Key mutations in the cylindrical inclusion involved in lettuce mosaic virus adaptation to eIF4E-mediated resistance in lettuce.

We previously showed that allelic genes mol¹ and mo1² used to protect lettuce crops against Lettuce mosaic virus (LMV) correspond to mutant alleles of the gene encoding the eukaryotic translation initiation factor 4E. LMV resistance-breaking determinants map not only to the main potyvirus virulence determinant, a genome-linked viral protein, but also to the C-terminal region of the cylindrical inclusion (CI), with a key role of amino acid at position 621. Here, we show that the propagation of several non-lettuce isolates of LMV in mo1¹ plants is accompanied by a gain of virulence correlated with the presence in the CI C terminus of a serine at position 617 and the accumulation of mutations at positions 602 or 627. Whole-genome sequencing of native and evolved isolates showed that no other mutation could be associated with adaptation to mo1 resistance. Site-directed mutagenesis pinpointed the key role in the virulence of the combination of mutations at positions 602 and 617, in addition to position 621. The impact of these mutations on the fitness of the virus was evaluated, suggesting that the durability of mo1 resistance in the field relies on the fitness cost associated with the resistance-breaking mutations, the nature of the mutations, and their potential antagonistic effects.

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Structure of sialyl-oligosaccharides isolated from bronchial mucus glycoproteins of patients (blood group O) suffering from cystic fibrosis.

The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group O were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 1H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra- up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. H. G. M., Van Halbeek H. & Vliegenthart J. F. G. (1984) Eur. J. Biochem. 143, 227-236]. The NeuAc residue was found to occur either in alpha (2----3)-linkage to Gal, or in alpha (2----6)-linkage to GalNAc-ol or Gal.

Action of trifluoromethanesulfonic acid on highly glycosylated regions of human bronchial mucins.

Highly glycosylated glycopeptides were prepared from human bronchial mucus. They were heterogeneous and contained an average of 45 residues of glycosylated hydroxyamino acid per 100 amino acid residues. The kinetics of deglycosylation of these glycopeptides by trifluoromethanesulfonic acid-anisole mixtures at 25 degrees was monitored by chemical analysis and by polyacrylamide gel electrophoresis. The peripheral sugars were almost completely cleaved in 45 min with 3:2 and 2:1 CF3SO3H-anisole. A maximum of 75% of the O-linked N-acetylgalactosamine residues were released and mixtures of glycopeptides and peptides were obtained. Increasing the reaction time caused peptide bond cleavage. Rather mild conditions (1.2:1 CF3SO3H-anisole at 25 degrees for 90 min) gave limited deglycosylation of highly glycosylated bronchial glycopeptides, allowing the uncovering of GalNAc-peptide linkages and peptide regions able to induce the formation of specific antibodies in the rabbit.