Effect of periodontal treatment on the activity of chitinase in whole saliva of periodontitis patients.

Human salivary chitinase could play a role in the defence against chitin-containing oral pathogens. The activity levels of chitinase in the whole saliva of periodontitis patients were significantly higher than those in saliva from controls. Periodontal treatment for a period of 5-6 months resulted in a three- to fourfold decrease in this enzyme activity. The activity of beta-N-acetylhexosaminidase, which is another enzyme that hydrolyses glycosidic linkages, also decreased as a result of treatment, although to a lesser extent. The decrease in chitinase activity upon treatment of the disease did not correlate with the decrease that was seen in clinical attachment loss and bleeding on probing, and only a weak correlation was observed with the changes in probing pocket depth and plaque index. No correlations were found between the above clinical parameters and the decrease in beta-N-acetylhexosaminidase activity.

Chitinase in whole and glandular human salivas and in whole saliva of patients with periodontal inflammation.

In recent studies the existence of a chitinase in various mammals, like man, was described. The aim of the present study was to find out whether salivas of periodontally healthy and inflamed humans also contain chitinase activity. Chitinase activity, assayed with the substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside, was shown to be present in human whole saliva, with an activity level and apparent molecular mass (35 kDa) that were comparable with those of the human serum enzyme. Both lysozyme and beta-N-acetylhexosaminidase could be separated from chitinase by means of Bio-Gel P-100 gel filtration chromatography. The enzyme was also present in glandular saliva of parotid, palatine, submandibular and sublingual glands. The chitinase activity was not of oral epithelial, bacterial or plaque bacterial origin and was not correlated with the activity of salivary amylase. A comparative study of whole salivas of periodontally healthy controls and gingivitis and periodontitis subjects showed that only in the case of periodontitis there was a significant increase of the specific chitinase activity. The latter enzyme showed a gel filtration pattern that was comparable with that of the enzyme from controls. The measured albumin levels in saliva and the absence of correlation between the chitinase activity levels in plasma and saliva from periodontitis patients indicated that the (increased) chitinase activities did not originate from blood leakage to the oral cavity.

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus.

The presence of chitinase activity in human serum has recently been described by us. On that occasion we speculated on the possible role of mammalian chitinases as a defense mechanism against chitin-containing pathogens. The results of the present study substantiate our hypothesis. We demonstrate and partially characterize the chitinase activities that are present in plasma of guinea pigs and in homogenates of A.fumigatus with the aid of the substrates MU-[GlcNAc]2,3 and also with glycol [3H]chitin. Upon infection with A.fumigatus the serum chitinase activity levels in the circulation of pathogen-free guinea pigs increased in a time-dependent manner. The increase was also dependent on the size of the infecting fungal inoculum. Antifungal treatment diminished the increases. The increased chitinase activity was of guinea pig origin. The activity of beta-hexosaminidase showed a very slight increase subsequent to the infection. The activities of three other enzymes of lysosomal origin (alpha-mannosidase, beta-galactosidase and beta-glucosidase) did not increase.

Partial purification and further characterization of the novel endoglucosaminidase from human serum that hydrolyses 4-methylumbelliferyl-N-acetyl-beta-D-chitotetraoside (MU-TACT hydrolase).

A novel endoglucosaminidase, originally described by Den Tandt et al. [Int. J. Biochem. 20 (1988), 713-719] and bearing the provisional name MU-TACT hydrolase, was purified from human serum 56,000-fold by means of ammonium sulphate precipitation, anion-exchange chromatography, Con A-Sepharose chromatography and gel filtration on Sepharose CL-6B followed by Superose 12 HR. Based on the latter technique the native apparent molecular weight of the enzyme appeared to be equal to that of myoglobin, being approx. 17 kD. The enzyme eluted clearly at a different volume than lysozyme. MU-TACT is a commercially available substrate for lysozyme. For unknown reasons two major peptides co-purify that give bands on SDS-PAGE of 55-60 and 31 kD, respectively.

Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase).

Since 1988 an endoglucosaminidase, provisionally named MU-TACT hydrolase, has been known that hydrolyses the artificial substrate 4-methylumbelliferyl-tetra-N-acetyl-chitotetraoside (MU-[GlcNAc]4, where GlcNAc is N-acetylglucosamine). The biological function of the enzyme was unknown. In this paper evidence is presented showing that this endoglucosaminidase from human serum is in fact a chitinase that is different from lysozyme. The facts sustaining this finding are: (i) the identification of the products formed from MU-[GlcNAc]3 and [GlcNAc]2;and [GlcNAc]3; (ii) chitin and ethylene glycolchitin can be degraded by the enzyme; (iii) the chitinase inhibitor allosamidin also inhibits the action of MU-TACT hydrolase from human serum; (iv) no hydrolysis of the lysozyme substrate Micrococcus lysodeikticus. The enzyme also occurs in rat liver. It was demonstrated that upon Percoll density gradient centrifugation the enzyme from this tissue distributed parallel to the lysosomal marker enzymes beta-N-acetylhexosaminidase and beta-galactosidase, indicating a lysosomal localization for this enzyme. It is proposed that the enzyme functions in the hydrolysis of chitin, to which mammals are frequently exposed during infection by pathogens.

Binding of lysosomal enzymes to the mannose 6-phosphate receptor: a novel binding assay that makes use of biotinylated receptor molecules, coupled to avidin-agarose.

Binding assay procedures for receptor-ligand interactions should meet requirements such as ease of operation, reproducibility and low costs. In the case of the mannose 6-phosphate receptor (MPR) for lysosomal enzymes, the earliest assay procedure made use of a crude membrane preparation containing MPR, that was sedimented after incubation with an enzyme solution. The bound enzyme activity was determined thereafter. With purification methods of MPR (CI and CD) available, we found it of interest to compare the binding of different lysosomal enzymes with these molecular MPR preparations. We therefore developed a method in which MPR was biotinylated, followed by coupling to avidin-agarose. Very small quantities of this gel (2 microliters) appear to be needed to bind sufficient amounts of lysosomal enzyme. The bound enzyme activity can be rapidly measured with high reproducibility, by incubating the agarose spheres directly with substrate solutions. We could demonstrate that the binding properties of MPR, although biotinylated and immobilized, were not different from those obtained with crude MPR-preparations from rat liver membranes.

Structural analysis of the carbohydrate chains of beta-N-acetylhexosaminidases from bovine brain.

The oligosaccharide structures of bovine brain beta-N-acetylhexosaminidases A and B (EC 3.2.1.30) were studied at the glycopeptide level by employing 500 MHz 1H-n.m.r. spectroscopy and methylation analysis involving g.l.c.-m.s. More than 90% of the chains were found to be of the oligomannoside type, containing, on average, five to six mannose residues. Biantennary N-acetyl-lactosamine-type chains terminated in N-acetylneuraminic acid were found to comprise the remaining 5-10% of the total carbohydrate. The isoenzyme forms A and B do not differ from each other in the structure of their carbohydrate moiety, but do deviate in carbohydrate content and, in consequence, in the number of carbohydrate chains per molecule.

Isolation and further characterization of bovine brain hexosaminidase C.

Hexosaminidase C (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was partially purified from bovine brain tissue. The resulting preparation, free of its lysosomal counterparts, was used for the characterization of the enzyme and for further purification (lectin affinity chromatography, hydrophobic interaction chromatography, substrate-ligand affinity chromatography, ion-exchange chromatography, chromatography on activated thiol-Sepharose 4B). Only ion-exchange chromatography on DEAE-Sephacel appeared to improve the purity. The Michaelis constant was 0.46 mM for the substrate 4-methyl-umbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. The enzyme was not inhibited by acetate or N-acetylgalactosamine. Inhibition by N-acetylglucosamine was competitive, with a Ki value of 8.0 mM. Inhibition by divalent metal ions increased in the order Fe less than Zn less than Cu. Dithiothreitol and beta-mercaptoethanol, at an optimum concentration of about 10 mM, stimulated the activity. The enzyme is apparently not a glycoprotein since it did not bind to various lectins, nor did sialidase change its isoelectric point.