The presence of an alpha4-fucosyltransferase in plants was first deduced from the characterization of Lewis-a glycoepitopes in some N-glycans. The first plant gene encoding an alpha4-fucosyltransferase was recently cloned in Beta vulgaris. In the present paper we provide evidence for the presence of an alpha4-fucosyltransferase in A. thaliana by measurement of this glycosyltransferase activity from a purified microsomal preparation and by immunolocalization of Le(a) epitopes on glycans N-linked to glycoproteins located to the Golgi apparatus and on the cell surface. The corresponding gene AtFT4 (AY026941) was characterized. A unique copy of this gene was found in A. thaliana genome, and a single AtFT4 transcript was revealed in leaves, in roots, and at a lower extent in flowers. The coding sequence of AtFT4 gene is interrupted by two introns spanning 465 bp and 84 bp, respectively. The putative 393-amino-acid protein (44 kDa, pI: 6.59) contains an N-terminal hydrophobic region and one potential N-glycosylation site, but AtFT4 has poor homology (less than 30%) to the other alpha3/4-fucosyltransferases except for motif II. When expressed in COS 7 cells the protein is able to transfer Fuc from GDP-Fuc to a type 1 acceptor substrate, but this transferase activity is detected only in the culture medium of transfected cells

The peptide-N4-(N-acetylglucosaminyl) asparagine amidase (PNGase Se) earlier described [Lhernould S., Karamanos Y., Bourgerie S., Strecker G., Julien R., Morvan H. (1992) Glycoconjugate J 9:191-97] was partially purified from cultured Silene alba cells using affinity chromatography. The enzyme is active between pH 3.0 and 6.5, and is stable in the presence of moderate concentrations of several other protein unfolding chemicals, but is readily inactivated by SDS. Although the enzyme cleaves the carbohydrate from a variety of animal and plant glycopeptides, it does not hydrolyse the carbohydrate from most of the corresponding unfolded glycoproteins in otherwise comparable conditions. The substrate specificity of this plant PNGase supports the hypothesis that this enzyme could be at the origin of the production of 'unconjugated N-glycans' in a suspension medium of cultured Silene alba cells.

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducing N-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man3(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and 1H- and 13C-NMR assignments for the oligosaccharide Man3(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).

No abstract

We studied the distribution of wall ingrowth (WI) polymers by probing thin sections of companion cells specialized as transfer cells in minor veins of Medicago sativa cv Gabès blade with affinity probes and antibodies specific to polysaccharides and glycoproteins. The wall polymers in the controls were similar in WIs and in the primary wall but differently distributed. The extent of labeling in these papillate WIs differed for JIM5 and JIM7 homogalacturonans but was in the same range for LM5 and LM6 rhamnogalacturonans and xyloglucans. These data show that WI enhancement probably requires arabinogalactan proteins (JIM8) mainly localized on the outer part of the primary wall and WIs. By comparison, NaCl-treated plants exhibited cell wall polysaccharide modifications indicating (1) an increase in unesterified homogalacturonans (JIM5), probably implicated in Na(+) binding and/or polysaccharide network interaction for limiting turgor variations in mesophyll cells; (2) enhancement of the xyloglucan network with an accumulation of fucosylated xyloglucans (CCRC-M1) known to increase the capacity of cellulose binding; and (3) specific recognition of JIM8 arabinogalactan proteins that could participate in both wall enlargement and cohesion by increasing the number of molecular interactions with the other polymers. In conclusion, the cell wall polysaccharide distribution in enlarged WIs might (1) participate in wall resistance to sequestration of Na(+), allowing a better control of hydric homeostasis in mesophyll cells to maintain metabolic activity in source leaves, and (2) maintain tolerance of M. sativa to NaCl.

We previously reported the occurrence of oligomannosides and xylomannosides corresponding to unconjugated N-glycans (UNGs) in the medium of a white campion (Silene alba) cell suspension.Attention has been focused on these oligosaccharides since it was shown that they confer biological activities in plants. In an attempt to elucidate the origin of these oligosaccharides, we studied two endoglycosidase activities, putative enzymes involved in their formation. The previously described peptide-N'-(N-acetyl-glucosaminyl) asparagine amidase activity and the endo-N-acetyl-8-Dglucosaminidase activity described in this paper were both quantified in white campion cells during the culture cycle with variable initial concentrations of sucrose. The lower the sucrose supply, the higher the two activities. Furthermore, endoglycosidase activities were greatly enhanced after the disappearance of sugar from the medium. The production of UNCs in the culture medium rose correlatively. These data strongly suggest that the production of UNCs in our white campion cell-suspension system is due to the increase of these endoglycosidase activities, which reach their highest levels of activity during conditions of carbon starvation.Xylomannosides Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAcMan~(Xyl)GlcNAc(Fuc)GlcNAc and the oligomannoside ManaGlcNAc have been isolated from the extracellular suspension-culture medium of white campion (Silene alba) (Priem et al., 1990b; Lhemould et al., 1992). The importance of these UNGs was recently emphasized (Priem et al., 1994). Several glycoproteins conjugated to these oligosaccharides have been reported (Kamerling, 1991). The excretion and accumulation of the UNGs in the medium at the end of the culture period could be explained by glycoprotein breakdown. When deprived of SUC, cells consume starch, protein, and lipid reserves (Joumet et al., 1986). We have recently demonstrated the presence of peptide-N4-(N-acetyl-glucosaminyl) Asn amidase, named PNGase Se, in the cell extract of white campion (Lhemould et al., 1992). This enzyme hydrolyzes the glycosylamine linkage. Oligomannosides and xylomannosides are effective substrates for PNGase Se only when they are carried by small peptides and not by proteins or glycoasparagines (S. Lhemould, Y . Karamanos, H. Morvan, unpublished data).In this paper we show the presence of endo-N-acetyl-8-D-* Corresponding author; fax 33-55-45-73-86. 779glucosaminidase, named ENGase Se, in cell extracts of white campion, which could explain the occurrence of the oligomannoside described earlier (Priem et al., 1990b), and we have also evaluated the influence of exogenous Suc concentration on the level of endoglycosidase activities. MATERIALS A N D METHODS Cell CultureWhite campion (Silene alba [Miller] E.H.L. Krause) suspension culture was obtained by the Dubois and Bourriquet (1973) procedure. Subcultures were produced in 500-mL flasks with 200 mL of medium as previously described (Morvan, 1982), except that the growth period was extended to 30 d. Suc (10, 20, or 40 g/L) was i...

No abstract

The wood polysaccharide composition, a new analytical method, based on ionic liquid dissolution of low amount of biomass coupled with an ELISA essay of polysaccharides. In the present work, we synthesized and tested several imidazolium and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) based ILs for their ability to solubilize Douglas-fir wood while preserving the wall polymer integrity. The couple times-temperatures have been essayed for wood dissolution. Then their efficiency for wood biomass dissolution was compared to the impact of IL on storing and/or destroy polysaccharides. Thanks to the ELISA technique with a set of mAbs against epitopes of the main hemicellulose, pectin, and protein families of cell wall components. Wood destructuration at 80˚C with the 1-ethyl-3-methylimidazolium bromide represents a good compromise of wood dissolution efficiency and low polysaccharide destruction.