Günther Mersmann

Sanfilippo B disease: Serum assays for detection of homozygous and heterozygous individuals in three families

Three assays for the determination of N-acetyl-α-D-glucosaminidase activity in the serum are described. In three families with patients suffering from Sanfilippo B disease, the affected individuals had a residual enzyme activity in the range of 2 to 16 per cent that of normal control subjects. Their obligate heterozygous parents had an activity diminished to 26 to 35 per cent. Nine other members of these families had enzyme activities lowered to the same extent and were therefore considered to be heterozygous carriers of the Sanfilippo B gene.

Mannosidosis. Storage of Mannose-containing Material in Cultured Human Mannosidosis Cells and Metabolic Correction by Pig Kidney α-Mannosidase

Skin fibroblasts from healthy individuals and a mannosidosis patient were cultured in the presence of [2-3H] mannose and the cell homogenates were fractionated by trichloroacetic acid precipitation into a precipitable and a non-precipitable portion. In uptake as well as in chase experiments the precipitable fractions show no significant difference in their content of radioactivity, while an increased level of radioactivity is found in the non-precipitable fraction of mannosidosis cells. This higher radioactivity content is due to a higher mannose content and is caused by a slower degradation of this fraction. The differences between the metabolisms of the two cell lines can be expressed by the ratio of radioactivity in the non-precipitable and the precipitable fractions. This value is about three times higher for mannosidosis than for control cells. Pig kidney alpha-mannosidase is taken up by both cell lines and is able to correct the impaired degradation of the non-precipitable material in mannosidosis cells, as shown by the normalization of the above defined ratio of radioactivity for this cell type.

Physical properties and biological activities of two forms of α-N-acetylglucosaminidase from bovine spleen

Abstract α - N -Acetylglucosaminidase (2-acetamido-2-deoxy-α- d -glucoside acetamido-deoxyglucohydrolase, EC 3.2.1.50) was purified 108-fold from bovine spleen homogenates and could be separated into two interconvertible forms (I and II) by gel filtration and disc electrophoresis, whereas separation was not possible by DEAE-cellulose chromatography or isoelectric focusing. The two forms have a molecular weight ratio of 2:1 and display identical enzyme kinetics and the same biological activity in the correction of the altered heparan sulfate catabolism of cultured human Sanfilippo B fibroblasts. The lower corrective activity of the bovine α - N -acetylglucosaminidase as compared with that of the human urinary enzyme is caused by a lower rate of uptake into the fibroblasts.

Influence of effectors of the complex-type-oligosaccharide biosynthesis on the formation of proteokeratan sulfate in bovine cornea

The structural similarity of the inner core of complex-type prosthetic oligosaccharides of N-asparagine glycoproteins and of the linkage region between the polysaccharide part and the protein chain of cornea proteokeratan sulfate makes their biosynthesis via a common route an attractive hypothesis. To test this, a tissue culture system was established to determine the rate of proteokeratan sulfate biosynthesis in bovine cornea and to measure the influence of several effectors of the dolichol pathway on this rate. Addition of dolichyl phosphate enhanced the formation of proteokeratan sulfate. Tunicamycin, 2-deoxy-D-glucose, bromoconduritol and deoxynojirimycin inhibited this process. Swainsonine probably led to the formation of a keratan sulfate with hybrid structure. The results support that the linkage region of cornea proteokeratan sulfate is synthesized via the assembly of a glucosylated dolichyl pyrophosphoryl oligosaccharide, its transfer to protein and subsequent processing by glycosidases.

Remarks on the differentiation of lysosomes from cultured human fibroblasts by silica gradient centrifugation.

Abstract Two bands of lysosomal marker enzyme activity from cultured skin fibroblasts can be obtained by silica gradient centrifugation (Rome, L H, Garvin, L H, Allietta, M M & Neufeld, E F, Cell 17 (1979) 143) [3]. It is shown that the distribution of lysosomes into the bands in the upper and lower part of the density gradient depends on the starting density and the shape of the self-forming gradient. A plot of marker enzyme activity vs density reveals that the two bands stem from a lysosomal population with unimodal density distribution.

The carbohydrate-protein binding region in keratan sulfate from bovine cornea structure of a major binding region oligosaccharide

Peptido-keratan sulfate from bovine cornea was degraded by a combination of desulfation, hydrazinolysis, nitrous acid deamination and NaB3H4 reduction. The tetrasaccharide fraction obtained by gel filtration was studied by degradation with specific exoglycosidases and methylation analysis. The existence of two different binding region oligosaccharide structures was established: The first structure contains one terminal fucose, two mannosine residues and N-acetylglucosamine at the reducing end. In the second structure one N-acetylglucosamine is bound to the protein backbone and substituted with branched 3,6-di-O-alpha-mannosyl-beta-mannose. Both terminal alpha-mannosine residues bear keratan sulfate chains in the 2-position.

The Carbohydrate-Protein Binding Region in Keratan Sulfate from Bovine Cornea. I. Isolation and Partial Characterization

We present a defined chemical method for the isolation of the oligosaccharide linking the protein to the disaccharide units of keratan sulfate from bovine corneas. 1) Hydrazinolysis removes the amino acids from the peptido keratan sulfate, and leads to deacetylation of the glucosamine residues but does not split off the sulfate groups. 2) NaB3H4 reduction selectively labels the reducing terminal glucosamine of the chain by converting it to [3H]glucosaminitol. 3) Nitrous acid deamination splits the glycosidic bonds of glucosamine and converts this sugar into 2,5-anhydromannose but also leads to several derivatives of the free terminal [3H]glucosaminitol. 4) Na12CN treatment stabilizes the reactive 2,5-anhydromannose and the terminal compounds containing aldehyde groups in a cyanhydrin reaction. 5) The oligosaccharide structure between the glucosamine residue at the reducing end and the first glucosamine of the disaccharide chain is not degraded by this procedure and is obtained intact and in labelled form. The data so far obtained on this part of the cornea keratan sulfate show that it is heterogeneous and contains besides the terminal glucosamine, mannose and fucose in a similar ratio as found in undegraded keratan sulfate. The predominant compound probably contains three neutral sugar residues.

Immunological cross-reactivity between α-mannosidases from different sources

Abstract 1. 1. α-Mannosidase with acidic pH-optimum was purified from pig kidneys more than 2000-fold to a specific activity of 13.2U/mg protein. The final product contains the multiple forms A and B and has a quarternary structure. 2. 2. The γ-globulin fraction of rabbit antiserum against the pure pig kidney α-mannosidase leads in immunodiffusion and precipitation experiments to precipitation not only with α-mannosidases from pig tissues but also (though with lower affinity) from bovine liver and human placenta, urine and skin fibroblasts.

Amount and properties of uptake forms in preparations of α-mannosidase from pig kidney

Abstract α-Mannosidase (α- d -mannoside mannohydrolase, EC 3.2.1.24) from pig kidney has been shown to exist in multiple forms differing in their capability to be endocytosed by α-mannosidase deficient cultured cells. A method is presented to evaluate the amount of “uptake” forms in different preparations of the enzyme. Preparations with different rates of uptake were shown to contain different amount of “uptake” forms and “non-uptake” forms. The content of “uptake” forms in a preparation was identical with that of enzyme molecules bearing a phosphorylated carbohydrate group necessary for the recognition by cell surface receptors.

Comparative studies of lysosomal enzyme uptake by cultivated fibroblasts, hepatocytes and non parenchymal liver cells: 52

Different cultured cell types posses receptors recognizing different carbohydrate moieties on lysosomal enzymes (glycoproteins) for adsorptive endocytosis. By using glycoproteins and sugars as inhibitors and by enzymatic modulation of lysosomal enzymes it could be shown that fibroblasts recognize on lysosomal enzymes terminal mannose 6-phosphate residues whereas non parenchymal liver cells recognize terminal N-acetylglucosamine residues. Hepatocytes recognize galactose, mannose 6-phosphate and possibly mannose/N-acetylglucosamine residues on lysosomal enzymes. As liver is the major organ for lysosomal enzyme uptake from blood a calculation was done on the basis of these experiments to predict the uptake of different lysosomal enzymes by hepatocytes and non parenchymal liver cells after in vivo administration for enzyme replacement therapy. Whereas α-N-acetylglucosaminidase is taken up by hepatocytes to 74 % the bulk of α-mannosidase (85 %) and of ß-hexosaminidase (65 %) is taken up by non parenchymal liver cells. The results show that the cell specific uptake depends on the carbohydrate structure of the enzymes and underlines the need for organ specific targeting of the lysosomal enzymes.