The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.Lipopolysaccharides (endotoxins) of gram-negative bacteria which consist of a heteropolysaccharide and a lipid component (termed lipid A) elicit multiple acute pathophysiological effects such as fever, lethality, Shwartzman reactivity, macrophage and B-lymphocyte activation, and other activities [I]. In 1954 it was proposed that for the induction of these effects the polysaccharide portion is dispensable and that the lipid A component represents the active center responsible for the endotoxic properties of lipopolysaccharides [2]. Evidence for this was then obtained in numerous investigations [2 -41 and this concept is now generally accepted.The chemical structure of the lipid A component of several enterobacterial lipopolysaccharides has been analysed during recent years in great detail (for reviews see [5, 61) and it was recognized that lipid A of Escherichiu coli possesses a comparatively simple structure. Free E. coli lipid A consists of a 8(1-6)-linked D-glucosamine disaccharide which is substituted by two phosphoryl groups, one being bound to position 4' of the nonreducing glucosamine residue (GlcN 11) and one being a-linked [7] to the glycosidic hydroxyl group of the reducing glucosaminyl group (GlcN I) (Fig.

GDP-L-Fuc:N-acetyl-␤-D-glucosaminide ␣136fucosyl-transferase (␣1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAc␤1-2Man␣1-6(GlcNAc␤1-2Man␣1-2)-Man␤1-4GlcNAc␤1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440,000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The ␣1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. ␣1-6FucT activity was found to be high in the transfected cells, as compared with non-or mocktransfected cells. Northern blotting analyses of rat adult tissues showed that ␣1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.It has been reported that the structures of glycopeptides change during the development and differentiation of embryos (1-4). Detailed analysis of specific antigens on the surface of various carcinoma cells revealed that carcinoma-specific sugar chains are expressed on the cell surface. A well documented phenotypic alteration of these specific sugar chains is the increase in the molecular weight of cell surface complex type N-linked glycan in transformed cells. This change has been observed regardless of the nature of the transforming agent: oncogenic viruses (5-9), chemical mutagens (10 -11), or DNA from unrelated tumor cells (12)(13)(14). This phenomenon was thought to reflect the deviation of carcinoma cells from the ordinary differentiation processes. ␣-Fucose residue attached to asparagine-linked GlcNAc also have some relationship with carcinogenesis. A difference in the binding pattern of serum ␣-fetoprotein with lentil lectin between hepatocellular carcinomas and benign liver diseases has been reported (15-17). Analyses of the carbohydrate structure of ␣-fetoprotein from hepatocellular carcinoma cell lines have indicated that almost all of the carbohydrates of ␣-fetoprotein are ␣1-6-fucosylated (18). ␣-Fetoprotein produced by germ cell tumors, such as yolk sac tumors, is also highly fucosylated (19). The activity of ␣1-6FucT 1 was higher in hepatocellular carcinoma tissue than in non-tumor tissue (20) and was induced by the transfection of the ras protooncogene into 3T3 fibroblast cells (21). Schachter et al. (22,23) first characterized ␣1-6FucT in porcine liver using a partially purified enzyme extract. The special release of ␣1-6FucT from platelets during blood clotting has been reported (24, 25), alteration of fucosylation has been reported in cystic fibrosis glycoproteins from different s...

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro.It was found that a synthetic P-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity.The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.

No abstract