Functional properties of glycosylated lysozyme secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

Structural and functional properties of hen egg-white lysozyme deamidated by protein engineering.

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (delta G) at 20 degrees C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Studies on the “Salvage” Synthesis of Ribonucleosides and their 5′-Phosphates by Microorganisms

The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (ΔG) at 20°C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.     

Functional Properties of Glycosylated Lysozyme Secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn¹⁹-Tyr²⁰-Thr²¹), G49N (Asn⁴⁹- Ser⁵⁰-Thr⁵¹), R21T/G49N (Asn¹⁹-Tyr²⁰-Thr²¹/Asn⁴⁹-Ser⁵⁰-Thr⁵¹), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc₂Man_9-11, while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc₄Man_27-32. The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

Redesign of the substrate-binding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes.

On the basis of the molecular evolution of hen egg white, human, and turkey lysozymes, three replacements (Trp62 with Tyr, Asn37 with Gly, and Asp101 with Gly) were introduced into the active-site cleft of hen egg white lysozyme by site-directed mutagenesis. The replacement of Trp62 with Tyr led to enhanced bacteriolytic activity at pH 6.2 and a lower binding constant for chitotriose. The fluorescence spectral properties of this mutant hen egg white lysozyme were found to be similar to those of human lysozyme, which contains Tyr at position 62. The replacement of Asn37 with Gly had little effect on the enzymatic activity and binding constant for chitotriose. However, the combination of Asn37----Gly (N37G) replacement with Asp101----Gly (D101G) and Trp62----Tyr (W62Y) conversions enhanced bacteriolytic activity much more than each single mutation and restored hydrolytic activity toward glycol chitin. Consequently, the mutant lysozyme containing triple replacements (N37G, W62Y, and D101G) showed about 3-fold higher bacteriolytic activity than the wild-type hen lysozyme at pH 6.2, which is close to the optimum pH of the wild-type enzyme.     

Fluctuations in free or substrate-complexed lysozyme and a mutant of it detected on x-ray crystallography and comparison with those detected on NMR

A mutant lysozyme in which Arg14 and His15 were deleted together exhibited higher activity toward glycol chitin than the wild-type lysozyme. Moreover, the mutant lysozyme, which is less stable than the wild-type lysozyme by 7 degrees C, showed a shift of temperature dependence of activity to the low temperature side compared with the wild-type lysozyme [Protein Eng. 7, 743-748 (1994)]. In the free enzyme, the internal motion of the mutant lysozyme was similar to that of the wild-type. The internal motions of the wild-type and mutant lysozymes in the enzyme-substrate complex increased more than those in the free enzymes. Moreover, the increased internal motions of the substrate-complexed mutant lysozyme were greater than those of the substrate-complexed wild-type lysozyme in several residues [J. Mol. Biol. 286, 1547-1565 (1999)]. The structure of the mutant lysozyme was very similar to that of the wild-type lysozyme. Both structures were also alike in the complex of the trimer of N-acetyl-D-glucosamine. The mobility from B-factors agreed to some degree with that from order parameters in the regions showing great mobility of the protein, but this was not the case in the regions showing fast motion. However, we came to the same conclusion that the increased activity of the mutant lysozyme is due to the increase in the fluctuation of the lysozyme molecule. B-factor and order parameter do not always exhibit harmony because the time-scale of the analysis of mobility is different. However, they are not incompatible but complementary for detecting precise protein motions.     

Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft

Substitution of Thr26 by Gln in the lysozyme of bacteriophage T4 produces an enzyme with greatly reduced activity but essentially unaltered stability relative to wild type. Spontaneous second-site revertants of the mutant were selected genetically; two of them were chosen for structural and biochemical characterization. One revertant bears (in addition to the primary mutation) the substitution Tyr18----His, the other, Tyr18----Asp. The primary mutant and both revertant lysozyme genes were reconstructed in a plasmid-based expression system, and the proteins were produced and purified. The two revertant lysozymes exhibit enzymatic activities intermediate between wild type and the primary mutant; both also exhibit melting temperatures approximately 3 degrees C lower than either the wild type or the primary mutant. Crystals suitable for X-ray diffraction analysis were obtained from both revertant lysozymes, but not the primary mutant. Structures of the double mutant lysozymes were refined at 1.8-A resolution to crystallographic residuals of 15.1% (Tyr18----His) and 15.2% (Tyr18----Asp). Model building suggests that the side chain of Gln26 in the primary mutant is forced to protrude into the active site cleft, resulting in low catalytic activity. In contrast, the crystal structures of the revertants reveal that the double substitutions (Gln26 and His18, or Gln26 and Asp18) fit into the same space that is occupied by Thr26 and Tyr18 in the wild-type enzyme; the effect is a restructuring of the surface of the active site cleft, with essentially no perturbation of the polypeptide backbone. This restructuring is effected by a novel series of hydrogen bonds and electrostatic interactions that apparently stabilize the revertant structures.     

Preparation of bioactive and surface functional oligomannosyl neoglycoprotein using extracellular pH-sensitive glycosylation of mutant lysozyme having N-linked signal sequence in yeast

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 (Man(310)GlcNAc(2)) chain-linked lysozyme was predominantly expressed accompanied by small amounts of a core-type oligomannose chain (Man(14)GlcNAc(2))-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 degrees 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.     

Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes

Both glycosylated amyloidogenic lysozymes I55T/G49N and D66H/G49N were expressed in wild-type and calnexin-disrupted Saccharomyces cerevisiae. The secretion amounts of mutant I55T/G49N were almost similar in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of mutant D66H/G49N greatly increased in calnexin-disrupted S. cerevisiae, while the secretion was very low in the wild-type strain. In parallel, the induction level of the molecular chaperones BiP and PDI located in the endoplasmic reticulum (ER) was investigated when these glycosylated amyloidogenic lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when mutant lysozyme D66H/G49N was expressed in calnexin-disrupted S. cerevisiae, while they were not so increased when I55T/G49N mutant was expressed. This observation indicates that the conformation of mutant lysozyme D66H/G49N was less stable in the ER, thus leading to the higher-level expression of ER molecular chaperones via the unfolded protein response pathway. This suggests that glycosylated amyloidogenic lysozyme I55T/G49N may have a relatively stable conformation in the ER, thus releasing it from the quality control of calnexin compared with mutant lysozyme D66H/G49N.     

An Analysis of the Interaction of Sodium Dodecyl Sulfate with Lysozyme

The interaction of SDS with lysozyme was analyzed with enzyme activity and with NMR, fluorescence, and UV difference spectroscopies using various alkyl sulfates and variously modified lysozymes. SDS formed a stable complex with lysozyme without causing a gross conformational change in the enzyme molecule. Some SDS molecules bound to the active site cleft of lysozyme and therefore strongly inhibited the activity of lysozyme. Hydrophobic regions and positive charges for protein side, and a hydrophobic tail (possibly more than 8 carbons in alkyl chain) and a negative charge for detergent side were required for the formation of the complex.     

Relationship between the Stability of Hen Egg-White Lysozymes Mutated at Sites Designed to Interact with α-Helix Dipoles and Their Secretion Amounts in Yeast

The positively charged lysine at the C-terminals of three long α-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change ΔG of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long α-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and ΔG of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long α-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.     

Purification, characterization and comparison of reptile lysozymes

Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle (Trionyx sinensis), ASTL from Asiatic soft shelled turtle (Amyda cartilagenea) and GSTL from green sea turtle (Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 degrees C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.     

Purification and characterization of the lysozyme from the gut of the soft tick Ornithodoros moubata

The gut of the adult soft ticks Ornithodoros moubata displays high lytic activity against the bacteria Micrococcus luteus. The activity differed in the range of two orders of magnitude among individual animals and increased on average 4 fold during the first week following ingestion. In homogenates of first instar nymphs the activity was much lower increasing exponentially as nymphs neared the first molt. The protein responsible for this activity was purified out of gut contents of adult ticks by means of affinity adsorption on magnetic-chitin followed by two chromatography steps on cation exchange FPLC column MonoS. The homogeneous active protein has a mass of 14006 +/- 20 Daltons as determined by MALDI-TOF mass spectrometry. The N-terminal amino-acid sequence of this protein is K-V-Y-D-R-C-S-L-A-S-E-L-R with the highest similarity to the lysozyme from liver of rainbow trout and to lysozymes from digestive tracts of several mammals. The motif DRCSLA is specific for the digestive lysozymes of several dipteran insects. Based on this evidence, we have identified the protein as the tick gut lysozyme. The tick gut lysozyme has a pI near 9.7 and retains its full activity after treatment at 60 degrees C for 30 minutes. The pH optimum of the tick lysozyme was in the range from pH 5-7. Only marginal activity could be detected at pH > 8 which raises the question about the function of lysozyme in anti-bacterial defense in the environment of the tick gut.     

Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with alpha-helix dipoles and their secretion amounts in yeast

The positively charged lysine at the C-terminals of three long alpha-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change Delta G of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long alpha-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and Delta G of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or C-terminal sites of the three long alpha-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability.     

Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.     

Amyloid fibril formation of hen lysozyme depends on the instability of the C-helix (88-99)

Stable and unstable mutant lysozymes in long helices B and C were constructed to evaluate the effect of the helices on amyloid fibril formation at pH 2. Stable mutant N27D and unstable mutant K33D in the B-helix did not change in amyloid fibril formation. In contrast, stable mutant N93D and unstable mutant K97D in the C-helix showed big differences in behavior as to amyloid fibril formation. Stable mutant N93D showed a longer lag phase of aggregation and suppressed the amyloid fibril formation, whereas unstable mutant K97D showed a shorter lag phase of aggregation and accelerated amyloid fibril formation. These results suggest that the long C-helix is involved mainly in the alpha-helix to beta-sheet transition during amyloid formation of lysozyme.     

Effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of glycosylated lysozymes.

Disruption of the calnexin gene in Saccharomyces cerevisiae did not lead to gross effects on the levels of cell growth and secretion of wild-type hen egg white lysozymes (HEWL). To investigate the function of calnexin in relation to the secretion of glycoproteins, we expressed both stable and unstable mutant glycosylated lysozymes in calnexin-disrupted S. cerevisiae. The secreted amounts of stable mutant glycosylated lysozymes (G49N and S91T/G49N) were almost the same in both wild-type and calnexin-disrupted S. cerevisiae. In contrast, the secretion of unstable mutant glycosylated lysozymes (K13D/G49N, C76A/G49N, and D66H/G49N) greatly increased in calnexin-disrupted S. cerevisiae, although their secretion was very low in the wild-type strain. This indicates that calnexin may act in the quality control of glycoproteins. We further investigated the expression level of the mRNA of the molecular chaperones BiP and PDI, which play a major role in the protein folding process in the ER, when glycosylated lysozymes were expressed in wild-type and calnexin-disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when the glycosylated lysozymes were expressed in calnexin-disrupted S. cerevisiae. This observation indicates that BiP and PDI may be induced by the accumulation of unfolded glycosylated lysozymes due to the deletion of calnexin.     

Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.     

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.     

Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme

X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a "safe" substitution.