Binding of a highly de-N-acetylated chitosan to Japanese pheasan lysozyme as measured by 1H-NMR spectroscopy.

Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114-->His), was investigated by 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gammaCH, as in the case of binding to HEWL. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23+/-0.05 mm (-31.5 kJ/mol), which is similar to that of HEWL (0.11+/-0.02 mm, -33.3 kJ/mol). Thus, the Arg-->His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantly affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.

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.

The amino acid sequence of wood duck lysozyme.

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.

The amino acid sequence of monal pheasant lysozyme and its activity.

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).

Preparation and partial structural characterization of alpha1T-glycoprotein from normal human plasma.

alpha1T-glycoprotein (alpha1T) was isolated from normal human plasma in the immunochemically homogeneous state. The partial amino acid sequence and carbohydrate chains of this glycoprotein were determined. To achieve this, the carboxymethylated alpha1T was analyzed by sequencing some of the lysylendoprotease, V8 protease, tryptic, and cyanogen bromide peptides as well as the N-terminal sequence of the protein. A large number of amino acid residues (460 amino acids) was determined by chemical procedure. The peptide sequences were compared with that of other proteins. A high degree of homology was found for proteins of the albumin family. Further, human alpha-albumin, a new member of this protein family, showed an amino acid sequence identical to that of alpha1T indicating that these two proteins are very similar in amino acid sequence and composition. These proteins are closely related to alpha-fetoprotein; however, five carbohydrate chains were found on alpha1T at Asn12, Asn88, Asn362, Asn381, and Asn467 as biantennary complex type chains and the chain on Asn362 possessed a rare consensus sequence of Asn-X-Cys. Thus, alpha1T distinguishes itself by possessing five N-glycans, a finding reported here for the first time for the ALB family.

Reptile lysozyme: the complete amino acid sequence of soft-shelled turtle lysozyme and its activity.

Soft-shelled turtle egg-white lysozyme was purified and sequenced. Lysozyme was reduced and carboxymethylated to fragment it with trypsin, V8 protease and CNBr. The peptides yielded were purified by RP-HPLC and sequenced. Every trypsin peptide was overlapped by V8 protease peptides and CNBr fragment. The amino acid sequence was compared with other lysozymes. This lysozyme has an extra Gly residue at N-terminus, which was found in pheasant lysozyme. Further, this lysozyme has an insertion of a Gly residue between 47 and 48 residues when compared with chicken lysozyme, as found in human lysozyme, therefore it proved that this lysozyme has the largest number of amino acids (131 aa) in chicken type lysozymes. The amino acid substitutions were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114 were replaced by His, Tyr, Arg, and Tyr, respectively. The time course using N-acetylglucosamine pentamer as a substrate showed a reduction of the rate constant for glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution of amino acids mentioned above for substrate binding at subsites E and F.

Positions of disulfide bonds in yam (Dioscorea japonica) acidic class IL (class IV) chitinase.

Yam acidic class I chitinase belongs to a low molecular weight subclass of class I (class IL; corresponds to class IV) chitinase. The positions of disulfide bonds in this chitinase were examined. Chitinase protein was digested with acid protease and thermolysin, and the resulting disulfide bond containing peptides were separated by reversed-phase HPLC and detected using the SBD-F (7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid ammonium salt) method. Four intradisulfide bonds containing peptides were purified and three disulfide bonds in the catalytic domain were identified as Cys-66 and Cys-115, Cys-128 and Cys-136, and Cys-218 and Cys-250. Location of disulfide bonds in the catalytic domain was identical to that of barley class II chitinase but different from rye class II chitinase at the C-terminal. Conservation of S-S bonds at the N-terminal half of the catalytic domain between class I and class II chitinases strongly suggests that this region is important for formation of the active site.

Structural classification of plant chitinases: two subclasses in class I and class II chitinases.

For the classification of plant chitinases, phylogenetic relationships were analyzed besides the established classification of domain structure and C-terminal extension sequences. Two genetically different subclasses (high and low molecular weight) were found for the main structure of class I and class II chitinases in their phylogenetic trees. The genetic distance of these subclasses showed that the high molecular weight subclass may be an ancestral molecule.

Binding mode of N,N',N",N"'-tetraacetylchitotetraitol to hen egg white lysozyme.

The binding of N,N',N",N"'-tetraacetylchitotetraitol [(GlcNAc)4-ol] to hen egg white lysozyme was investigated by fluorescence and 1H NMR spectroscopy. From observation of changes in the fluorescence intensity, the association constants of (GlcNAc)4-ol and (GlcNAc)3 were found to be 0.70 x 10(5) and 1.07 x 10(5) M-1, respectively, at pH 5.0 and 30 degrees C. The lack of a substantial difference between the association constants suggests that the binding mode of the (GlcNAc)3 moiety of (GlcNAc)4-ol is basically similar to that of (GlcNAc)3, but that the N-acetylglucosaminitol residue of (GlcNAC)4-ol does not interact significantly with lysozyme. On the other hand, 1H NMR spectroscopy revealed a minor difference in the binding modes of the two saccharides. For most of the 1H signals responding to saccharide binding, such as those of Trp 63 H2, Trp 28 H5, and Ile 98 H gamma 1, the chemical shift changes induced by (GlcNAc)4-ol were almost identical to those induced by (GlcNAc)3. However, the effect of binding on the signals of Asn 59 H alpha and Trp 108 indole N1H, which are located near subsite C, was different for (GlcNAc)4-ol and (GlcNAc)3. Thus it is inferred that the binding mode of the first sugar residue of (GlcNAc)4-ol to subsite C is somewhat different from that of (GlcNAc)3.

The amino acid sequence of copper pheasant lysozyme.

The amino acid sequence of copper pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 20, 77, and 113 for Lady Amherst's pheasant lysozyme and seven amino acid substitutions at positions 3, 15, 20, 41, 113, 121, and 124 for hen lysozyme. Phenylalanine at position 20 was newly detected in avian lysozyme.

The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase.

The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber.

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.

1H-NMR study on the structure of lysozyme from guinea hen egg white.

The structure of lysozyme from guinea hen egg white (GEWL), which differs from hen egg white lysozyme (HEWL) by ten amino acid substitutions, was investigated by nuclear magnetic resonance (NMR) spectroscopy. GEWL and HEWL were very similar to each other in their tertiary structure as judged from the profile of 1H-NMR spectra, pH titration, and an N-acetylglucosamine trisaccharide [(GlcNAc)3 binding experiment. However, we have noticed several characteristics which distinguish GEWL from HEWL. The signal of Trp 108 indole N1H of GEWL was shifted upfield by about 0.3 ppm when compared with that of HEWL, and its hydrogen exchange was faster than that of HEWL. The pKa values of Glu 35 estimated from the pH titration curve of Trp 108 indole N1H were different between GEWL and HEWL. From a careful examination of spectral changes caused by (GlcNAc)3 binding, the changes in the chemical shift values of Trp 28 C5H and Asn 59 alpha CH of GEWL were found to be slightly larger than those of HEWL. Ile 55 of HEWL is replaced by valine in GEWL. Such a replacement may affect the neighboring hydrogen bonding between the main chain C = O of Leu 56 and Trp 108 indole N1H, resulting in a change in the microenvironment of the substrate-binding site near Trp 108.

The amino acid sequence of lysozyme from kalij pheasant (Lophura leucomelana) egg-white.

The amino acid sequence of kalij pheasant lysozyme has been analyzed. From the comparison of the tryptic peptide pattern of kalij pheasant lysozyme and maps from other bird lysozymes followed by the sequencing of tryptic peptides, the amino acid sequence of kalij pheasant was found to be: KVYGRCELAAAMKRLGLDNYRGYSLGNWVCAAKYESNFNTHATNRNTDGSTDYGIL- QINSRWWCNDGKTPGSRNLCHIPCSALLSSDITASVNCAKKIVSDGNGMNAW- VAWRNRCKGTDVSVWTRGCRL. This sequence had 9 amino acid substitutions compared with hen egg-white lysozyme. Two of these substitutions, positions 34 and 121, were newly detected in phasianid birds. The protein genealogy of phasianid bird lysozymes showed some discordance with the morphological classification of these birds.

The amino acid sequence of reeves' pheasant (Syrmaticus reevesii) lysozyme.

The amino acid sequence of reeves' pheasant lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and resulting peptides were analyzed using the DABITC/PITC double coupling manual Edman method. The established amino acid sequence had seven substitutions, Tyr3, Leu15, His41, His77, Ser79, Arg102, and Asn121, compared with hen egg-white lysozyme. Ser79 was the first found in a bird lysozyme. A substitution in the active site was found in position 102 which has been considered to participate in the substrate binding at subsites A-C.

The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes.

The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group.

State of binding subsites in Asp 101-modified lysozymes.

The environments of the binding subsites in Asp 101-modified lysozyme, in which glucosamine or ethanolamine is covalently bound to the carboxyl group of Asp 101, were investigated by chemical modification and nuclear magnetic resonance spectroscopy. Trp 62 in each of the native and the modified lysozymes was nitrophenylsulfenylated. The yield of the nitrophenylsulfenylated derivative from the lysozyme modified with glucosamine at Asp 101 (GlcN-lysozyme) was considerably lower than those from native lysozyme and from the lysozyme modified with ethanolamine at Asp 101 (EtN-lysozyme). These results suggest that Trp 62 in GlcN-lysozyme is less susceptible to nitrophenylsulfenylation. Kinetic analyses of the [Trp 62 and Asp 101]-doubly modified lysozymes indicated that the nitrophenylsulfenylation of Trp 62 in the native lysozyme, EtN-lysozyme, or GlcN-lysozyme decreased the sugar residue affinity at subsite C while increasing the binding free energy change by 2.7 kcal/mol, 1.5 kcal/mol, or 0.1 kcal/mol, respectively. Although the profile of tryptophan indole NH resonances in the 1H-NMR spectrum for EtN-lysozyme was not different from that for the native lysozyme, the indole NH resonance of Trp 62 in GlcN-lysozyme was apparently perturbed in comparison with that of native lysozyme. These results suggest that the environment of subsite C in GlcN-lysozyme is considerably different from those in native lysozyme and EtN-lysozyme. The glucosamine residue attached to Asp 101 may contact the sugar residue binding site of the lysozyme, affecting the environment of subsite C.

Enzymatic activity of avian egg-white lysozymes.

The experimental time-courses of eight avian lysozymes, seven hen-type lysozymes and one goose-type lysozyme, were measured with a substrate of chitopentaose (GlcNAc)5 at pH 5.0 and 50 degrees C. Chitooligosaccharides in the reaction mixture were analyzed by high-performance gel-filtration. From the experimental time-courses, the overall reaction rates represented by the disappearance of the initial substrate and the values of reaction parameters were estimated by computer analysis. With taking hen lysozyme as the reference, the values of reaction parameters estimated were correlated to the replaced amino acid residue in the binding site of the lysozyme, and the roles of some amino acid residues in the binding site were discussed.

Human lysozyme-catalyzed reaction of chitooligosaccharides.

The time-courses of the human lysozyme-catalyzed reaction of chitopentaose were measured by high-performance gel-filtration in comparison with those of hen egg-white lysozyme. Human lysozyme has considerably larger rate constants for the cleavage of glycosidic linkages and transglycosylation than those of hen lysozyme, in agreement with the fact that human lysozyme exhibits a large lytic activity. It has been reported that binding subsite D in human lysozyme has negative free energy on substrate binding, whereas subsite D in hen lysozyme has unfavorable positive free energy due to the distortion of a sugar residue. The time-courses calculated under the assumption that subsite D in human lysozyme has negative free energy on substrate binding did not fit the experimentally obtained time-courses, even though the combination of values of rate constants in the enzymatic reaction widely varied in the calculation of the time-courses. Thus, it was concluded that subsite D in human lysozyme may not have negative binding free energy, but positive values similar to hen lysozyme.

Estimation of rate constants in lysozyme-catalyzed reaction of chitooligosaccharides.

The rate constants of the cleavage of glycoside linkage, hydration (hydrolysis) and transglycosylation in a lysozyme-catalyzed reaction of substrate chitooligosaccharides were evaluated by computer analysis of the experimentally obtained reaction time-courses. In the computer analysis, the rate equation was numerically solved by use of the known binding constants for each subsite. Because of the complexity of the lysozyme-catalyzed reaction, optimal values of rate constants were determined by checking the sensitivity of each rate constant to the computed time-courses. It was not possible to estimate uniquely the rate constants for transglycosylation and hydration, owing to the nature of the enzymatic reaction, but it was possible to estimate accurately their ratio. The estimated values were 0.94 s-1 for the rate constant for the cleavage of glycosidic linkage and 133 for the ratio of rate constants of transglycosylation and hydration.