Studies on Fungal Polysaccharides: The Immunological Determinant of the Serologically Active Substances from Absidia cylindrospora, Mucor hiemalis and Rhizopus nigricans

Abstract

Six manno-oligosaccharides were isolated from the partial acid hydrolysate of pronasetreated extracellular glycan (pt-ACE-11) of Absidia cylindrospora by gel filtration on Bio Gel P-2 and descending paper chromatography. The pentasaccharide and the higher molecular weight oligosaccharides significantly inhibited the precipitation reaction of intracellular glycans from Absidia cylindrospora (ACI-B), Mucor hiemalis (MhI-B) and Rhizopus nigricans (RnI-B) with anti-ACE serum. Structural analysis showed that these six oligosaccharides were composed of linear ( 1 + 6)-a-linked mannopyranosyl residues. INTRODUCTIONPreviously (Hayashi et al., 1978a, b) we have described the isolation, purification and partial characterization of the serologically active fucomannan-peptide of Absidia cylindrospora, a member of the Mucorales. Rabbit antiserum (anti-ACE serum) against the extracellular non-dialysable fraction of A. cylindrospora reacted not only with this fucomannanpeptide but also with antigenic fractions from four Mucor spp. and Rhizopus nigricans. However, anti-ACE serum did not cross-react with the purified mannans of Candida albicans and Saccharomyces cerevisiae, so it would appear that the chemical structure of the serologically active determinant might be different from those of the mannans of C. albicans and S. cerevisiae.In the present paper, the purification of six manno-oligosaccharides and the results of precipitation inhibition tests with these oligosaccharides are described. The common determinant in the serologically active substances of A. cylindrospora, M. hiemalis and R. nigricans is discussed. METHODSAnalytical methods. Quantitative analyses of carbohydrate, protein and phosphate were performed by the methods of Dubois et al. (1956( ), Lowry et al. (1951 and of Fiske & Subbarow (1925), respectively, using mannose, bovine serum albumin and KHzPOB as the standards.For paper and thin-layer chromatography (t.1.c.) the following solvent systems were used: A, ethyl acetate/pyridine/acetic acid/water (5: 5: 1 : 3, by vol.); By 1-butanol/ethanol/water (3 : 1 : 1 , by vol.). Carbohydrates on paper and thin-layer chromatograms were detected with the alkaline silver nitrate reagent (Trevelyan et al., 1950) or the periodate-benzidine reagent (Cifonelli & Smith, 1954). (The use of benzidine in the UK is prohibited under the Carcinogenic Substances Regulations, 1968). Gas-liquid chromatography (g.1.c.) was performed using a Shimadzu GC-6A chromatograph (Shimadzu, Tokyo, Japan) as described previously (Hayashi et al., 1978a). A glass column packed with 3 % (w/w) ECNSS-M on Chromosorb W (AW-DMCS, 60 to 80 mesh) was used for identification of the partially methylated alditol acetates. G. T. M I Y A Z A K I A N D OTHERScolumn packed with 3 % (w/w) silicone OV-225 on Chromosorb W (AW-DMCS, 80 to 100 mesh) at 170 "C and operated at an ionization voltage of 70 eV, a trap current of 60 ,uA and an ion source at 310 "C. Infrared spectra were recorded with a Hitachi-215 spectrometer. Preparation of ACI-By MhI-B and RnI-B.The s...

Heterogeneity and Antigenic Properties of Mannoheteroglycan from Absidia cylindrospora

“…Because growth of A . cylindrospora is slow in dialysable Sabouraud's medium (Miyazaki et al, 1977), it was cultured for 28 d in the present study. Thus it was possible that the heterogeneity was a result of the long period of incubation.…”

“…Absidia cylindrospora I F 0 4000 was obtained from the Institute for Fermentation, Osaka, Japan. The cells were cultured in modified Sabouraud's liquid medium at 27 " C until the stationary growth phase was reached (Miyazaki et al, 1977). Extracellular polysaccharides from A. cylindrospora were prepared according to the procedure previously described (Miyazaki et al, 1977;Hayashi et al, 1978a).…”

“…Antiserum was prepared by immunization of male New Zealand white rabbits with extracellular polysaccharides of A. cylindrospora as previously described (Miyazaki et at., 1977; Hayashi et al, 1 9 7 8~) . Antisera to whole cells, to cell walls and to extracellular polysaccharides all reacted with the Con A-binding fraction, and their precipitin lines fused with each other completely (Hayashi et al, 1978a).…”

“…Previous work has shown that mechanical disruption of mycelium of Absidia cylindrospora, a species of the Mucorales, releases a water-soluble antigenic substance( s) (Miyazaki et al, 1977(Miyazaki et al, , 1980Hayashi et al, 1978a). This antigen is probably a common antigen among Mucorales such as Rhizopus nigricans, Mucor mucedo and Mucor hiemalis (Hayashi et al, 1978b), and may be distributed on the cell surface, because the mycelium surface was stained by fluorescein-conjugated antiserum (Hay ashi et al, 1979).…”

Partial purification and characterization of mold antigens commonly found in foods

“…Polysaccharides found in fungal mycelia are immunologically active (5,11,31,34,42,48). Galactose, mannose, and glucose are the dominant sugars in these fungal polysaccharides (4, 10, 48); therefore, cross-reactions of the antigenantibody among mold species might be due to the presence of these common sugars.…”