Detlef Grunow

Isolation and partial characterization of basic proteinases from stem bromelain.

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosine→serine) and position 20 (asparagine→ glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.0∶2.0∶1.0∶2.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (Km 2.30 mM,kcat 0.87 sec−1 and F5 (Km 2.42 mM,kcat 0.68 sec−1), and differed greatly from F9 (Km 0.40 mM,kcat 3.94 sec−1).

Enzymatic Sequence Analysis of N -Glycans by Exoglycosidase Cleavage and Mass Spectrometry – detection of Lewis X Structures

Enzymatic sequencing of oligosaccharides gives structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can be applied to oligosaccharide mixtures as well to evaluate the occurrence of distinct oligosac-charide motives of functional and/or structural interest. Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from alpha1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosi-dase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans by use of the respective specific exoglycosidases.

Carbohydrate structures of soluble human L-selectin recombinantly expressed in baby-hamster kidney cells.

A soluble form of L‐selectin was recombinantly produced, which might be an effective therapeutic agent in inflammatory disorders, acting as an inhibitor for leucocyte endothelium adhesion. In the present study the oligosaccharide structures of soluble human L‐selectin, recombinantly expressed in baby‐hamster kidney cells, were determined. The N‐linked glycans were enzymically released and fluorescently labelled with 2‐aminobenzamide. Sialylation of the N‐glycans was analysed by anion‐exchange chromatography followed by rechromatography of the resulting fractions on amino‐phase HPLC after release of the sialic acid residues. Desialylated oligosaccharides were separated using two‐dimensional HPLC and characterized by digestion with exoglycosidases and MS. More than 30 oligosaccharide structures representing at least 95% of the overall glycosylation of this protein were determined. The results revealed that recombinant soluble human L‐selectin carries bi‐, tri‐ and tetra‐antennary sugar chains, which are fucosylated on the innermost residue of N‐acetylglucosamine. The number of sialic acid residues linked to these glycans ranges from 0 (neutral glycans) to 4 (tetrasialylated oligosaccharides). The sialic acid is found exclusively in the α2–3 linkage to galactose. In addition to the main glycans, different minor structures containing terminal N‐acetylgalactosamine, or the H (O) blood‐group determinant were also identified. O‐Glycosylation of mucin‐type sugar chains was not detected in recombinant soluble human L‐selectin.

Incorporation of the hexose analogue 2-deoxy-d-galactose into membrane glycoproteins in HepG2 cells

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.

Synthesis of 2-deoxy-d-galactose containing gangliosides in vivo

Incorporation of 2‐deoxy‐d‐galactose into the oligosaccharide moieties of different gangliosides of rat liver was examined. After intraperitoneal administration of 2‐deoxy‐d‐galactose it was shown by GLC/MS analysis that this hexose analogue is metabolized and incorporated into all the gangliosides investigated, and predominantly into GM3 and GD3. In both of these gangliosides, 25–55% of the galactose residues were substituted by 2‐deoxy‐d‐galactose. The epimer, 2‐deoxy‐o‐glucose, was not detectable.