Bernard Overdijk

Distribution of chitinase in guinea pig tissues and increases in levels of this enzyme after systemic infection with Aspergillus fumigatus.

Intravenous infection of guinea pigs with the fungus Aspergillus fumigatus resulted in increased levels of chitinase in serum and tissues of the animals. The molecular properties of the enzyme were demonstrated to be different from those of the fungal chitinase, but also from guinea pig lysozyme and beta-N-acetylhexosaminidase. Bio-Gel P-100 gel filtration showed that in liver, spleen, heart and lung tissue of control animals there were two molecular mass forms present with apparent molecular masses of 35 kDa and 15 kDa. In brain and serum, only the 35 kDa form was detectable. Kidney showed only the 15 kDa form. Upon infection the 35 kDa form appeared in kidney and increased in the other tissues. When a less pathogenic form of the fungus was used the 35 kDa form remained absent in kidney. In contrast to human serum chitinase, the enzyme from guinea pig serum and tissues did bind to concanavalin A-Sepharose. This was the case for both molecular mass forms. The mode of cleavage of the substrate 4-methylumbelliferyl-tri-N-acetylchitotrioside (MU-[GlcNAc]3, where GlcNAc is N-acetylglucosamine) by the two forms of the enzyme was the same: both [GlcNAc]2 and [GlcNAc]3 were released. The chitinase activity levels in the control tissues showed a large variation in this order: spleen > lung, kidney > liver > heart > brain. The fact that spleen showed the highest chitinase level is in agreement with its major role as a lymphoid organ in cases of systemic infections. The relative increases upon infection were the highest for the tissues that showed low control values.

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.

Purification and partial characterization of the carbohydrate structure of lysosomal N-acetyl-beta-D-hexosaminidases from bovine brain.

1. The lysosomal forms A and B, and an intermediate form I of N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) were isolated from bovine brain, resulting in the following purification factors and specific activities: hexosaminidase A 20255, 103 U mg-1; hexosaminidase B 34715, 134 U mg-1; hexosaminidase I 15241, 78 U mg-1. 2. The molecular weights of the polypeptide chains were identical for each isoenzyme: two bands of 50 and 53 k daltons were found. 3. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine and sialic acid. This composition, and the absence of N-acetylgalactosamine, indicated that only N-glycosidically linked oligosaccharide chains are present. 4. The amino-acid composition showed no substantial differences for the three isoenzymes.

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid α-l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kDa and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz1H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 3∶1. While the oligomannoside-type chains show some heterogeneity in size (Man5–8GlcNAc2), theN-acetyllactosaminetype chains are exclusively bi-α(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of α-l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, β-N-acetylhexosaminidase, α-l-fucosidase and β-galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.

Primary structure of O- and N-glycosylic carbohydrate chains derived from murine submandibular mucin (MSM)

The carbohydrate moiety of mouse submandibular mucin (MSM) contains mainly D-mannose and 2-acetamido-2-deoxy-D-glucose together with sialic acid, D-galactose, and 2-acetamido-2-deoxy-D-galactose. O-Glycosylically bound saccharides, obtained by treatment of MSM with alkaline borohydride, were shown by methylation analysis to have the structure: alpha-NeuAc-(2----3)-beta-Gal-(1----3)-GalNAc-ol. N-Glycosylically bound saccharides obtained from MSM by hydrazinolysis, and analysed by 500-MHz 1H-n.m.r. spectroscopy, were shown to have the following comprehensive structures. (Formula: see text).

Partial purification and further characterization of the novel endoglucosaminidase from human serum that hydrolyses 4-methylumbelliferyl-N-acetyl-β-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.

The carbohydrate structures of β-galactosidase from human liver

AbstractAcid β-galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (Mr63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz1H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the1H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man5-6GlcNAc2Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of β-galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.

Lysosomal β-hexosaminidase is highly resistant towards proteolytic degradation in vitro

1. A partially purified enzyme preparation of beta-hexosaminidase from human fibroblasts was treated with proteases and the effect on its molecular weight and enzymatic activity was studied. 2. Both the forms A and B of the enzyme appeared to be resistant to a protease treatment that degraded the majority of the contaminating proteins to a large extent. 3. The same result was obtained with enzyme preparations from cells treated with tunicamycin. 4. Also the molecular weights of the individual polypeptide chains of the enzyme were not decreased, as was shown by SDS-PAGE, followed by immuno-blotting.

β-Galactosidase and α-Fucosidase of Human Fibroblasts Show Hardly Binding to the Mannose 6-Phosphate Receptor in Comparison with β-Hexosaminidase

In I-cell disease the mannose 6-phosphate (Man6P) receptor-mediated routing of lysosomal enzymes is impaired, due to the deficiency of N-acetyl-glucosamine- 1-phosphotransferase (1,2). Normally, this cis-Golgi-localized enzyme is involved in the phosphorylation of mannose residues that are present in the oligomannoside-type carbohydrate chains of lysosomal enzymes, thereby forming the proper ligand for recognition by the Man6P receptor. The absence of the phosphotransferase in I-cell disease patients results in multiple deficiencies of lysosomal enzyme activities.

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.