B. Smestad Paulsen

Immunologically active (1→3)-(1→4)-α-D-Glucan from Cetraria islandica

Summary A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean Mr of Ci-3 was determined to be 2000 kD, compared to 6–8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1→3)- and (l→4)-α-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.

Immunologically active O6-branched (1»3)-β-glucan from the lichen Thamnolia vermicularis var. subuliformis

A lentinan-type gel-forming beta-glucan, Ths-2, has been isolated in about 1.5% yield from the alkali extract of the lichen Thamnolia vermicularis var. subuliformis, using ethanol fractionation, dialysis and gel filtration. The mean Mr of Ths-2 was determined by GP-HPLC to be 67 kD, and the optical rotation was measured to be -14 degrees. The structure of Ths-2 was further elucidated by methylation analysis by GC-MS, 1H- and 13C-NMR spectroscopy and selective enzymatic hydrolysis with exo-(1 --> 3)-beta-D-glucanase followed by analysis of oligosaccharides by HPAEC-PAD. Ths-2 was found to be consisting of a (1 --> 3)-beta-D-glucopyranosyl main chain with branches of a (1 --> 6) linked glucopyranosyl unit on every third unit of the main chain. Similar polysaccharide structures have been described from fungi, but this is the first report of a lentinan-type (1 --> 3)-beta-D-glucan from a lichen species. The immunomodulating activity of Ths-2 was tested in an in vitro anti-complementary assay, and proved to be strongly active.

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.

Rhamnopyranosylgalactofuranan,a new immunologically active polysaccharide from Thamnolia subuliformis

A complex polysaccharide, Ths-3, consisting mainly of rhamnopyranosyl and galactofuranosyl units, has been isolated from the water extract of the lichen Thamnolia subuliformis using ethanol fractionation, dialysis, ion-exchange chromatography, gel filtration and preparative GP-HPLC. The mean M(r) of Ths-3 was determined to be 1450 kD, and the monosaccharide composition is gal/rha/glc/xyl/man in the ratio of 40:31:13:10:6. The structure of Ths-3 was further elucidated by methylation analysis by GC-MS and NMR spectroscopy and found to be basically composed of (1-->3)-linked beta-D-galactofuranosyl units with branches on C6, and rhamnosyl units being predominantly (1-->2)-linked with branches on C3 and C4, while some units are (1-->3)-linked. Glucose, mannose and galactofuranose are found as terminal units and glucose and mannose are also (1-->4)-linked, while xylose is only present as terminal units. The trisaccharide xylglcglc was detected after partial hydrolysis of the polysaccharide. The immunomodulating activity of Ths-3 was tested in an in vitro phagocytosis assay and the classical anticomplementary assay, and proved to be active in both tests. The authors suggest the trivial name thamnolan for Ths-3.

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting.

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.

Comparison of IgE-binding antigens in horse dander and a mixture of horse hair and skin scrapings

Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS‐PAGE and IEE, both combined with immunoblotting, and CIE/CRIE showed the IgE‐binding ability of the proteins/glycoproteins present in the two extracts. SDS‐PAGE/immunoblotting showed the presence of mainly the same IgE‐binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins. The most important allergen was a glycoprotein with molecular mass of 27 kDa showing IgE‐binding ability to the 16 patient sera available. Another important allergen with molecular mass of 67 kDa is probably horse albumin, IEF/immunoblotting showed the presence of a glycoprotein with IgE‐binding ability at pH 3.2 in both extracts. Tandem CRIE showed that both extracts contained the same four allergens as major allergens. In addition, as we observed when analyzing both extracts in CRIE against 16 different patient sera, each extract contained allergens not present in the other extract. We may conclude from this investigation that commercial allergen extracts to be used in diagnosis and immunotherapy should include both horse dander and horse hair and skin scrapings in the starting material for the preparation of extracts. If not, allergens which may be of importance for some patients will be missing.

Further characterization of a high molecular weight glycoprotein antigen from the yeast Saccharomyces cerevisiae

Abstract A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae . The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al. , 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N -acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O -glycosidic and that a minor type is of N -glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points. Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Manα1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Manα1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.

Further characterization of IgE-binding antigens in horse dander, with particular emphasis on glycoprotein allergens

IgE‐binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS‐PAGE under reducing conditions and blotting, several of the glycoprotein IgE‐binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked α2→6 to galactose, and galactose linked β1→ 4 to N‐acetylglucosamine, respectively. Carbohydrate analysis after SDS‐PAGE and blotting showed the presence of mannose, galactose, N‐acetylglucosamine, and N‐acetyl neuraminic acid in the 27‐kDa band. The 14.4‐kDa glycoprotein component lost its IgE‐binding activity after periodate oxidation. Minor differences in allergenic activity in the other glycoprotein allergenic components were also detected, indicating that the carbohydrate part of the molecule seems to play a role in the IgE‐binding activity. Two‐dimensional electrophoresis and subsequent immunoblotting were performed to estimate the approximate number, molecular mass, and pI of IgE‐binding components. This resulted in a cluster of such components on the blot membrane.