Characterization of the binding of alpha-L-Fuc (1-->2)-beta-D-Gal (1-->), a xyloglucan signal, in blackberry protoplasts.

Previous work showed that the fucose-->galactose moiety of the xyloglucan nonasaccharide XXFG is responsable for its biological activity. We used this side chain of XXFG (alpha-L-Fuc (1-->2)-beta-D-Gal (1-->)) in ligand-binding experiments to demonstrate its role as a signal molecule in plant cells. Proteins solubilized from plasma membrane enriched fractions isolated from Rubus fruticosus protoplasts were tested for their ability to bind the side chain of XXFG, using a digoxigenin- or biotin-conjugated neoglycoprotein specific for 2'-fucosyl-lactose in blots and k-ELISA tests. A putative receptor for the signaling sugar was identified, and the ligand specificity is reported. The role of structural elements important for biological activities was investigated using compounds structurally related to xyloglucan, and a variety of phytohormones such as 2,4-D.

Two rhamnogalacturonide tetrasaccharides isolated from semi-retted flax fibers are signaling molecules in Rubus fruticosus L. cells.

Water extraction of semi-retted flax (Linum usitatissimum L.) fiber bundles yielded a mixture of pectic oligosaccharides and two acidic rhamnogalacturonide tetrasaccharides that were separated by size-exclusion chromatography. One- and two-dimensional nuclear magnetic resonance studies and fast atom bombardment-mass spectrometry experiments indicated that the two tetrasaccharides have a common primary structure, i.e. alpha-D-delta GalpA(1-->2)-alpha-L- Rhap(1-->4)-alpha-D-GalpA-(1-->2)-L-alpha,beta-Rhap, with a rhamnopyranose as terminal reducing end, and a 4-deoxy-beta-L-threo-hex-4-enopyranosiduronic acid at the nonreducing end. However, the two tetrasaccharides differ by an acetyl group located at the O-3 position of the internal galacturonic acid residue. These two tetrasaccharides induce the activation of D-glycohydrolases of Rubus fructicosus L. cells or protoplasts within minutes.

Membrane binding sites for the human blood group H-type 2 determinant, an inducer of laminarinase activity in protoplasts of Rubus fruticosus L.

The human blood-group determinants H-type 1 (α-L-Fuc-(1 → 2)-ß-D-Gal-(1 → 3)ß-D-GlcNAc), or type 2 (α-L-Fuc(1 → 2)-ß-D-Gal(1 → 4)-ß-D-GlcNAc) and their mono- and disaccharidic precursors, have been reported to induce D-glycanase (laminarinase) activity in Rubus cells (Y. Liénart et al. 1990, Plant Science 68, 197-202) and protoplasts (Y. Liénart et al. 1991, Plant Science 77, 41-45). Using immunoadsorbent H-type 1 as a matrix for the affinity purification of membrane proteins, and the H-type 2 trisaccharide neoglycoprotein as ligand in kinetic-dependent enzyme-linked-immunosorbent assay for measuring binding, we were able to show that Rubus microsomes contain high-affinity binding sites for the laminarinase inducers. The N-acetyl glucosamine (GlcNAc) eluate was found to contain a saturable, high affinity binding activity for GlcNAc, compatible with the presence of a single class of binding sites Kd = 2 nM, Bmax = 400 pmol · (mg protein)(-1). In contrast, the Scatchard plot of proteins in the lactose eluate was nonlinear. In competition studies, the precursors of H-type 1 (GlcNAc-OCH3, ß-D-Gal-(1 → 3)-ß-D-GlcNAc-OCH3) or of H-type 2 (GlcNAc, N-acetyl lactosamine) trisaccharides inhibited the binding of the proteins in the GlcNAc eluate by H-type 2 neoglycoprotein with respective IC50 values of 0.6, 0.6 or 2, 0.4 nM. These data, and the binding of the H-type 2 trisaccharide by a protein of Mr 260 kDa in a ligand-blot process, are indicative of the general properties exhibited by receptors.

Isolation from Rubus cell-suspension cultures of a lectin specific for glucosamine oligomers.

A lectin specific for glucosamine oligomers has been purified by chitosan affinity chromatography from cultured cells of Rubus. The lectin, eluted by a glucosamine oligomer of degree of polymerization 4 in the presence of l-α-phosphatidylserine dipalmitoyl, was found by sodium dodecyl sulfate-polyacrylamide gel electroploresis to be homogeneous and to have a molecular weight of 67 kilodaltons; it could best bind the tetrasaccharide, as shown by ligand-blot processing. Data from kinetic-dependent enzyme-linked immunosorbent assays showed that the lectin has two apparent binding sites which better accommodate the tetrasaccharide and the hexasaccharide, respectively, of the glucosamineoligomer series. The affinity of the lectin for glucosamine oligomers was shown to decrease for chain lengths greater than six glucosaminyl residues.

ß-D-glucan-hydrolase activities in pure cell-wall-enriched fractions from Valerianella olitoria cells.

An effective method for the preparation of purified cell walls from mesophyll cells of Valerianella olitoria has been developed. Cells were isolated by a mechanical procedure only and crude cell walls were prepared from cell homogenates. Crude wall suspensions were fractionated in a discontinuous sucrose gradient and the wall fragments recovered were examined by scanning electron microscopy. An evaluation of the degree of purity and physiological integrity of the wall fragments showed that the material found at the 50-60% (w/w) interface consisted mostly of wall particles of high purity. Some characteristics of the purified walls are reported, especially the following enzyme activities: ß-D-glucosidase (EC 3.2.1.21) and the ß-D-glucanases, 1,4-ß-glucan glucanohydrolase (EC 3.2.1.4), 1,4-ß-glucan cellobiohydrolase (EC 3.2.1.91), 1,3-ß-glucan glucanohydrolase (EC 3.2.1.39), 1,3-ß-glucan glucohydrolase (EC 3.2.1.58). The results provided evidence for the microlocalization of some hydrolases and indicated that enzymes extracted only with a high-salt-concentration buffer were confined to walls whereas the 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-solubilized enzymes could have multiple sites, e.g. walls and membranes of the endoplasmic reticulum.