Rolf Nuck

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).

Isolation and Characterization of Two Forms of an Acidic Bromelain Stem Proteinase

Two forms of an acidic bromelain proteinase isolated from crude bromelain, an extract from pineapple stem, were found by a two-step FPLC purification procedure. The basic main components were removed by cation exchange chromatography and the breakthrough fraction was further resolved by anion exchange chromatography into 15 protein fractions, only two of which, called SBA/a and SBA/b, were proteolytically active. These components were characterized by electrospray mass spectroscopy (ESMS), isoelectric focusing, N-terminal amino acid sequence analysis, monosaccharide analysis, and enzymatic parameters. The molecular masses of SBA/a and SBA/b were determined by ESMS to be 23,550 and 23,560, respectively. The isoelectric points (pI) of the two bands of SBA/a were 4.8 and 4.9; SBA/b focused as a single band at pI = 4.8. Partial N-terminal amino acid sequences (11 residues) were identical to SBA/a and SBA/b and identical with those of stem bromelain, the basic main proteinase of the pineapple stem, and fruit bromelain, the acidic main proteinase of the pineapple fruit. Both components are highly glycosylated; hydrolysis of SBA/a yielded about twofold more monosaccharide per protein than SBA/b. The comparison of the catalytic properties of SBA/a with those of SBA/b revealed no relevant differences in the hydrolysis of three peptidyl-NH-Mec substrates and in the inhibition profiles using chicken cystatin and E-64, indicating that these components can be considered as two forms of a single enzyme. Both forms are scarcely inhibited by chicken cystatin and slowly inactivated by E-64, hence are nontypical cysteine proteinases of the papain superfamily.

N-Glycosylation of the carcinoembryonic antigen related cell adhesion molecule, C-CAM, from rat liver: detection of oversialylated bi- and triantennary structures

Abstract Rat C-CAM is a ubiquitous, transmembrane and carcino-embryonic antigen related cell adhesion molecule. The human counterpart is known as biliary glycoprotein (BGP) or CD66a. It is involved in different cellular functions ranging from intercellular adhesion, microbial receptor activity, signaling and tumor suppression. In the present study N-glyco-sylation of C-CAM immunopurified from rat liver was analyzed in detail. The primary sequence of rat C-CAM contains 15 potential N-glycosylation sites. The N-glycans were enzymatically released from glycopeptides, fluorescently labeled with 2-aminobenzamide, and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by enzymatic sequencing and MALDI-TOF-MS. Mainly bi- and triantennary complex structures were identified. The presence of type I and type II chains in the antennae of these glycans results in heterogeneous glycosylation of C-CAM. Sialylation of the sugars was found to be unusual; bi- and triantennary glycans contained three and four sialic acid residues, respectively, and this linkage seemed to be restricted to the type I chain in the antennae. Approximately 20% of the detected sugars contain these unusual numbers of sialic acids. C-CAM is the first transmembrane protein found to be over-sialylated.

H.p.l.c. of 4-nitrophenyl-α-d-malto-oligosaccharides

Abstract The distribution of 4-nitrophenyl-α- d -malto-oligosaccharides produced by phosphorolytic synthesis was analyzed by an amine-modified and a C 18 -bonded silica gel. With both systems high resolution of components up to d.p.40 can be achieved by gradient elution using acetonitrile-water and water-methanol, respectively. The use of water-acetonitrile in reversed-phase (C 18 ) chromatography allows only a separation up to d.p. 9. The elution behaviour of oligomers in the d.p. range of 1–10 glucosyl residues per molecule eluted by water-methanol is discussed. The observation of rearrangement products of 4-nitrophenyl-α- d -malto-oligomers was successful by normal-phase chromatography.

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.

Phosphorolytische synthese von Niedermolekularen amylosen mit Modifizierten Endgruppen

Abstract The preparation of low-molecular-weight amyloses with modified terminal groups in the range d.p. 10–20 was achieved by enzymic synthesis using phosphorylase from a potato crude extract. 4-Nitrophenyl α- d -maltooligoglycosides with a minimum chain length of five d -glucose units served as primers, providing good yields in a 10–12 fold molar excess of α- d -glucopyranosyl phosphate. Up to 89% conversion was obtained after 4 h at pH 5.8, 40°, and a phosphorylase activity of 30 units. The reaction products were analyzed by h.p.l.c. Elution profiles always revealed oligomers shorter than the primer. The preferred appearance of the maltotrioside after incubation of the primer with the enzyme crude extract indicated the presence of a disproportionation enzyme.

H (0) blood group determinant is present on soluble human L‐selectin expressed in BHK‐cells

In the present study we show that the H (0) blood group determinant Fucα1‐2Galβ1‐4GlcNAcβ1‐R is present on N‐linked glycans of soluble human L‐selectin recombinantly expressed in baby hamster kidney (BHK) cells. The glycans were isolated using complementary HPLC techniques and characterized by a combination of exoglycosidase digestion and mass spectrometry. The linkage of the fucose residues was determined by incubation of the glycans with specific fucosidases. The H blood determinant Fucα1‐2Galβ1‐4GlcNAcβ1 was detected for bi‐, 2,4 branched tri‐ and tetraantennary structures. To our knowledge, the proposed oligosaccharide structures represent a new glycosylation motif for recombinant glycoproteins expressed on BHK cells.

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.

Occurrence of α1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.

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.