Preparation of Bioactive and Surface Functional Oligomannosyl

Bioconjugate Chem. 2006, 17, 1170−1177

1170

Preparation of Bioactive and Surface Functional Oligomannosyl Neoglycoprotein Using Extracellular pH-Sensitive Glycosylation of Mutant Lysozyme Having N-Linked Signal Sequence in Yeast Soichiro Nakamura,*,† Masanori Ban,# and Akio Kato# Department of Biosciences and Biotechnology, Shinshu University, Nagano 399-4598, Japan, and Department of Biochemistry, Yamaguchi University, Yamaguchi 753, Japan. Received April 19, 2006; Revised Manuscript Received July 21, 2006

Bioactive oligomannosyl lysozyme with improved surface functionalities was successfully prepared by using an extracellular pH-sensitive glycosylation system for heterogeneous protein in yeast cell. A recombinant Saccharomyces cereVisiae carrying a mutant lysozyme gene encoding the signal sequence of an N-linked glycosylation site at position 49 was cultivated in various pH conditions to investigate the effects of extracellular pH on the glycosylation patterns and the expression of the protein. A large polymannose (Man310GlcNAc2) chainlinked lysozyme was predominantly expressed accompanied by small amounts of a core-type oligomannose chain (Man14GlcNAc2)-linked lysozyme in the yeast medium where the extracellular pH was kept at 3.5 or above, while an oligomannose chain lysozyme was preferentially expressed in the yeast medium where the pH was less than 3. The lytic activities of the oligomannosyl and the polymannosyl lysozymes were found to be 70.4 and 5.1%, respectively, of the wild-type lysozyme when Micrococcus lysodeikticus cells were used as the substrate. The enzymatic activity of the oligomannosyl lysozyme was totally conserved for the glycolysis assay with a soluble substrate, glycol chitin, whereas that of the polymannosyl lysozyme was not. After heating the sample up to 95 °C at pH 7.0 where no visible protein coagulation was observed, thermostability of the enzymatic activity of the oligomannosyl lysozyme was drastically improved with more than 60% of residual lytic activity. Emulsifying properties of the protein also were highly improved by the oligomannosylation, in which the emulsifying activity was 3.2 times higher than that of the wild-type protein. Corresponding to the increase of the surface functionalities, the surface tension of the oligomannosyl protein exhibited a significantly (p < 0.05) lower value compared to that of the wild-type. By using the lower pH medium at 3.0, it was revealed that a substantial amount (0.31 mg/L) of the oligomannosyl lysozyme was successfully obtained in the culture medium. Therefore, the extracellular pH-sensitive glycosylation system can be used to obtain bioactive and surface functional neoglycoproteins.

INTRODUCTION Yeasts are considered cost-effective host organisms for producing high titers of recombinant heterologous proteins (1). The yeast Saccharomyces cereVisiae has been expected to be a promising candidate to produce therapeutic human glycoproteins (2). Yeasts and mammalian cells share the features of asparagine-linked (N-linked) oligosaccharide processing in the endoplasmic reticulum (ER), including the attachment of the common dolichol-linked precursor oligosaccharide (Dol-PPGlcNAc2-Man9-Glc3) and subsequent truncation to GlcNAc2Man8. However, the oligosaccharide processed in the ER is subsequently modified in the Golgi apparatus in different glycosylation systems when comparing mammalian cells and yeasts. In S. cereVisiae, the core oligosaccharide is elongated in the Golgi cisternae through stepwise addition of mannose residues catalyzed by several mannosyltransferases, leading to highly branched outer chains consisting of 50-150 mannose residues (3, 4). Although the yeast expression system can be used to increase the stability during heating or protease attack, hyperglycosylation in S. cereVisiae is a problem for therapeutic glycoprotein production. Indeed, the large molecular size of N-glycosylated lysozyme with a few hundred mannose residues was expressed in the yeast carrying the mutant lysozyme expression plasmid (5). The unusual polymerization of mannose * To whom correspondence should be addressed. Tel & Fax: +81265-77-1609. E-mail: [email protected]. † Shinshu University. # Yamaguchi University.

outer chains in mutant lysozyme may be due to the time interval during which the protein molecule is present in Golgi cisternae. There is little information on the effects of culture condition on the expression and glycosylation patterns of heterologous proteins in yeast cells, while much work has been accomplished on the production of human therapeutics requiring glycosylation using mammalian cells (6-14). Mammalian expression studies revealed that culture pH, ammonia concentration, temperature, and carbon source influenced the glycosylation level and yield of recombinant glycoproteins (13-16). The existence of extracellular ammonia has been shown to decrease the amount of glycosylation and degree of sialylation in the mammalian expression system, indicating that this is due to local intracellular pH alterations as a result of importing ammonia into the cell (13, 15, 16). An extracellular ammonia concentration of 1015 mM can increase the trans-Golgi pH to 7.0-7.2, while β-1,4galactosyltransferase has been identified as having optimum activity at pH 6.5 (15). The shift in culture pH can alter the enzymatic activity of the transferase involved in protein processing, resulting in changes in glycosylation of the protein (14). It has been postulated that the extracellular pH of yeast transformants indirectly affects glycosylation during processing. In the present study, we assumed that the glycosylation pattern of mutant lysozyme could be affected by the culture condition, especially by the culture pH. Although it has been anticipated that the attachment of carbohydrate chains to protein brings about its stability and surface functionalities, polyglycosylation appears to be inappropriate to use for the production of biologically active proteins

10.1021/bc0600970 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/17/2006

Preparation of Bioactive Oligomannosyl Neoglycoprotein

because of the steric hindrance of excessively long chains. The lytic activity of lysozyme was drastically diminished by the polyglycosylation with dextran through Maillard-type glycosylation (17), while the conjugation with small carbohydrate molecules such as glucose or lactose caused insoluble aggregates with poor surface properties (18). Accordingly, if a conjugation with oligosaccharide were to be applied, then a desirable active neoglycoprotein with improved surface functionalities could be created. The objective of this study was to synthesize bioactive and surface functional oligomannosyl lysozyme using an extracellular pH-sensitive glycosylation system of heterogeneous protein in yeast cells.

EXPERIMENTAL PROCEDURES Materials. The yeast expression plasmid pYG-100 was supplied from Dr. K. Matsubara, Osaka University. The recombinant plasmid pKK-1, which contains a full-length hen egg-white lysozyme cDNA (19), was from Dr. I. Kumagai, University of Tokyo. T4 DNA ligase, alkaline phosphatase, and restriction enzymes were purchased from Takara Shuzo (Kyoto). The 7-DEAZA sequence kit for sequencing and the kit for blunting were also purchased from Takara Shuzo. The oligonucleotide-directed in vitro mutagenesis system (version 2) for site-directed mutagenesis and [R-32P] dCTP (800 Ci/mmol) were purchased from Amersham Pharmacia Biotech. CM-Toyopearl 650M resin was from Tosoh (Tokyo). Concanavalin ASepharose and R-methylmannoside were from Amersham Pharmacia Biotech and Wako Pure Chemical Industries Ltd. (Tokyo), respectively. Sephadex G-50 was from Amersham Pharmacia Biotech. Micrococcus lysodeikticus dried cells and ethylene glycol chitin for lysozyme assay were from Sigma and Nacalai Tesque Inc. (Kyoto), respectively. Endo-β-N-acetylglucosaminidase (endo-H) was from Genzyme. All other chemicals were of analytical grade for biochemical use. Construction of Yeast Expression Plasmid Carrying a Mutation of the Lysozyme Gene. A mutant lysozyme cDNA was constructed to introduce the potential asparagine-linked (Nlinked) glycosylation sites (Asn-Ser-Thr) on the molecular surface at position 49, as described before (5). The conversion of Gly49 codon to Asn was carried out by site-specific mutagenesis using the bacteriophage vector M13mp19. The EcoRI/HindIII fragment of pKK-1 plasmid was subcloned into the EcoRI/HindIII site of the bacteriophage vector M13mp19. The mutant hen egg-white lysozyme cDNA was constructed in the M13mp19 vector by the Amersham oligonucleotide-directed mutagenesis system (version 2). A mutagenic oligonucleotide primer, 5′-AACACCGATAACAGTACCGA-3′, which was synthesized by the phosphoamidate method using a Pharmacia DNA synthesizer, was used to convert the Gly49 (GGG) codon to Asn (AAC). The presence of the mutation was confirmed by dideoxy DNA sequencing analysis (20). For construction of the vector, the mutant lysozyme cDNA was inserted into SalI site of a yeast expression plasmid pYG-100, as previously described (19, 21). Yeast Transformation and Cultivation. The expression vector carrying a mutant lysozyme gene was introduced into S. cereVisiae AH22 (MATa, Leu2, His4, Cir() according to the lithium acetate procedure (22). Leu+ transformants were screened by subculturing in the modified Burkholder minimum medium (23) plates supplemented with histidine (20 µg/mL) at 30 °C. After cultivation, well-growing colonies were then replicacultivated in the yeast medium on a small scale (5 mL), and the overexpression subclones with the highest levels of lysozyme activity were screened and propagated from single colonies. The overexpression colonies were directly subcultured on a large scale (6 L) at 30 °C in the yeast minimum medium, in which ammonium sulfate or L-asparagine was used as a nitrogen

Bioconjugate Chem., Vol. 17, No. 5, 2006 1171

source. Extracellular pH of the culture was controlled at the given pHs by addition of 1 N sodium hydroxide solution. Duplicate 10 mL samples were removed for monitoring pH and measuring turbidity of the culture mediums. The culture pH for all experiments was controlled within (0.1 pH units. Isolation and Purification of the Mutant Lysozyme from Yeast Culture Medium. The supernatant of yeast culture medium was directly applied to a CM-Toyopearl 650M opened column (1.8 × 5 cm) equilibrated with 50 mM Tris-HCl buffer (pH 7.5), and the column was washed with the same buffer until the washing solution was free from proteins. The adsorbed lysozyme was eluted with 0.5 M NaCl in 50 mM Tris-HCl buffer (pH 7.5). The protein solution was diluted with deionized water at least five times and again applied to the regenerated CM-Toyopearl 650M column. The eluted lysozyme was collected and then lyophilized to measure the secretion amount and to analyze SDS-polyacrylamide gel electrophoresis. For the chemical analysis and assay of enzymatic activity, further purification was carried out using the gel filtration on a Sephadex G-50 equilibrated and eluted with 50 mM Tris-HCl buffer, pH 7.5. The protein content in each fraction was detected by measuring the absorbance at 280 nm, and the carbohydrate content was determined by measuring the absorbance at 490 nm after color development with the phenol-sulfuric acid reaction. All fractions containing glycoprotein were collected and concentrated using a regenerated cation-exchange column. The glycoprotein samples were further applied to a concanavalin A-Sepharose column (0.9 × 3 cm), which was prewashed with 10 bed volumes of 20 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl and then washed with the same buffer until the washing solution was free from proteins. The adsorbed glycoprotein was subsequently eluted with 100 mM R-methylmannoside in the 20 mM Tris-HCl buffer containing 0.5 M NaCl. The glycoprotein solution was diluted with deionized water at least five times and again applied to the regenerated CMToyopearl 650M opened column (1.8 × 2 cm). SDS-Polyacrylamide Gel Electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the method of Laemmli (24) using a 15% (w/v) acrylamide separating gel and a 5% (w/v) stacking gel containing 1% (w/v) SDS. Samples were heated at 100 °C for 5 min in Tris-glycine buffer (pH 8.8) containing 1% SDS and 1% (v/ v) 2-mercaptoethanol. Electrophoresis was carried out at a constant current of 10 mA for 5 h using an electrophoretic buffer of Tris-glycine containing 0.1% (w/v) SDS. After electrophoresis, the gels were stained for protein and carbohydrate with 0.025% (w/v) Coomassie Brilliant Blue R-250 solution and 0.5% (w/v) periodic acid-Fuchsin solution (25), respectively. Endo-H Treatment. The digestion of the glycosylated lysozymes with endo-H was carried out according to the slightly modified method of Tarentino and Maley (26). The glycosylated lysozymes (0.1 mg/mL) were boiled in 50 mM sodium citrate buffer (pH 5.5) containing 1% (w/v) SDS and 200 µg/mL of phenylmethylsulfonyl fluoride for 5 min. After cooling, samples were supplemented with an equal volume of either 0.02 unit of endo-H in 50 mM sodium citrate buffer (pH 5.5) or the same buffer without the enzyme and were subsequently incubated at 37 °C for 20 h. After incubation, the samples were analyzed by SDS-PAGE. Analysis of the N-Linked Oligosaccharide Length of Mutant Lysozyme. The total sugar content of the glycosylated lysozymes was estimated using the phenol-sulfuric acid reaction employing mannose as a standard. HPLC analysis serves as a method of identification of the hexose liberated from glycosylated lysozymes by hydrolysis with 2 N HCl at 100 °C for 3 h in a sealed glass ample. The hydrolysates were dried, dissolved in water, and chromatographed on an Asahipak NH2P-

1172 Bioconjugate Chem., Vol. 17, No. 5, 2006

50 column (Asahi Chemical, Tokyo) in 75% acetonitrile using the Hitachi HPLC system equipped with an RI detector. For the analysis of hexosamine, the hydrolysates with 3 N HCl at 100 °C for 4 h were dried and then analyzed using an amino acid analyzer (Tosoh, HLC 805). Enzymatic Assay. Lysozyme activity was measured by a lysis and glycolysis assays using M. lysodeikticus cells and ethylene glycol chitin as substrates, respectively. Bacteriolytic activity of lysozyme was assayed by the method of Parry et al. (27) with a slight modification. The suspensions of M. lysodeikticus cells (OD450 ) 0.7) were prepared in 100 mM acetic acid-sodium acetate buffer at pH 6.0. After its absorbance at 280 nm was first adjusted to 0.050, the assay lysozyme solution (0.1 mL) was added to 2.4 mL of the cell suspension in sodium acetate buffer (pH 6.0). The initial decrease in the absorbance at 450 nm of the mixture caused by lysis of M. lysodeikticus cells was measured at 20 °C for 1 min with a Hitachi U-2000 spectrophotometer. Hydrolytic activity with glycolysis was measured by following the reducing procedure (28). 0.5 mL of the lysozyme solution in 10 mM acetate buffer (pH 4.5) was added into a 1.0 mL of 0.05% (w/v) solution of ethylene glycol chitin. The mixture was incubated at 40 °C for 30 min. After the reaction, 2.0 mL of the color reagent (made by dissolving 0.5 g of potassium ferricyanide in 1 L of 0.5 M sodium carbonate) was added, and the mixture was immediately boiled for 15 min to estimate the reducing power resulting from hydrolysis of ethylene glycol chitin. Emulsifying Properties and Surface Tension. Emulsifying activity and emulsion stability were estimated according to the modified method of Pearce and Kinsella (29). An emulsion was prepared by homogenizing the mixture of 1.0 mL of corn oil and 3.0 mL of a 0.1% (w/v) protein solution in a Polytron homogenizer PT 10-35 (Kinematica Co., Switzerland) at 12 000 rpm for 1 min at 20 °C. A total of 100 µL of emulsion was taken from the bottom of the test tube after the sample stood for 0, 1, 2, 3, 5 and 10 min and was diluted with 5.0 mL of 0.1% (w/v) SDS solution. The absorbance of the diluted emulsion was then measured at 500 nm. The relative emulsifying activity was represented as the absorbance at 500 nm measured immediately after emulsion formation (0 min). The emulsion stability was estimated by measuring the half-life of the turbidity detected immediately after the emulsion had formed. Surface tension was measured by an interfacial tensiometer (Kyowa Kagaku Co., Tokyo), a duNouy tensiometer, which is essentially a torsion balance with platinum ring (2 cm diameter) suspended from the beam. The platinum ring was put into 2 mL of a 0.05% (w/v) protein solution, and then the force required to release the ring from the surface was measured using the apparatus. The exhibitions of the steady surface tension required 5 min after preparation of the interface. Therefore, the measurement was carried out at 5 min intervals. Thermal Denaturation. The apparent heat stability was estimated by measuring the developed turbidity when 0.1% (w/ v) protein concentration of native and glycosylated lysozymes were heated to 95 °C from 30 °C at a heating rate of 1 °C/min in 1/15 M sodium phosphate buffer (pH7.0). After a given temperature, the heated sample was immediately replaced into a cuvette and the turbidity was measured at 500 nm. The residual lytic activity of the heated samples was determined by using M. lysodeikticus cells as substrate.

RESULTS Effect of Extracellular pH on the Glycosylation and Secretion of Mutant Lysozyme. Ammonium sulfate as a nitrogen source is not used for the yeast minimum medium because the pH of the medium is lowered by the increases in sulfate during the growth of yeast. However, the nitrogen source

Nakamura et al.

Figure 1. Effects of extracellular pH on expression of mutant lysozyme in the recombinant yeast S. cereVisiae AH22. (A) pH profiles and growth curves when the recombinant yeast cells carrying mutant lysozyme gene were cultured at 30 °C for 8 days in Burkholder’s yeast minimum medium with L-asparagine (4, 2) or ammonium sulfate (O, b) as sole nitrogen sources. The solid and open symbols indicate pH and the turbidity of culture, respectively. Duplicate 10 mL samples were removed and used for monitoring pH and turbidity of the culture mediums. (B) Time course of secretion amount of the mutant lysozyme. The yeast cells were grown in Burkholder’s yeast minimum medium with ammonium sulfate as a sole nitrogen source at 30 °C for 4 days (at final pH 2.0) and then incubated continuously at 30 °C for 4 days in the pH-controlled medium at pH 4.0 by the addition of NaOH. The secretion amounts of the mutant lysozyme at pH 2.0 (O) and pH 4.0 ()) were determined by measuring the dry weight of the mutant proteins purified by two steps of cation-exchange chromatography with CMToyopearl from the cultivation medium. The small letters a, b, and c in the panel indicate the position and the secreted amount of mutant lysozymes obtained 0, 2, and 4 days after raising the pH to 4.0, respectively.

was very useful for investigating the effect of pH on the glycosylation and expression of the heterologous lysozyme. When ammonium sulfate was used as a nitrogen source, the extracellular pH during yeast growth was reduced to 2.0, as shown in Figure 1A, whereas the pH in a culture of S. cereVisiae AH22 was kept at 4.6-4.7 in the yeast minimum medium in which L-asparagine was used as a nitrogen source. Although the growth of yeast was slightly suppressed in the medium containing ammonium sulfate, compared to that in the medium containing L-asparagine, almost the same order (108 cells/mL) of cell growth was kept in the stationary phase despite the extreme lowering of the extracellular pH. To investigate the effect of the extracellular pH on the expression of mutant lysozyme, the pH regulation of yeast culture was carried out as follows. After the cultivation of the recombinant yeast in the medium of ammonium sulfate for 4 days at 30 °C, the pH of the medium was raised to 4.0 by addition of 1 N NaOH and then kept to the same pH for another 4 days. As shown in Figure 1B, the secreted amount of lysozyme was greatly increased with the recovery of pH to 4.0. The SDS-polyacrylamide gel electrophoretic patterns of mutant lysozyme at each culture stage are shown in Figure 2. After incubation in the culture medium



Figure 3. Effect of extracellular pH on the expression rates of mutant lysozyme. The yeast cells were cultured in the yeast minimum medium with ammonium sulfate as a sole nitrogen source at 30 °C for 4 days. To maintain pH conditions at 4.0, 3.5, and 3.0, 1 N NaOH was added in constantly maintaining by pH controller. The final pH became 2.0 without pH control. The secreted lysozymes were purified by cationexchange chromatography with CM-Toyopearl, and the concentration was determined based on the absorbance at 280 nm. Represented values are means of three independent experiments, and the bars indicate the standard deviation.

Figure 2. Changes in the SDS-polyacrylamide gel electrophoretic patterns of mutant lysozymes secreted from the recombinant yeast S. cereVisiae AH22 in the pH-controlled condition. The yeast cells were cultured in the yeast minimum medium with ammonium sulfate as a sole nitrogen source at 30 °C for 4 days and then incubated continuously at 30 °C for 4 days in the controlled medium at pH 4.0. The secreted lysozyme in the culture supernatant after 4 (lane 3), 6 (lane 4), and 8 days (lane 5) of incubation were purified by cation-exchange chromatography with CM-Toyopearl and subjected directly to SDS-PAGE. Electrophoresis was carried out at a constant current of 10 mA for 5 h in an electrophoresis of Tris-glycine buffer (pH 8.8) containing 0.1% SDS. The gels were stained for proteins and carbohydrates with Coomassie Brilliant Blue (A) and periodic acid-Fuchsin (B), respectively. Arrows indicate the position of the boundary between the stacking (upper) and separating (lower) gels. Lane 1, molecular weight markers (94 000, phosphorylase b; 67 000, bovine serum albumin; 43 000, ovalbumin; 30 000, carbonic anhydrase; 20 100, trypsin inhibitor; 14 300, R-lactalbumin); lane 2, wild-type lysozyme; lane 3, 4-day culture product shown in Figure 1B, a; lane 4, 6-day culture product shown in Figure 1B, b; lane 5, 8-day culture product shown in Figure 1B, c.

for 4 days at 30 °C (pH 2.0), most lysozymes were not glycosylated, but a small amount of oligomannosyl protein was observed (Figure 2, lane 3). The subsequent incubation at pH 4.0 resulted in dramatic increases in the expression of polymannosyl and oligomannosyl lysozymes (Figure 2, lanes 4 and 5), although cell growth was not observed during incubation at pH 4.0. This result suggests that the glycosylation of the mutant lysozyme in yeast was regulated by the extracellular pH. To confirm the above result, the effects of the extracellular pH in the yeast culture medium on the glycosylation of the mutant lysozyme were further examined. In the medium containing ammonium sulfate as a nitrogen source, the extracellular pHs were constantly adjusted to 4.0, 3.5, and 3.0 using 1 N NaOH, and yeast cell growth was monitored by the measurement of the culture turbidity. The cell growth increased in proportion to the incubation time at each pH and attained stationary phase after a 60 h incubation. No significant differences in the cellular growth were observed at each pH. On the other hand, remarkable increases in the secretion amounts of the lysozyme were observed with the increase in extracellular pH (Figure 3). The secretion amounts from the cultured yeast cells incubated at pH 2.0, 3.0, 3.5, and 4.0 were 0.10, 0.31, 0.91, and 3.6 mg/L of medium, respectively. When the extracellular pH was maintained at 4.0, the yield of the mutant lysozyme was 36% of that in the medium (pH 4.6-4.7) using L-asparagine as a nitrogen source (5). Figure 4 shows the SDSPAGE patterns of the secreted lysozymes in the supernatant of the pH-controlled media at 4.0, 3.5, and 3.0. Polymannosyl lysozyme was secreted in proportion to increases in extracellular pH. It is interesting that pH regulation of the yeast medium greatly affected the glycosylation patterns of the mutant lysozyme. The first step in glycosylation (oligomannosylation) of the mutant lysozyme was greatly suppressed at pH 2.0 (lane 3), although protein synthesis proceeded normally. On the other hand, the mutant lysozyme was predominantly oligomannosylated at pH 3.0 (lane 4) and mostly polymannosylated at pH 3.5 (lane 5) or above (lane 6). These observations suggest that the glycosylation of proteins in yeast is affected by the extracellular pH. These data indicate that a substantial amount (0.31 mg/L) of the oligomannosyl lysozyme could be obtained in the culture medium by using the lower pH medium, i.e., at 3.0. The glycosylated lysozymes were treated with endo-H which cleaves the high mannose-type asparagine-linked oligosaccharide

1174 Bioconjugate Chem., Vol. 17, No. 5, 2006

Nakamura et al. Table 1. Carbohydrate Analysis of Glycosylated Lysozymes contents (mol/mol lysozyme) of lysozyme

N-acetylglucosaminea

mannoseb

polymannosylc

2 2

310 ( 7.5 14 ( 0.4

oligomannosyld

a Determined with an amino acid analyzer using the hydrolysates with 3 N HCl at 100 °C for 4 h. b Determined by HPLC analysis using an NH2P50 column and by the phenol-sulfuric acid method. Each value is the means ( standard deviation of three replications. c Obtained from the medium at pH 4.0. d Obtained from the medium at pH 3.0.

Table 2. Enzymatic Activity of Oligomannosyl and Polymannosyl Lysozymes enzymatic activity (%)a determined by

Figure 4. Electrophoretic patterns of mutant lysozymes secreted from the recombinant yeast S. cereVisiae AH22 cultured at various pH. The secreted lysozymes in the various pH-controlled conditions were purified by cation-exchange chromatography with CM-Toyopearl and treated with endo-H as described in Experimental Procedures and then subjected to SDS-PAGE. The gels were stained with Coomassie Brilliant Blue. Arrow indicates the position of the boundary between the stacking (upper) and separating (lower) gels. Lane 1, molecular weight markers; lane 2, wild-type lysozyme; lane 3, product obtained from the medium without pH control (final pH 2.0); lane 4, product obtained from the medium controlled at pH 3.0; lane 5, product obtained from the medium controlled at pH 3.5; lane 6, product obtained from the medium controlled at pH 4.0.

(30). Oligomannosyl and polymannosyl lysozyme were completely digested by endo-H, suggesting that these glycosyl lysozymes are a type of N-linked glycosylation (data not shown). The cell growth of the yeast may affect the glycosylation pattern of mutant lysozyme. To elucidate this point, the glycosylation pattern of the mutant lysozyme in various growth stages was investigated. The polymannosylation occurred in the early or mid-log phase, and no differences in the polymannosylation of mutant lysozymes were observed in each growth stage. On the other hand, as shown in Figures 2 and 4, the polymannosylation in extremely acidic medium (less than pH 3.0) did not occur in the stationary phase where the yeast growth was almost the same as that in normal medium. These results suggest that the polymannosylation was affected only by extracellular pH, independent of the yeast cell growth. The extracellular pH may affect the sorting and secretion of the foreign lysozyme in yeast cells. The polymannosyl lysozyme may remain in the cytosol or cell membrane at extremely low pH. Therefore, the presence of polymannosyl lysozyme in the yeast cell membrane and the cytosol was investigated. No polymannosyl lysozyme was observed in the cytosol or cell membrane at pH 2.0, although considerable amounts of glycosylated lysozymes were obtained at pH 4.0 or 4.6. Except for the presence of mutant lysozymes, there are no differences in the SDS-PAGE patterns of membrane proteins under various medium conditions (data not shown). Thus, it was confirmed that the glycosylated lysozyme did not remain in the yeast cell membrane or cytosol at low pH 2.0. Chemical and Enzymatic Analysis of Glycosylated Lysozymes. The chemical composition analysis revealed that the polysaccharide chain of the polymannosyl lysozyme consisted of 310 mol of mannose and 2 mol of N-acetylglucosamine, while that of oligomannosyl one consisted of 14 and 2 mol, respec-

types of protein

M. lysodeikticusb

glycol chitinc

wild-type lysozyme oligomannosyl lysozyme polymannosyl lysozyme

100 70.4 ( 2.7 5.1 ( 2.8

100 100 ( 1.5 88.7 ( 3.1

a Enzymatic activity was represented as the percentages of wild-type lysozyme. Each value is the means ( SD of independent experiences performed in triplicate. b The lytic activity was measured in 0.1 M acetate buffer (pH 6.0) with M. lysodeikticus as substrate. c The glycosis activity was measured in 0.01 M acetate buffer (pH 4.5) with glycol chitin as substrate.

tively (Table 1). The composition indicates that poly- and oligomannosyl lysozymes carry Man310GlcNA2-linked and Man14GlcNAc2-linked forms, respectively. This result corresponds to an increase in apparent molecular mass in SDSPAGE (Figure 2). The carbohydrate composition of oligomannosyl lysozyme secreted at acidic pH 3.0 is almost the same as that of the core type N-linked oligosaccharide, Man8GlcNAc2, while the length of the polymannosyl chains secreted at pH 4.0 was almost the same as that secreted in normal medium (5). These results suggest that the acidic extracellular pH 3.0 did not affect the processing in the ER membrane but affected the subsequent elongation of the outer chains in the Golgi cisternae. Thus, it was confirmed that the lysozyme glycosylated with N-linked oligosaccharide and polysaccharide chains were separately secreted in the recombinant yeast and controlled by the extracellular pH. A similar dependency of the glycosylation of the mutant lysozyme on the extracellular pH was also observed in the medium in which asparagine was used as a nitrogen source. The lysozyme activities were measured by lysis and glycolysis assays for oligomannosyl lysozyme and polymannosyl lysozyme secreted in the medium at pH3.0. The enzymatic activities of oligomannosyl and polymannosyl lysozymes were, respectively, 70.1 and 5.1% of the wild-type protein when M. lysodeikticus cells were used as substrate; therefore, long-chain glycosylation may be unsuitable for producing biologically active proteins (Table 2). These lysozyme activities were recovered by treating with endo-H. On the other hand, when glycol chitin was used as the substrate, the enzymatic activity of the oligomannosyl lysozyme was almost the same as that of unglycosylated (wildtype) lysozyme, while that of the polymannosyl lysozyme was 88.7% (Table 2). These results indicate that mature oligomannosyl lysozyme was correctly processed during the secretion in the recombinant yeast at lower pH (3.0), suggesting the inhibition of oligomannosylation was not caused by the structural changes in protein structure under the acidic condition. The biological activity of lysozyme was not appreciably deteriorated by oligomannosylation in the yeast expression system. The inhibitory activity was highly conserved in the oligomannosylated lysozyme, whereas it was significantly decreased in the fully polymannosylated lysozyme. This result indicates that the long polymannosyl chain may interfere with


Figure 5. Thermal stability of oligomannosyl lysozyme. The thermal stability was determined by measuring the turbidity and residual activity. Samples were heated to 95 °C from 30 °C at a rate of 1 °C/min in 1/15 M sodium phosphate buffer, pH 7.0. After a given temperature, the heated sample was immediately replaced into a cuvette, and the residual activity was determined based on the lytic activity using M. lysodeikiticus as substrate (solid symbols). The turbidity of the sample solution was measured at the absorbance of 500 nm (open symbols). Data shown are from a representative experiment repeated three times with similar results. bO, wild-type lysozyme; 24, oligomannosyl lysozyme; ][, polymannosyl lysozyme.

effective interaction between enzymatic pockets of the protein moiety and the substrate. Heat Stability of Recombinant Lysozymes. Figure 5 shows the heat stability of oligomannosyl lysozyme, which was assessed by monitoring its thermal denaturation when a 0.1% protein solution was heated to 95 °C at a rate of 1 °C/min. As a result, the heating assay revealed that polymannosylation greatly improved the heat stability of the protein to an extent that no coagulation was observed. Under the same conditions, unglycosylated protein had coagulated. Almost the same phenomenon was observed in the heating system coexisting with oligomannosyl lysozyme. During heating, the protein molecules are partially unfolded, resulting in aggregation due to the heatinduced disruption of a delicate balance of various noncovalent interactions. This process was reversible in the glycosylated cystatins because of the inhibition of aggregation by poly and oligomannosyl chains through interaction of unfolded proteins, whereas that of wild-type lysozyme was irreversible. In addition, as shown in Figure 5, considerably high residual lytic activity of lysozyme toward M. lysodeikticus cells was kept in the oligomannosyl lysozyme after heating to 95 °C. After heating of the sample to 95 °C, the lytic activity of the polymannosyl lysozyme was 4.5% that of the native wild-type lysozyme, whereas that of oligomannosyl lysozyme was 63.3%. Thus, we have succeeded in obtaining a bioactive and stable lysozyme with an oligomannose chain. The oligomannosyl lysozyme was used for further investigation. Improved Surface Functionalities of Oligomannosyl Lysozyme. The emulsifying properties of the oligomannosyl lysozyme constructed by genetic modification were measured under various solution systems (Figure 6). The turbidity of emulsion is plotted as the ordinate, and standing time after emulsion formation is plotted as the abscissa. The value of the ordinate at zero time is relative emulsifying activity, and the half-life of initial turbidity reflects the stability of the emulsion. Better emulsifying properties were observed in the emulsion of the oligomannosyl lysozyme. Emulsifying activity was 3.2 times higher than that of wild-type lysozyme in the neutral pH system (Figure 6A). The emulsifying properties of the oligo-


Figure 6. Emulsifying properties of oligomannosyl lysozyme under a neutral pH (1/15 M sodium phosphate buffer, pH 7.0) condition (A), an acidic pH (1/15 M sodium citrate buffer, pH 3.0) condition (B), a high-salt concentration (1/15 M sodium phosphate buffer, pH 7.0, containing 0.2 M NaCl) condition (C), and in a heating (at 65 °C for 30 min in the above neutral pH solution) condition (D). Representative data are obtained from three independent experiments with similar results. O, oligomannosyl lysozyme; b, wild-type lysozyme.

mannosyl lysozyme were still excellent in the high-salt condition containing 0.2 M NaCl compared to those of wild-type. The emulsifying activity and the emulsion stability of the oligomannosyl lysozyme were respectively 1.8- and 3.1-fold higher in the wild-type lysozyme (Figure 6B). The emulsifying properties of lysozymes were substantially improved in the acidic (1/15 M acetate buffer, pH 3.0) system. In particular, further improvement was demonstrated in the oligomannosyl lysozyme (Figure 6C). The emulsifying activity of the oligomannosyl lysozyme was increased 1.1 times that of the native form by preheating to 95 °C from 30 °C at a rate to 1 °C/min in the neutral pH system (Figure 6D). As shown in Figure 5, the oligomannosyl lysozyme was heat-stable, and no coagulate was observed during heat treatment at 95 °C. The resulting unfolded form of the oligomannosyl lysozyme was kept by the attached oligomannosyl outer chains without coagulation of the protein portion. The amphiphilic balance between the protein and the carbohydrate chain may play an important role for better emulsifying properties of glycoproteins. It is assumed that the hydrophobic residues of the protein moiety partially denaturated during emulsion formation at the oil-water interface may be anchored to the surface of oil droplets in emulsion, where the hydrophilic residues of the extended branched oligomannosyl chains oriented to water may cover oil droplets to inhibit the coalescence of oil droplets, resulting in higher emulsion formation. Indeed, the emulsifying properties of the oligomannosyl lysozyme were deteriorated compared to those of wildtype lysozyme after deglycosylation with endo-H (data not shown). The measurement of surface tension supplied us reasonable data to elucidate the mechanisms of the improved surface functional properties of the oligomannosyl lysozyme. As shown in Table 3, the surface tension of a 0.05% protein concentration of lysozyme solution was significantly (p < 0.05) reduced by the oligomannosylation compared to that of wild-

1176 Bioconjugate Chem., Vol. 17, No. 5, 2006 Table 3. Surface Tension of Oligomannosyl and Polymannosyl Lysozymesa types of protein

surface tension (dynes/cm)

wild-type lysozyme oligomannosyl lysozyme polymannosyl lysozyme

71.5 ( 1.7a 65.5 ( 1.5b 60.1 ( 1.3 c

a The surface tension was determined in room temperature (20 °C) where the value of distilled water without any protein was 75.6 ( 0.9. Values are the means ( SD of ten times replications, and values with different letters are significantly different (P < 0.05).

type lysozyme. The oligomannosylation allows lysozyme to convert to a surface-active protein without a fatalloss of the biological activity.

DISCUSSION We have shown that the polymannosyl lysozyme was preferentially secreted in the medium when the mutant lysozyme gene encoding the N-linked signal sequence at the position 49 was expressed in the yeast S. cereVisiae AH22 (5). The length of the mannose chain was found to be widely distributed from 200 to 350 residues per mole of protein, as reported in the previous paper (5). The length of the mannose residues of the polymannosyl lysozyme was above twice that of the general mannoproteins in yeast. This observation suggests that extensive glycosylation of the outer chain occurs in the secretion system of the heterogeneous lysozyme in yeast, probably due to its specific structural factors. In the yeast S. cereVisiae cells, the polymannose chain can be elongated by a highly branched outer chain that has an R-1,6-linked backbone attached to numerous side chains with one to two R-1,2-mannoses and terminal residues in an R-1,3 linkage (31). In addition, the mannose outer chains are extended by the different elongation specific R-1,6mannosyltransferases (31). The chain length of the carbohydrate is reported to be strain specific for glycoproteins in yeast (32). The presence of various sizes of mannose outer chains in the yeast seems to be dependent on the characteristic of the protein structure, the degree of phosphorylation of the side chains, and the action of mannosyl transferase, etc. In addition, the size of the outer chains may also be determined by the time interval in which the corresponding proteins are present in the Golgi cisternae. Therefore, the unusual polymannosylation of lysozyme may arise from the activation of these factors to secrete harmful heterogeneous protein into the exterior of the yeast cell. However, the secretion amounts of the polymannosyl lysozyme were greatly dependent on the conditions used in the yeast culture as described in this paper. The results in Figure 1 demonstrate that the glycosylation and expression of the recombinant N-glycosylated lysozyme are reversibly recovered by raising the extracellular pH after the suppression of the glycosylation at pH 2.0. When the cultured medium pH was raised from 2.0 (extremely acidic condition) to 4.0, the yeast cells began to produce significant quantities of oligomannosyl or polymannosyl lysozyme, indicating that the recovery of the culture environment to normal pH caused the activation of the glycosylation events in the ER membrane and Golgi cisternae. This suggests that the glycosylation of protein in the ER membrane is inhibited, and further modification in the Golgi apparatus is suppressed by pH stress (the extremely low pH). No polymannosyl lysozyme was secreted; only a core-type oligomannosyl protein was secreted in a yeast culture medium at pH 3.0. A core-type oligosaccharide (Man14GlcNAc2) is formed in the initial steps in the elongation pathway in the Golgi. The selective secretion of core-type oligomannosyl lysozyme at pH 3.0 is important for elucidating the mechanism of the polymannosylation of lysozyme. The possible explanations are due

Nakamura et al.

to inhibition of the subsequent elongation events of enzymes, alteration of the steric accessibility in the core oligosaccharide, and/or shortened period of time in the Golgi in the low pH (3.0) condition. The internal pH in yeast cells may be lowered to less than 6.0 when the extracellular pH is less than 3.0, although it is well-known that yeasts are able to maintain their internal pH between 6 and 7.5 when the extracellular pH varies from 3.5 to 9.0 (33). The decrease in the internal pH of yeast cells may affect the various events in the elongation of mannose outer chains in the Golgi cisternae. It is most likely that the enzymes involved in the elongation of polymannosyl residues may be inhibited at an extremely low pH. The results obtained in this study also suggest that the glycosylated lysozymes could be subjected to the transport and sorting mechanisms, which regulate their direction into regulated or constitutive secretion pathways even at an extracellular pH of less than 3.0. Defects in the polymerization of the outer chains lead to a clumped morphology, reduced viability, and distortion of the yeast cell wall (34), although a functional explanation is not possible at the moment. The secretion of polymannosyl lysozyme was observed at the normal extracellular pH but not at lower pH. However, the membrane proteins were secreted at lower pH as well as at the normal pH. It seems that the elongation of the outer chains in the heterologous lysozymes is sensitive to pH stress and is uniquely inhibited in the yeast culture at the low pH. Hence, the heterologous lysozyme gene could be used as a reporter in studies of the intracellular traffic of proteins through the ER and Golgi apparatus. It was also revealed that the processing of the N-linked glycosylation of heterologous proteins in yeast is greatly affected by the extracellular pH. In this paper, we demonstrated that oligomannosylation in yeast using lower extracellular pH is able to carry an excellent emulsifying properties to lysozyme without serious loss of biological activity. The mechanism was supported by the measurement of surface tension of the oligoglycosyl lysozyme solution. It has been confirmed that the reduction of surface tension of protein solution induces excellent surfactant activity (35). The excellent emulsifying properties of the oligoglycosyl lysozyme were not reduced even in a high-salt solution system (1/15 M phosphate buffer, pH 7.4, containing 0.2 M NaCl) nor in an acidic (1/15 M sodium citrate buffer, pH 3.0) system and in a heating (at 65 °C for 30 min in the above neutral pH solution) system. Since high-salt, acidic pH, and heating conditions are commonly used in industrial applications, the oligomannosyl lysozyme constructed by the genetic modification could be a suitable ingredient for food agents. The oligomannosyl lysozyme revealed much better emulsifying properties, especially emulsion stability, than those of the wild-type protein, suggesting the special role of the carbohydrate chain in emulsion stability. It is well-known that amphiphilic proteins such as casein, serum albumin, and β-lactoglobulin show good emulsifying properties (36). By the natural attachment of the carbohydrate chain, the amphiphilicity of the protein increased in the oligomannosyl lysozyme. The enzymatic activity of lysozyme was highly conserved in the oligomannosylation in the yeast expression system; therefore, the novel oligomannosyl lysozyme with improved emulsifying properties suggests the direction of the design of new functional proteins. There is accumulating evidence that the N-linked oligosaccharide serves to improve the folding and function of bioactive proteins including Erthrina corallodendron lectin, soybean agglutinin, RNase B, protein aquaporin 2, and so on (37). We previously demonstrated that anti-rotavirus activity of human cystatin C was substantially enhanced by site-directed oligomannosylation using the yeast expression system. The highly efficient action of oligomannosyl cystatin was interpreted as a binding effect of the oligomannosyl chain to rotavirus (38). With the improved



surface functionalities by oligomannosylation, proteins are capable of acquiring extended biological activities. The characteristics can be exploited for various health benefits, for example, in pharmaceuticals, nutraceuticals, and food ingredients. Thus, the present study will be further appreciated from many fields.

ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research from the Minister of Education, Science and Culture of Japan (No. 02660143).

LITERATURE CITED (1) Bergquist, P. L., Te’o, V. S., Gibbs, M. D., Cziferszky, A. C., De Faria, F. P., Azevedo, M. O., and Nevalainen, K. M. (2002) Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts. Appl. Biochem. Biotechnol. 98100, 165-176. (2) Chiba, Y., Sakuraba, H., Kotani, M., Kase, R., Kobayashi, K., Takeuchi, M., Ogasawara, S., Maruyama, Y., Nakajima, T., Takaoka, Y., and Jigami, Y. (2002) Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease. Glycobiology 12, 821-828. (3) Kukuruzinska, M. A., Bergh, M. L., and Jackson, B. J. (1987) Protein glycosylation in yeast. Annu. ReV. Biochem. 56, 915-944. (4) Tanner, W., and Lehle, L. (1987) Protein glycosylation in yeast. Biochim. Biophys. Acta 906, 81-99. (5) Nakamura, S., Takasaki, H., Kobayashi, K., and Kato, A. (1993) Hyperglycosylation of hen egg white lysozyme in yeast. J. Biol. Chem. 268, 12706-12712. (6) Rademacher, T. W., Parekh, R. B., and Dwek, R. A. (1988) Glycobiology. Annu. ReV. Biochem. 57, 785-838. (7) Miller, W. M., Blanch, H. W., and Wilke, C. R. (1988) A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Biotechnol. Bioeng. 32, 947-965. (8) Rothman, R. J., Warren, L., Vliegenthart, J. F., and Hard, K. J. (1989) Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybridomas. Biochemistry 28, 1377-1384. (9) Ozturk, S. S., and Palsson, B. O. (1991) Growth, metabolic, and antibody production kinetics of hybridoma cell culture. 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor. Biotechnol. Prog. 7, 481-494. (10) Doyle, C., and Butler, M. (1990) The effect of pH on the toxicity of ammonia to a murine hybridoma. J. Biotechnol. 15, 91-100. (11) Goochee, C. F., and Monica, T. (1990) Environmental effects on protein glycosylation. Biotechnology (N Y). 8, 421-427. (12) Wang, X. B., Sato, N., Greer, M. A., Greer, S. E., and McAdams, S. (1990) Role of extracellular calcium and calmodulin in prolactin secretion induced by hyposmolarity, thyrotropin-releasing hormone, and high K+ in GH4C1 cells. Acta Endocrinol. (Copenh). 123, 218224. (13) Goochee, C. F., Gramer, M. J., Andersen, D. C., Bahr, J. B., and Rasmussen, J. R. (1991) The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Biotechnology (N Y) 9, 1347-1355. (14) Borys, M. C., Linzer, D. I., and Papoutsakis, E. T. (1993) Culture pH affects expression rates and glycosylation of recombinant mouse placental lactogen proteins by Chinese hamster ovary (CHO) cells. Biotechnology (N.Y.) 11, 720-724. (15) Gawlitzek, M., Ryll, T., Lofgren, J., and Sliwkowski, M. B. (2000) Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. Biotechnol. Bioeng. 68, 637-646. (16) Yang, M., and Butler, M. (2000) Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation. Biotechnol. Bioeng. 68, 370-380.

(17) Nakamura, S., Kato, A., and Kobayashi, K. (1991) New antimicrobial characeteristics of lysozyme-dextra conjugate. J. Agric. Food Chem. 39, 647-650. (18) Kato, A., Sasaki, Y., Furuta, R., and Kobayashi, K. (1990) Functional protein-polysaccharide conjugate prepared by controlled dry-heating of ovalbumin-dextran mixture. Agric. Biol. Chem. 54, 107-12. (19) Kumagai, I., and Miura, K. (1989) Enhanced bacteriolytic activity of hen egg-white lysozyme due to conversion of Trp62 to other aromatic amino acid residues. J. Biochem. (Tokyo) 105, 946-948. (20) Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. (21) Kato, A., Tanimoto, S., Muraki, Y., Kobayashi, K., and Kumagai, I. (1992) Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering. Biosci. Biotechnol. Biochem. 56, 1424-1428. (22) Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163-168. (23) Toh-e, A., and Wickner, R. B. (1981) Curing of the 2 mu DNA plasmid from Saccharomyces cereVisiae. J. Bacteriol. 145, 14211424. (24) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (25) Zacharius, R. M., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 30, 148-152. (26) Tarentino, A. L., and Maley, F. (1974) Purification and properties of an endo-beta-N-acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem. 249, 811-817. (27) Parry, R. M., Jr., Chandan, R. C., and Shahani, K. M. (1965) A Rapid and Sensitive Assay of Muramidase. Proc. Soc. Exp. Biol. Med. 119, 384-386. (28) Imoto, T., and Yagishita, K. (1971) A simple activity measurement of lysozyme. Agric. Biol. Chem. 35, 1154-1156. (29) Pearce, K., and Kinsella, J. (1978) Emulsifying properties of protein:Evaluation of a turbidimetric technique. J. Agric. Food Chem. 26, 716-723. (30) Kobata, A. (1979) Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal. Biochem. 100, 1-14. (31) Ballou, L., Hernandez, L. M., Alvarado, E., and Ballou, C. E. (1990) Revision of the oligosaccharide structures of yeast carboxypeptidase Y. Proc. Natl. Acad. Sci. U.S.A. 87, 3368-3372. (32) Ballou, C. E., and Raschke, W. C. (1974) Polymorphism of the somatic antigen of yeast. Science 184, 127-134. (33) Eraso, P., and Gancedo, C. (1987) Activation of yeast plasma membrane ATPase by acid pH during growth. FEBS Lett. 224, 187192. (34) Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) Saccharomyces cereVisiae mutants that make mannoproteins with a truncated carbohydrate outer chain. J. Biol. Chem. 255, 5986-5991. (35) Kato, A., and Yutani, K. (1988) Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase alpha-subunits substituted at the same position. Protein Eng. 2, 153-156. (36) Kato, A., and Nakai, S. (1980) Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochim. Biophys. Acta. 624, 13-20. (37) Mitra, N., Sinha, S., Ramya, T. N., and Surolia, A. (2006) N-linked oligosaccharides as outfitters for glycoprotein folding, form and function. Trends Biochem. Sci. 31, 156-163. (38) Nakamura, S., Hata, J., Kawamukai, M., Matsuda, H., Ogawa, M., Nakamura, K., Jing, H., Kitts, D. D., and Nakai, S. (2004) Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast. Bioconjugate Chem. 15, 1289-1296. BC0600970

Preparation of Bioactive and Surface Functional Oligomannosyl

Recommend Documents