muramidase and chitotriose

Although information about invertebrate lysozymes is scarce, these enzymes have been described as components of the innate immune system, functioning as antibacterial proteins. Here we describe the first thermodynamic and structural study of a new C-type lysozyme from a Pacific white shrimp Litopenaeus vannamei (LvL), which has shown high activity against both Gram (+) and Gram (-) bacteria including Vibrio sp. that is one of the most severe pathogens in penaeid shrimp aquaculture. Compared with hen egg-white lysozyme, its sequence harbors a seven-residue insertion from amino acid 97 to 103, and a nine-residue extension at the C-terminus only found in penaeid crustaceans, making this enzyme one of the longest lysozyme reported to date. LvL was crystallized in the presence and absence of chitotriose. The former crystallized as a monomer in space group P6

Topics: Amino Acid Sequence; Animals; Calorimetry; Chickens; Ducks; Gram-Negative Bacteria; Gram-Positive Bacteria; Immunity, Innate; Molecular Docking Simulation; Muramidase; Penaeidae; Protein Binding; Protein Structure, Tertiary; Trisaccharides; Vibrio

It has become clear that the binding of small and large ligands to proteins can invoke significant changes in side chain and main chain motion in the fast picosecond to nanosecond timescale. Recently, the use of a "dynamical proxy" has indicated that changes in these motions often reflect significant changes in conformational entropy. These entropic contributions are sometimes of the same order as the total entropy of binding. Thus, it is important to understand the connections amongst motion between the manifold of states accessible to the native state of proteins, the corresponding entropy, and how this impacts the overall energetics of protein function. The interaction of proteins with carbohydrate ligands is central to a range of biological functions. Here, we examine a classic carbohydrate interaction with an enzyme: the binding of wild-type hen egg white lysozyme (HEWL) to the natural, competitive inhibitor chitotriose. Using NMR relaxation experiments, backbone amide and side chain methyl axial order parameters were obtained across apo and chitotriose-bound HEWL. Upon binding, changes in the apparent amplitude of picosecond to nanosecond main chain and side chain motions are seen across the protein. Indeed, binding of chitotriose renders a large contiguous fraction of HEWL effectively completely rigid. Changes in methyl flexibility are most pronounced closest to the binding site, but average to only a small overall change in the dynamics across the protein. The corresponding change in conformational entropy is unfavorable and estimated to be a significant fraction of the total binding entropy.

Topics: Animals; Binding Sites; Chickens; Entropy; Ligands; Models, Molecular; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Trisaccharides

High-sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg-white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15 degrees C -45 degrees C temperature range. Using a structural-energetic parameterization derived previously for lectin-carbohydrate associations, both binding enthalpies and entropies for the present systems and for the complex of chitobiose with turkey egg-white lysozyme from the literature were correctly accounted for. These observations suggest that both lysozymes and lectins follow the same structural-energetic behavior in the binding to their ligands. From the analysis of lysozyme data in conjunction with other binding data reported in the literature, an ad hoc parameterization of DeltaCp for protein-carbohydrate complexes was derived for the first time. The novel parameters for both polar and apolar surface areas differed significantly from correlations obtained previously from model compounds and protein-folding data. As DeltaCp is extremely sensitive to changes in solvent structure, this finding indicates that protein-carbohydrate complexes have distinctive hydration properties. According to our analysis, the dehydration of polar groups is the major cause for the observed decrease in DeltaCp, which implies that these groups behave hydrophobically. The contribution of apolar surface areas was found of the expected sign, but their specific weight is much smaller than those obtained in other correlations. This small contribution to DeltaCp is consistent with Lemieux's hypothesis of a low degree of hydration of apolar surfaces on carbohydrates.

Topics: Animals; Calorimetry; Chickens; Disaccharides; Egg White; Enzyme Inhibitors; Hydrophobic and Hydrophilic Interactions; Muramidase; Protein Binding; Protein Folding; Temperature; Thermodynamics; Trisaccharides; Water

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the beta- agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation-contraction coupling in sepsis.

Topics: Adrenergic Antagonists; Adrenergic beta-Agonists; Animals; Dogs; Escherichia coli Infections; Heart; Isometric Contraction; Isoproterenol; Muramidase; Myocardial Contraction; Shock, Septic; Spleen; Trisaccharides

A mutant of alpha-lactalbumin was expressed and purified, in which His32, Thr33, Glu49, Ile59, Val99, and Tyr103 were substituted by Leu32, Glu33, Asp49, Trp59, Asn99, and Ala103, respectively, to create a catalytic site of lysozyme in alpha-lactalbumin. The mutant catalyzed hydrolysis of the synthetic substrate, pNP-(NAcGlc)(3), with a K(M) and k(cat) of 0.160 +/- 0.00986 mmol/L and 3.39 +/- 0. 0456 x10(-5) min(-1), respectively, which was comparable with those of chicken lysozyme of 0.137 +/- 0.0153 mmol/L and 5.25 +/- 0.115 x10(-4) min(-1). By using the Isothermal Titration Calorimetre (ITC), the average binding enthalpy of the mutant or chicken lysozyme with the substrate (chitopentaose) was measured, which was 49.22 KJ/mol for the mutant and 105.47 KJ/mol for chicken lysozyme. In conclusion, the six point mutations occurring in alpha-lactalbumin could be converted into an enzyme that was 17.5-fold less efficient than chicken lysozyme but nevertheless capable of hydrolyzing the glycosidic bond.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Calorimetry, Differential Scanning; Catalytic Domain; Cattle; Chickens; Evolution, Molecular; Hydrolysis; Kinetics; Lactalbumin; Molecular Sequence Data; Muramidase; Mutagenesis, Site-Directed; Protein Binding; Sequence Alignment; Substrate Specificity; Thermodynamics; Trisaccharides

N,N',N"-Tri(monochloro)acetylchitotriose prepared by N-monochloroacetylation of chitotriose trihydrochloride was successfully polymerized into higher-molecular-weight oligomers by a lysozyme-catalyzed transglycosylation reaction, and a following base-catalyzed N-demonochloroacetylation gave a chitosan oligomer mixture mainly composed of oligomers with dp > 6. Partially N-deacetylated chitin oligomers (DAC oligomers) with dp 4-12 were synthesized by the enzyme reaction using N,N',N"-tri(monochloro)acetylchitotriose and N,N',N"-triacetylchitotriose (chitin trimer) as initial substrates followed by N-demonochloroacetylation. The structures of synthetic oligomers were analyzed by 1H NMR spectroscopy, enzymatic hydrolysis and nitrous acid deamination-NaBH4 reduction treatment. The dp of synthetic oligomers was measured by MALDI TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) using per-N-acetylated derivatives. The synthetic chitosan and DAC oligomers were strong elicitors for phytoalexin induction in Pisum sativum and Phaseolus vulgaris. This chemo-enzymatic method utilizing N-acylated chitotrioses as substrates is a novel approach to the synthesis of high-molecular-weight chitosan oligomers and DAC oligomers of biological importance.

Topics: Acetylation; Benzopyrans; Carbohydrate Sequence; Chitin; Chitosan; Fabaceae; Glycosylation; Isoflavones; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Muramidase; Oligosaccharides; Phytoalexins; Pisum sativum; Plant Extracts; Plants, Medicinal; Pterocarpans; Sesquiterpenes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Terpenes; Trisaccharides

Gaucher disease (GD; glucosylceramidosis) is caused by a deficient activity of the enzyme glucocerebrosidase (GC). Clinical manifestations are highly variable and cannot be predicted accurately on the basis of the properties of mutant GC. Analysis of secondary abnormalities, such as elevated plasma levels of some hydrolases, may help to increase insight into the complicated pathophysiology of the disease and could also provide useful disease markers. The recent availability of enzyme supplementation therapy for GD increases the need for markers as early predictors of the efficacy of treatment. We report the finding of a very marked increase in chitotrisidase activity in plasma of 30 of 32 symptomatic type 1 GD patients studied: the median activity being > 600 times the median value in plasma of healthy volunteers. In three GC-deficient individuals without clinical symptoms, only slight increases were noted. Chitotriosidase activity was absent in plasma of three control subjects and two patients. During enzyme supplementation therapy, chitotriosidase activity declined dramatically. We conclude that plasma chitotriosidase levels can serve as a new diagnostic hallmark of GD and should prove to be useful in assessing whether clinical manifestations of GD are present and for monitoring the efficacy of therapeutic intervention.

Topics: Adolescent; Adult; Aged; Alkaline Phosphatase; Child; Child, Preschool; Female; Gaucher Disease; Hexosaminidases; Humans; Male; Middle Aged; Muramidase; Trisaccharides

Interaction between hen egg white lysozyme and chitotrisaccharide was investigated by 1H-NMR spectroscopy using partially acetylated chitotrisaccharides and chemically modified lysozyme. Monoacetyl (GlcN-GlcN-GlcNAc), diacetyl (GlcN-GlcNAc-GlcNAc), or triacetyl chitotrisaccharide [(GlcNAc)3] was added to the lysozyme solution, and the changes in the 1H-NMR signals of the lysozyme were analyzed. Although many of the resonances were affected by addition of the saccharide, the most remarkable effect was seen on the signal of Trp28 C5H which is in a hydrophobic box adjacent to the saccharide-binding site. The signal shifted upfield by 0.2 ppm upon (GlcNAc)3 binding, whereas the chemical shift change of the signal resulting from binding of GlcN-GlcNAc-GlcNAc or GlcN-GlcN-GlcNAc was smaller than that resulting from (GlcNAc)3 binding. When the Asp101-modified lysozyme was used instead of the native lysozyme, the chemical shift change of the Trp28 C5H signal resulting from (GlcNAc)3 binding was also smaller than that for the native lysozyme. The chemical shift change of the signal reflects the conformational change of the hydrophobic box region which should synchronize with the movement of the binding site resulting from the saccharide binding. Therefore, the conformational change resulting from the saccharide binding might be reduced when the sugar residues located at binding subsites A and B of the lysozyme are deacetylated, as well as when Asp101 interacting with the sugar residues at the same subsites is modified.

Topics: Animals; Binding Sites; Carbohydrate Sequence; Chickens; Egg White; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Muramidase; Protons; Trisaccharides

In a previous report from this laboratory (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985), evidence was presented to suggest that the bactericidal actions of both reduced (i.e., muramidase-inactive) human placental lysozyme and the synthetic cationic homopolymer poly-D-lysine involved the activation of a bacterial endogenous activity that was inhibitable by N,N',N"-triacetylchitotriose (chitotriose). In the present investigation however, we found that the bactericidal and bacteriolytic action of poly-D-lysine could be prevented only by some commercially available chitotriose preparations and not by others. Analysis by physical and chemical methods failed to distinguish protective chitotriose (CTa) and nonprotective chitotriose (CTi) preparations. CTi and CTa preparations displayed equal capacities to competitively inhibit binding of [3H]chitotriose by immobilized lysozyme and were indistinguishable in their abilities to block the lytic activity of lysozyme against Micrococcus lysodeikticus cells. Elemental analysis revealed significantly higher levels of phosphorus, calcium, iron, sodium, manganese, and copper in CTa. Removal of metals from CTa by chelate chromatography completely abolished the poly-D-lysine-protective capacity. Of the metals detected, only ferric iron (5 to 10 microM) mimicked the protective action of CTa. A Fe(III) concentration of 50 microM was required to inhibit lysozyme (5 micrograms/ml). Both Fe(III) and CTa (but not CTi) quantitatively blocked the labeling of poly-D-lysine by fluorescamine, suggesting that the primary amino groups of the lysine residues participate in iron binding. Thus, it appears that the poly-D-lysine-protective capacity of certain chitotriose preparations was due not to the chitotriose itself but to contaminating metal ions which interact directly with the polycationic agent. In contrast, Fe(III) cannot account for inhibition of either the bactericidal or bacteriolytic activity of lysozyme by chitotriose.

Topics: Bacteria; Bacteriolysis; Cell Wall; Iron; Muramidase; Polylysine; Trisaccharides

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Binding, Competitive; Calcium; Cell Membrane; Cytidine Monophosphate N-Acetylneuraminic Acid; Female; G(M1) Ganglioside; Gangliosides; HeLa Cells; Hexosyltransferases; Horseradish Peroxidase; Humans; Intracellular Membranes; Isoenzymes; Kinetics; Ligands; Microchemistry; Muramidase; Sialyltransferases; Trisaccharides; Uridine Diphosphate Galactose

The basis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidase-inactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of muramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe.

Topics: Bacteriolysis; Disaccharides; Enterococcus faecalis; Glucans; Humans; Muramidase; Oligosaccharides; Peptides; Streptococcus sanguis; Trisaccharides

Differences in the binding of the substrate analogue chitotriose to lysozymes correlate with amino acid substitutions in the binding site and not with substitutions elsewhere. This is evident from binding studies done with an immunological method as well as a conventional spectroscopic method. The immunological technique, based on the microcomplement fixation assay, required thousands of times less lysozyme than did the conventional technique. For eight bird lysozymes of known amino acid sequence, the immunologically and physically measured association constants were in approximate agreement. Five of the eight lysozymes have about the same affinity for chitotriose and have identical amino acids at the sites of contact between substrate and enzyme. In contrast, the three lysozymes that have altered affinities have amino acid substitutions in the binding site. Some of the lysozymes with similar affinities for chitotriose differ greatly in amino acid sequence outside the binding site. This suggests that evolutionary substitutions do not generally have long-range effects on the active site region of lysozyme.

Topics: Animals; Binding Sites; Birds; Chickens; Complement Fixation Tests; Ducks; Egg White; Kinetics; Muramidase; Protein Binding; Quail; Species Specificity; Spectrophotometry, Ultraviolet; Trisaccharides; Turkeys