated and extracted from a 1% agarose gel using the QIAquick gel extraction kit. Both fragments were combined with 1 x PCR buffer, 2 mL 10 mM dNTP, and sterile water up to 100 mL and heated at 94°C for 10 min. The reaction was then cooled to 37°C over 60 min and held for an additional 10 min. One unit of Pwo polymerase was added to the mixture, heated for 3 min at 72°C, cooled to 4°C, and 1 mL 20 pmol primers XhoI-F and R-primer was added. A final 25-cycle PCR reaction was completed and the final fragment purified. The newly formed fragment along with the pHIL-S1 vector was digested with Xho I and EcoR I for 2 h at 37°C, ligated, and transformed into E. coli TOP10F' using calcium chloride competent cells (Tomley, 1996Go). Transformants containing the pHIL-S1 plasmid were screened on Luria Bertani agar containing ampicillin (150 mg/mL). Plasmid DNA was purified and PCR analysis completed using a set of AOX primers (supplied with the Pichia kit). The AOX1 site was located upstream of the Xho I site. The amplified DNA fragment was then sequenced to confirm the nucleotide changes (Laboratory Services Division, University of Guelph).

Confirmed isolates were linearized with Sal I, purified, and transformed into the P. pastoris genome through electroporation according to the manufacturer's instructions (Invitrogen). Transformants were screened on minimal dextrose plates without histidine to ensure pure clonal isolates. Cultures were examined for extracellular expression as will be described, and pepsinogen was detected through western blotting. Genomic DNA from P. pastoris integrants was extracted and PCR analysis completed using primers supplied by the kit to confirm the presence of the insert. Nucleotide sequence analysis was also performed on the PCR product (Laboratory Services Division, University of Guelph).

Extracellular expression of pepsinogen
A recombinant clone of P. pastoris was inoculated into 2 mL minimal dextrose media and incubated at 30°C in a gyratory platform shaker at 200 rpm overnight. The culture was transferred into 1 L buffered glycerol-complex media in a 2-L baffled flask and incubated in an environmental shaker at 30°C, 200 rpm for 24 h. Cells were harvested by centrifugation at 5000 x g for 5 min at room temperature, resuspended in 1 L buffered methanol-complex medium (BMMY) and incubated for an additional 72 h with the addition of 0.75% methanol after every 24 h to maintain induction conditions. The culture was centrifuged at 5000 x g for 5 min at room temperature, and the supernatant was harvested.

For expression of nonglycosylated recombinant proteins, tunicamycin (20 mg/mL) was incorporated into BMMY media prior to the addition of the culture. Cultures followed the same incubation protocol as described.

Purification of recombinant pepsinogen
Ammonium sulfate was slowly added to the supernatant to 60% saturation and stored overnight at 4°C. The protein solution was centrifuged at 15,000 x g for 20 min at 4°C, the pellet resuspended in 20 mM Tris–HCl buffer, pH 7.5, and dialyzed (12,000 molecular weight cut-off) overnight at 4°C. The protein was applied onto a DEAE Sepharose CL-6B column (5 cm x 25 cm; flow rate 1.5 mL/min), washed with 20 mM Tris–HCl buffer, pH 7.5, and bound protein eluted using a linear gradient of 0.15–0.5 M NaCl solution. Fractions were collected, fractionated on SDS–PAGE (12.5% gel), and analyzed via western blotting using anti-pepsinogen polyclonal antibodies. Fractions containing recombinant pepsinogen were pooled, concentrated using a stirred ultrafiltration cell, model 8200 (Millipore, Bedford, MA) with a 30,000 molecular weight cut-off membrane (Millipore) and applied onto a 10 mL Concanavalin A Sepharose 4B column (2 cm x 10 cm syringe barrel). The column was washed with 20 mM Tris–HCl buffer, pH 7.5, containing 0.5 M NaCl, and bound glycosylated pepsinogen was eluted with 20 mM Tris–HCl buffer, pH 7.5, containing 0.4 M methyl {alpha}-D-mannopyranoside. Elution buffer containing the glycosylated recombinant pepsinogen was concentrated and washed with 20 mM Tris–HCl, pH 7.5, using a Centriplus-30 filter device (Millipore). Concentrates were filtered through a 13-mm-diameter (0.22 µm pore size) nitrocellulose syringe filter and protein concentration determined using the BioRad DC assay. Standard curves were generated using commercial pepsinogen (Sigma). Purified recombinant pepsinogen was stored at –20°C in the presence of 50% glycerol.

Deglycosylation using N-glycosidase F
Deglycosylation of glycosylated pepsinogen was carried out using N-glycosidase F. Briefly, 5 mg pepsinogen was mixed with 0.25 M sodium phosphate buffer, pH 7.2, containing 0.5% SDS and brought up to a final volume of 20 µL. The mixture was heated for 5 min at 100°C, cooled to room temperature, and divided into two 10-µL aliquots. Each aliquot was mixed with 20 µL 0.25 M sodium phosphate buffer, pH 7.2, containing 2% Triton X-100. One unit of N-glycosidase F was added to one of the aliquots. Samples were incubated overnight at 30°C, heated for 5 min at 100°C in the presence of 2.5% SDS and applied onto SDS–PAGE followed by western blotting.

Activation of pepsinogen
Activation was initiated by mixing 0.1 mg/mL of recombinant pepsinogen with 0.1 volumes of 0.8 M HCl and incubating for 1 h at room temperature. The sample was buffered to pH 5.3 through the addition of 0.1 volumes of 1 M sodium acetate, pH 5.3, and the prosegment was removed using a Centricon YM30 filtration unit (Millipore). The buffer was exchanged with 20 mM sodium acetate, pH 5.3, during filtration and protein concentration determined through the BioRad DC assay.

pH dependence of activation
Recombinant pepsinogen was diluted to 20 nM in 20 mM Tris–HCl buffer, pH 7.5, and mixed with an equal volume of 100 mM sodium citrate buffer at pH 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0. After incubation for 30 min at room temperature, activation was terminated through the addition of 0.1 volumes of 1.0 M Tris–HCl buffer, pH 8.0, and stored overnight at 4°C to denature any activated pepsin. The remaining pepsinogen was activated through the addition of 0.2 volumes of 0.8 M HCl for 60 min at room temperature and buffered to pH 5.3 through the addition of 0.1 volumes of 1.5 M sodium acetate, pH 5.3. The activity of each mixture was determined in triplicate using a synthetic octapeptide substrate, Lys-Pro-Ala-Glu-Phe-Phe(NO2)-Ala-Leu diluted to 0.1 mM in 100 mM citrate buffer, as described in the Kinetic measurements section.

pH optimum for activity and stability
To evaluate the effect of pH on activity, activated recombinant pepsin was diluted to 20 nM and activity determined with the synthetic substrate mentioned (0.1 mM in 100 mM sodium citrate) at the following pH values: 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0. To assess pH stability, the active sample was diluted to 20 nM and incubated with 2 volumes of 0.4 M sodium citrate at pH 5.0, 5.5, 6.0, and 6.5 and 0.4 M phosphate buffer at pH 7.0 and 7.5 for 10 min. After incubation, samples were adjusted to pH 5.3 with 0.2 volumes of 1.5 M sodium acetate, pH 5.3, and activity determined with the synthetic substrate (0.1 mM in 100 mM sodium citrate, pH 2.5).

Temperature stability
To assess temperature stability, activated recombinant pepsin was diluted to 20 nM and 100-µL aliquots were heated at 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75°C for 10 min. Mineral oil was placed on top of the enzyme solution to prevent evaporation. Samples were then placed on ice and activity determined with the synthetic substrate (0.1 mM in 100 mM sodium citrate, pH 2.5).

Gdn-HCl denaturation
Grade I Gdn-HCl (Sigma) was twice recrystallized according to the method of Nozaki (1972)Go and molar concentration determined using a refractive index technique (Nozaki, 1972Go). Activated enzyme sample was diluted to 250 nM in 0.1 M sodium acetate, pH 5.3, in the presence of increasing concentrations of Gdn-HCl and incubated for 9 h at 25°C. After incubation, samples were diluted to 50 nM in 0.1 M sodium acetate, pH 5.3, and activity determined using the synthetic substrate (0.1 mM in 100 mM sodium citrate, pH 2.5).

Using enzyme activity as an index for denaturation, the native fraction, fN, was determined by

where yU, yN, and y were the relative enzyme activity of the denatured enzyme, native enzyme, and enzyme equilibrated in Gdn-HCl, respectively. Activity for the denatured enzyme (yU) was determined though linear extrapolation of the posttransition region. Because enzyme activity in the pretransition region was considered to be nonlinear due to increased enzyme activity in the presence of low concentrations of Gdn-HCl (Pain et al., 1985Go), a baseline of slope equal to zero at the threshold value was used to approximate yN. The equilibrium constant, K, and the free energy change, {Delta}G, were calculated from the transition region.


Values of K within the range 0.1 to 10 were used in the analysis of the transition phase (Tanford, 1968Go). The limiting values of transition were determined through regression analysis of the line given by

where m is a measure of the dependence of {Delta}G on Gdn-HCl concentration and {Delta}G(H2O) is the free energy change in the absence of denaturant.

Kinetic measurements
Kinetic measurements were performed on a DU640 spectrophotometer (Beckman Instruments, Fullerton, CA). For the synthetic substrate, a change in absorbency was measured at 300 nm in 100 mM sodium citrate buffer, pH 2.0 (Lin et al., 1992Go). A minimum of 10 synthetic substrate concentrations ranging from 0.01 to 0.25 mM were used to determine initial rates. The enzyme was activated and diluted to 20 nM in 20 mM sodium acetate buffer, pH 5.3; kinetic experiments performed immediately. Initial slopes of progress curve were measured to give {Delta}A/min. A nonlinear least-squares fit of the data was used to calculate the kinetic constants Km and Vmax (Sakoda and Hiromi, 1976Go). To calculate kcat, protein concentration was determined through pepstatin titration. All results were done in triplicate.

Circular dichroism spectroscopy
Far-UV CD spectra were generated using 200 µL 0.2 mg/mL active enzyme sample in 20 mM sodium acetate, pH 5.3, with a Jasco 600 spectropolarimeter (Japan Spectroscopic, Tokyo). Samples were placed in a 0.1-cm path length cuvette and scanned four times from 190 to 250 nm at room temperature under continuous nitrogen flush. For near-UV CD, 1 mL 1.0 mg/mL activated enzyme was placed in a 1.0 cm pathlength cuvette and scanned four times from 320 to 240 nm. All samples and buffers were filtered with a 13 mm diameter (0.22 µm pore size) nitrocellulose syringe filter and degassed prior to analysis. Molar ellipticity was calculated based on the method of Yada and Nakai (1986)Go using a mean residue weight of 107 for pepsin.

Differential scanning calorimetry
Calorimetric measurements were conducted on a MicroCal MC-2 differential scanning calorimeter (MicroCal, Northhampton, MA) using 0.5 mg/mL active enzyme sample. Samples were diluted in 20 mM sodium acetate, pH 5.3 and scanned from 20 to 80°C at a heating rate of 1.5°C/min. All buffers were degassed prior to use. Data analysis was performed using the Origin DSCITC software (MicroCal).

SDS–PAGE and western blotting
SDS–PAGE was performed according to the method of Laemmli (1970)Go using a Mini-Protean II Electrophoresis Cell (BioRad). Protein samples were dissolved in sample buffer in the presence of ß-mercaptoethanol, heated for 5 min at 100°C, and loaded onto a 12.5% polyacrylamide gel. Gels were run at a constant current (20 mA/slab gel), after which they were either developed or transferred onto a polyvinylidene difluoride membrane (BioRad) for western blotting according to the method of Towbin et al. (1979)Go. Electroblotting was carried out overnight at constant voltage (10 V) on a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad).

Hydrolytic specificity
The hydrolytic specificity of recombinant pepsin was based on the cleavage profile of oxidized insulin B-chain (Kervinen et al., 1993Go). Briefly, 100 mg oxidized insulin B-chain was dissolved in 1 mL 0.1 M sodium citrate, pH 2.5, and incubated at 37°C with activated recombinant pepsin at a ratio of 100:1 w/w. Aliquots were taken at 1, 3, 7, and 24 h and placed at –28°C to stop the reaction. Samples were then analyzed by HPLC using a Bondapak C18 column (Waters, Milford, MA) with a linear gradient of acetonitrile from 3 to 80% over 75 min containing 0.1% trifluroacetic acid at 0.7 mL/min with detection at 215 nm.


    Acknowledgements
 
We thank the Natural Sciences and Engineering Research Council of Canada for providing financial support and to Drs. Jordan Tang and Xin-Lin for supplying the porcine pepsinogen cDNA. We are also grateful to Angela Holliss for technical expertise in nucleotide sequencing (LSD, University of Guelph), students Marvin Dyck and Allison Barber for protein preparation, and Dr. Nicholas Low for reviewing the manuscript.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: ryada{at}uoguelph.ca


    Abbreviations
 
BMMY, buffered methanol-complex medium; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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