We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the u-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The u-1,6 arm shows a remarkable heterogeneity: in addition to a-1,2-Man and a-1,6-Man, the presence of a terminal mannose a-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant Nglycans on cellobiohydrolase I can be represented as follows : GlcMan,GIcNAc,, GlcMan,GlcNAc,, Man,GlcNAc,, ManPGlcMan,GlcNAc,, GlcMan,GlcNAc, and Man,GlcNAc,.Keywords: cellobiohydrolase I ; fungus ; N-glycans ; NMR ; Trichoderma reesei.Trichoderma reesei is a filamentous fungus capable of secreting high amounts of cellulose-degrading enzymes (up to 40 g/l by manipulated strains). This remarkable secretory capacity led to the idea to use this organism as a candidate host for the production of heterologous proteins [I]. However, it is not clear whether T. reesei can be used for production of pharmaceutically important glycoproteins since post-translational modifications by the fungus, such as glycosylation, could be a major problem for application. Oligosaccharides lacking sialic acid as terminal sugar residues are easily recognized by lectins circulating in blood and present on cells of the reticulo-endothelial system. This recognition leads to fast clearance, which prevents the therapeutic proteins reaching their target organs [2, 31. Furthermore, it has been demonstrated that foreign glycosyl structures often elicit an antigenic response [4].Only few data concerning glycosylation in filamentous fungi are available. They indicate high-mannose-type glycans of limited sizes compared with these synthesized by yeast [S-121. The identification by Salovuori et al. 1121 of Man,GlcNAc, and Man,GlcNAc, as the major N-glycans on 7: reesei cellobiohydrolase I (CBH I) suggested a possible analogy between fungal and mammalian oligosaccharide structures. For identical primary structures, in vitro and eventually in vivo extension of oligosaccharides on proteins secreted from this fungus to the complex type should be possible.Correspondence to R. Contreras Enzyme. a-mannosidase (EC 3.2.1.24).During the eukaryotic secretion process, proteins receive in the endoplasmic reticulum a common precursor oligosaccharide, namely Glc,Man,GlcNAc,. This precursor is transferred by an oligosaccharyl transferase to asparagine residues in the consensus sequence Asn-Xaa-(Ser or Thr), with Xaa representing any amino acid except proline. A battery of glycosidases and gl...

We investigated estrogen-like properties of five perfluorinated compounds using a combination of three in vitro assays. By means of an E-screen assay, we detected the proliferation-promoting capacity of the fluorotelomer alcohols 1H,1H,2H,2H-perfluorooctan-1-ol (6:2 FTOH) and 1H,1H,2H,2H-perfluoro-decan-1-ol (8:2 FTOH). The more widely environmentally distributed compounds perfluoro-1-octane sulfonate, perfluorooctanoic acid, and perfluorononanoic acid did not seem to possess this hormone-dependent proliferation capacity. We investigated cell cycle dynamics using flow cytometric analyses of the DNA content of the nuclei of MCF-7 breast cancer cells. Exposure to both fluorotelomer alcohols stimulated resting MCF-7 cells to reenter the synthesis phase (S-phase) of the cell cycle. After only 24 hr of treatment, we observed significant increases in the percentage of cells in the S-phase. In order to further investigate the resemblance of the newly detected xenoestrogens to the reference compound 17β-estradiol (E2), gene expression of a number of estrogen-responsive genes was analyzed by real-time polymerase chain reaction. With E2, as well as 4-nonylphenol and the fluorotelomer alcohols, we observed up-regulation of trefoil factor 1, progesterone receptor, and PDZK1 and down-regulation of ERBB2 gene expression. We observed small but relevant up-regulation of the estrogen receptor as a consequence of exposures to 6:2 FTOH or 8:2 FTOH. The latter finding suggests an alternative mode of action of the fluorotelomer alcohols compared with that of E2. This study clearly underlines the need for future in vivo testing for specific endocrine-related end points.

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The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae a-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10±12 glycosyl residues.Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD 50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.

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