Glycoinositolphosphosphingolipids (basidiolipids) of higher mushrooms.

The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0-trihydroxysphinganine and an alpha-hydroxy long-chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem. 259, 331--338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of alpha-DManp-2Ins1-[PO(4)]-Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, alpha-Manp-4Ins1-[PO(40]-Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Man alpha-3 or -6Man alpha; Cc2, Man alpha-3(Man alpha-6)Man alpha-. All other species investigated show extension of the alpha-mannoside in the 6-position by beta-galactoside, which, in some instances, is alpha-fucosylated in 2-position (Fuc alpha-2)Gal beta-6Man alpha-. Further sugar chain elongation at the beta-galactoside may be in 3- and/or 6-position by alpha-galactoside, e.g. Ce4, Po2, Gal alpha-3-(Gal alpha-6)(Fuc alpha-2)Gal beta-6Man alpha-, whereas A. virosa, Av-3, has a more complex, highly alpha-fucosylated terminus, Gal alpha-3 (Fuc alpha-2)(Fuc alpha-6)Gal alpha-2(Gal alpha-3)Gal beta-6Man alpha-. L. edodes basidiolipids show further elongation by alpha-mannoside, e.g. Le3, Man alpha-2Man alpha-6Gal alpha-3(Fuc alpha-2)Gal beta-6Man alpha-, C. exipuliformis glycolipid by alpha-glucoside, i.e. Ce3, Glc alpha-6Gal beta-6Man alpha-. Basidiolipid Ls1 from L. scabrum, notably, has a 3-alpha-mannosylated alpha-fucose, i.e. Gal alpha-6(Man alpha-3Fuc alpha-2)Gal alpha-6Gal beta-6Man alpha-. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.

Novel glycoinositolphosphosphingolipids, basidiolipids, from Agaricus.

From the edible mushroom, the basidiomycetes Agaricus bisporus and Agaricus campestris, a novel carbohydrate-homologous series of four glyco-inositol-phospho-sphingolipids, designated basidiolipids, was isolated and the constituents purified. The chemical structures of the basidiolipids were elucidated to be: Manpbeta1-2inositol1-phospho-ceramide, Galpalpha-6[Fucpalpha-2]Galpbeta-6Manpbeta-2i nositol1-phospho-ceramide, Galpalpha-6Galpalpha-6[Fucpalpha-2]Galpbeta- 6Manpbeta-2inositol1-phospho-ceramide and Galpalpha-6Galpalpha-6Galpalpha-6[Fucpalpha-2] Galpbeta-6Manpbeta-2ino sitol1-phospho-ceramide. All four glycolipids contained a ceramide which was composed of phytosphingosine and predominantly alpha-hydroxy-behenic and alpha-hydroxy-lignoceric acid.

Glycosylation of glycoprotein 55 encoded by the anaemia-inducing strain of Friend spleen focus-forming virus.

Normal rat kidney cells, non-productively infected with the anaemia-inducing variant of Friend spleen focus-forming virus (F-SFFVA), were metabolically labelled with [2-3H]mannose. The primary translation product of the viral envelope gene (env), representing a glycoprotein with an apparent molecular M(r) of 55,000 (gp55), was isolated from cell lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Radiolabelled oligosaccharides, released from tryptic glycopeptides by treatment with endo-beta-N-acetylglucosaminidase H, were characterized chromatographically, by enzymic digestion and by acetolysis. The results revealed that F-SFFVA gp55 obtained from this source carried predominantly oligomannose type sugar chains with five to nine mannoses. As a characteristic feature, glycans with seven to nine mannoses contained, in part, an additional glucose residue. Although the amount of glucosylated species found was higher in F-SFFVA gp55 (about 25% of total endo-H-sensitive oligosaccharides) than in gp55 of the corresponding polycythaemia-inducing variant (F-SFFVP, 16.3%), the overall glycosylation pattern of the F-SFFVA env product closely resembled that of F-SFFVP gp55 [Strube et al. (1988) J Biol Chem 263:3762-71]. Hence, our results demonstrate that the different intracellular processing and transport of the primary F-SFFVA env product cannot be attributed to aberrant trimming of its oligomannose type glycans.

Influence of phospholipid structure on sterol efflux induced by albumin-phospholipid complexes.

Sterol release from strain L fibroblasts was measured in serum-free medium supplemented with delipidated human serum albumin and various phospholipids. The sterol molecule appears to preferentially interact with the sn-2-acyl chain of the phospholipid. The carbonyl oxygen of the phospholipid acyl ester linkage is not required for sterol-phospholipid interactions, while the phosphate and choline groups are required. In the presence of the human serum albumin-phospholipid complex, phospholipids containing trans-acyl groups are significantly more effective at removing cellular sterol than the corresponding cis-acyl group.

The effects of serum albumin and phospholipid on sterol excretion in tissue culture cells.

Serum albumin was as effective as whole serum or alpha-globulins in facilitating sterol release from strain L mouse fibroblasts. Commercial bovine serum albumin preparations, however, had markedly different absolute effects in this regard. These differences were attributable to the variation in phospholipid content of these products. All but one of these albumins enhanced sterol release when supplemented with phospholipid. The exception was fatty acid-poor albumin which contained an adequate amount of phospholipid. Among the phospholipids examined, lecithin proved to be most effective, while phosphatidylethanolamine had little potentiating influence. As the unsaturation of the test lecithins increased, enhancement of sterol release decreased. The potentiating effect of the phospholipid was in turn dependent on the protein used, since the phenomenon was not observed with non-serum proteins like ovalbumin or with non-transport serum proteins such as gamma-globulins. The results of these studies raise the possibility that serum albumin together with phospholipid can play an important role in sterol release in tissue culture cells.

Stimulating effect of hydrocortisone on the catabolism of endogenous fatty acyl groups by fatty acid-supplemented mouse L fibroblasts.

Hydrocortisone stimulated the catabolism of prelabeled fatty acyls in mouse L fibroblasts supplemented with exogenous fatty acid. Both oxidation to 14CO2 and release as free fatty acid from prelabeled lipids increased up to 20-fold under the described experimental conditions. The stimulating effect of hydrocortisone was observed even at concentrations as low as 1 microgram/ml. Neither the hormone nor exogenous fatty acid alone had a significant effect on endogenous fatty acyl catabolism. The activity of a cellular pH 6.5 lipase was unaffected by exogenous fatty acid, slightly increased by hydrocortisone, but increased 2-fold when both supplements were present. It is suggested that since exogenous fatty acids shunt phospholipid acyls to triglycerides, the latter induce the formation of more pH 6.5 lipase. Hydrocortisone is either needed for this induction to occur, or activates the newly formed enzyme. The lipase acts on the triglycerides to liberate free fatty acids that are then oxidized to 14CO2 or lost into the medium.

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts.

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.