Jens K. Wold

Characterization of a biologically active pectin from Plantago major L.

Abstract PMII isolated from the leaves of Plantago major L. is a pectin type polysaccharide with anti-complementary activity. It is highly esterified and partly O-acetylated with regions of 1,4 linked polygalacturonic acid and at least two different hairy regions. The galactose side chains are linked to position 4 of rhamnose in the main chain. The structure of the galactan side chains is complex, but 1,3,6 linkages are dominating in one of the isolated hairy regions. Arabinose is attached to position 3 and 6 of galactose. In the other hairy region arabinose is attached to position 3 of galacturonic acid. De-esterification and de-acetylation do not alter the anti-complementary activity of PMII. Different parts of PMII were shown to have different activities. The smooth regions are only slightly active in contrast to the hairy regions which had significantly higher activity. The hairy regions of highest molecular weight (PVa) with 1,3,6 linked galactose side chains were found to be the most active fraction. The importance of arabinose for the activity seems to depend on the site of substitution. Removal of arabinose terminally linked to galactose increases the activity slightly while removal of arabinose linked to the galacturonic acid backbone decreases the activity.

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

Abstract PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, 13C-NMR, 1H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.

Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae

Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohns disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohns patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manα1→3Manα1→2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.

Allergens in Aspergillus fumigatus. I. Characterization of two different allergen extracts and evaluation of their stability and the importance of carbohydrate for IgE binding.

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS‐PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE‐binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE‐binding components. The surface extract contained about the same number of IgE‐binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE‐binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE‐binding ability of the 20‐kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4°C for 2, 7, and 21 d and then analyzed by SDS‐PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20‐kDa and 30‐kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract. When the same experiment was performed on samples in a 50% glycerol solution, the results strongly indicated that glycerol had a stabilizing effect on allergens in both extracts. The enzyme content, estimated by the API ZYM‐test, showed that both extracts contained several protein‐ and carbohydrate‐degrading enzymes. The presence of these enzymes may explain the lability of the extracts.

Glycoproteins in the skin mucus of the char (Salmo alpinus L.)

1. 1. Two glycoprotein fractions were obtained from char skin mucus by gel chromatography on Sepharose 4B. 2. 2. The amino acid and carbohydrate composition of the two fractions indicate that they are both of the mucin type, containing a high proportion of threonine and serine, and of fucose, galactose, N-acetylgalactosamine and N-acetylglucosamine. 3. 3. Treatment of the fraction of higher molecular weight with NaOH-NaBH4 caused degradation of the glycoprotein with concomitant release of carbohydrate residues terminating in N-acetylgalactosaminitol, as expected for a mucin.

Allergens in pollen from mugwort (Artemisia vulgaris L.). I. Partial characterization of allergen preparations from mugwort pollen with emphasis on the carbohydrate moiety.

The results obtained in these studies show that certain mugwort allergens are extracted after 5 min extraction time and that there is little difference whether phosphate-buffered saline or NH4HCO3 is used as extraction buffer. An extraction time of 1 h appears to be sufficient for a quantitative extraction of the main part of the allergens present. When the extracts are dialyzed, cathodic antigens/allergens are lost. Addition of thiourea leads to a reduction in the coloured matter of the extract, but not in a sufficient degree to recommend the compound to be included in the extraction procedures of allergens from mugwort pollen. A few allergens are heat-resistant, and as these fractions are richer in carbohydrate than equivalent fractions not being heat-treated, these allergens might be of glycoprotein nature. Structural features of the carbohydrate moiety are also described as well as the total amino acid composition of the protein moiety.

Immunological Studies of the Glycoprotein Allergen Ag-54 (Cla h II) in Cladosporium herbarum with Special Attention to the Carbohydrate and Protein Moieties

The glycoprotein allergen Ag-54 (Cla h II) isolated from Cladosporium herbarum was split into its carbohydrate and protein moieties by using alkaline-borohydride treatment and deglycosylation, respectively. The native Ag-54, the deglycosylated protein and the protein-free carbohydrate moieties were tested for IgE-binding activity by radioallergosorbent test inhibition using selected sera from individuals with C. herbarum allergy. The deglycosylated material had a stronger allergenic activity than the native Ag-54 with all the sera tested. The Ag-54 carbohydrate moieties were not found to possess any IgE-binding activity. The galactoglucomannan part of the carbohydrate moiety was isolated and it was demonstrated to precipitate IgG rabbit antibody. Removal of the galactofuranose units by mild acid hydrolysis did not alter the IgG-binding capacity of the polysaccharide, indicating that galactofuranose was not immunodominant.

Purification of a basic glycoprotein allergen from pollen of timothy by high-performance liquid chromatography.

The use of high-performance ion-exchange and size-exclusion chromatography in the purification of the basic timothy pollen allergen antigen 30 (Ag 30) was investigated. The most efficient purification was achieved when an initial purification step on a CM-Sepharose CL-6B column was followed by chromatography on Mono S and TSK G 2000 SW columns. This procedure was highly reproducible and well suited for semi-preparative scale purification of the allergen. The purified allergen gave one band on isoelectric focusing, corresponding to a pI of 9.30. On fused rocket immunoelectrophoresis a single precipitate was obtained that coincided with the allergenic activity.

Purification and Partial Characterization of the Allergen Ag-54 from Cladosporium herbarum

One important allergen, Ag-54, was extracted from Cladosporium herbarum and purified by a combination of diafiltration, gel filtrations and isoelectric focusing. The allergen, when examined by gel electrophoresis and high-performance liquid chromatography, was found to be essentially homogeneous with a molecular weight of about 20,000 daltons. Ag-54 is of glycoprotein nature. It contained 19.5% protein, calculated from its amino acid composition and 80.2% carbohydrate, quantified by methanolysis, trimethylsilyl derivatization and gas-liquid chromatography. The carbohydrate moiety was composed only of mannose, galactose and glucose in the ratio 1.0:0.6:1.3. Frequently occurring amino sugars like glucosamine and galactosamine could not be detected. Mass spectrometry of trimethylsilyl-derivatized methylglycosides demonstrated that sialic acid was not present.

Water-soluble glycoproteins from Cannabis sativa (South Africa)

Abstract The water-soluble glycoproteins obtained from Cannabis leaves of plants grown from South African seeds have been further studied. Treatment of the glycoprotein fractions with NaOH in the presence of NaBH 4 , resulted in a significant decrease in the serine content and a corresponding increase in alanine. The carbohydrate side chains released contained the sugar alcohol, galactitol. By treatment of the glycoprotein fractions with NaOH in the presence of Na 2 SO 3 , and subsequent acid hydrolysis, cysteic acid was formed. These data indicate that carbohydrate and protein are connected via serine- O -galactoside linkages. Further investigation of the structure of the carbohydrate part of the glycoproteins was carried out by methylation analysis, Smith-degradation and enzyme incubation. The present glycoprotein material of plants grown from South African seeds is similar to the material previously investigated, but in contrast to the latter, it is devoid of hexosamine.