Jan J.W. Lisman

Demonstration and partial characterization of endo-N-acetyl-β-d-glucosaminidase in human tissues

It has been postulated that the biological degradation of the oligosaccharide part of the serum type glycoproteins would be initiated by the splitting of the di-N-acetyl chitobiose moiety by an endo-Nacetyl-/3-D-glucosaminidase (EC 3.2.1.96 endoglucosaminidase) [ 1,2]. This hypothesis is sustained by the finding of oligosaccharide chains of various lengths and compositions in urine and tissues of patients, suffering from lysosomal storage disorders as in sialidosis 131. These oligosaccharides share a single N-acetylglucosamine residue at the reducing terminus. At the other end of the chain the sugar is present for which the hydrolysis is blocked due to the enzyme deficiency. Endoglucosaminidase activity towards oligomannosidic glycans was reported in rat and pig tissues [2]. This finding was confirmed [4], localizing this type of enzymatic activity in the cytosolic fraction of rat liver and kidney, with an oligomannosidic substrate derived from ovalbumin. In the same tissue activity was also shown towards a glycopeptide isolated from asialotransferrin, a substrate of the N-acetyl-lactosaminic type [5]. To obtain support for the hypothesis [ 1,2], it was of interest to see whether endoglucosaminidase activity could be detected in human tis-

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).

Evidence for the presence of β-subunit of hexosaminidase in a case of Sandhoff disease using a blotting technique

SummaryHexosaminidases, lysosomal enzymes whose deficiency is responsible for several genetic disorders, exist as two major forms: form A, containing two types of subunits α and β; and form B, containing only β subunits.We have used a technique involving successively electrophoresis of denatured proteins, transfer (blotting) onto nitrocellulose, and labelling by appropriate antibodies raised against the dissociated forms of hexosaminidases A and B. This technique allows the detection of α and β subunits in crude extracts of normal tissues.The presence of β chains was demonstrated in the liver of a fetus affected with Sandhoffs disease, deficient in functional hexosaminidases A and B.

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.

Sialylation in vitro of purified human liver β--N-acetylhexosaminidase☆

In order to study structure-function relationships of lysosomal enzymes, human liver β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-d-hexoside acetamidodeoxyhexohydrolase, EC 3.2.1.52) has been purified by an extraction/affinity chromatography/ion-exchange procedure. The isoenzymes A and B, native as well as neuraminidase-treated, were incubated with a partially purified preparation of bovine colostrum sialyltransferase (CMP-N-acetylneuraminate : d-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1). Native β-N-acetylhexosaminidases were found to be poor acceptors for the sialyltransferase used. However, incorporation of sialic acid into neuraminidase-treated β-N-acetylhexosaminidase A and B amounted to a 58 and 72% saturation of the theoretical acceptor sites, respectively. The acceptor specificity of the sialyltransferase suggests that Galβ(1 → 4)-GlcNAc units may be present on at least part of the β-N-acetylhexosaminidase A and B molecules. However, oligomannosidic-type chains may also occur on the lysosomal enzyme, as shown by sugar composition of the enzyme. The presence and/or amount of sialic acid residues does not appear to affect the kinetic properties of β-N-acetylhexosaminidase A and B towards 4-methylumbelliferyl glycoside substrate.

Isolation of lysosomes from bovine brain tissue. A new zonal centrifugation technique.

The isolation of lysosomes from brain, more so than from such other tissues as liver, encounters difficulties (1). Despite these difficulties lysosome enriched fractions can be prepared from brain homogenates by a combination of differential pelleting and density gradient centrifugation. Several reports dealing with the isolation of subcellular particles in brain tissue of different mammalia have been published (1,2).