Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds

Abstract

1. Methylation analysis of potato (Solanum tuberosum) lectin and thorn-apple (Datura stramonium) lectin confirmed previous conclusions that both glycoproteins contained high proportions of l-arabinofuranosides and lesser amounts of d-galactopyranosides. The arabinofuranosides are present in both lectins as short unbranched chains containing 1-->2- and 1-->3-linkages, which are known to be linked to hydroxyproline. Galactopyranosides are present as monosaccharides, which are known to be attached to serine, in potato lectin and as both the monosaccharide and the 1-->3-linked disaccharide in Datura lectin. 2. Alkaline digestion of potato lectin and subsequent separation of the components by gel filtration led to the isolation of four fractions corresponding to the mono-, di-, tri- and tetra-arabinosides of hydroxyproline. The latter two fractions accounted for over 70% of the total hydroxyproline. 3. Methylation analysis was used to show that the triarabinoside contained only 1-->2-linkages between sugars, but that the tetra-arabinoside contained both 1-->2- and 1-->3-linkages. Direct-insertion mass spectrometry of these compounds using electron impact and chemical ionization, in a comparison with other known structural patterns, was used to determine the sequences of the sugars, which were Araf1-->2Araf1-->2Araf1-->Hyp and Araf1-->3Araf1-->2Araf1-->2Araf 1-->Hyp. 4. On the basis of optical rotation it had previously been suggested [Allen, Desai, Neuberger & Creeth (1978) Biochem. J.171, 665-674] that all the arabinose of potato lectin was present as the beta-l-furanoside. However, measurement of the optical rotations of the hydroxyprolyl arabinosides showed that whereas the diarabinoside had a molar rotation ([m]) value close to that predicted, the triarabinoside was more dextrorotatory and the tetra-arabinoside was less dextrorotatory than expected. Possible explanations for these findings are that, although the di- and tri-arabinosides contain exclusively beta-arabinofuranosides, in the tri-arabinoside, interactions between pentose units lead to an enhanced positive rotation. The tetra-arabinoside, however, is proposed to contain a single alpha-arabinofuranoside residue, which is responsible for the lower than expected positive rotation. The observed rotation of the tetra-arabinoside was found to be close to the theoretical value predicted on that basis. Furthermore, the action of a specific alpha-arabinofuranosidase on the tetrasaccharide was to remove a single arabinose residue, presumably the terminal non-reducing sugar, and to produce a product that was indistinguishable on electrophoresis from the triarabinoside. Changes in rotation were compatible with this assumption. 5. It is concluded that the structures of the hydroxyprolyl tri- and tetra-arabinosides of potato lectin are: betaAraf1-->2betaAraf1-->2betaAraf1-->Hyp and alphaAraf1-->3betaAraf1-->2betaAraf 1-->2betaAraf1-->Hyp. These are identical with compounds that have been isolated from the insoluble hydroxyproline-rich glycoproteins of plant cell walls.

Molecular Cloning and Characterization of a β-l-Arabinobiosidase in Bifidobacterium longum That Belongs to a Novel Glycoside Hydrolase Family

“…Ashford et al (5) reported that fungus ␣-L-arabinofuranosidase hydrolyzed Ara 4 -Hyp to Ara 3 -Hyp. Because HypBA2 cannot hydrolyze Ara 4 -Hyp, ␣-L-arabinofuranosidase will be needed for effective degradation of ␤-L-arabinofuranosides.…”

A New Lectin from Meadow Saffron (Colchicum automnale)

“…Cystine was determined as cysteic acid after performic-acid oxidation of the protein and subsequent hydrolysis at 10°C in constant-boiling HCI (6). Amino acids were measured on a Locarte Mini amino acid analyzer by reaction with ninhydrin; details of the elution programs which were used have been given elsewhere (2,3). Neutral sugars were measured by GLC on a Perkin-Elmer gas chromatograph fitted with flame ionization detectors after methanolysis (4 h at 85C) and trimethylsilylation ofthe glycoproteins (5).…”

Functional Analysis of Degradative Enzymes for Hydroxyproline-linked ^|^beta;-L-Arabinofuranosides in Bifidobacterium longum

“…Extensin and Solanaceae lectins contain repetitive Ser-Hyp 4 motifs with Hyp residues that are O-glycosylated with 1-4 arabinofuranosyl (Araf) residues with β-L-arabinofuranosyl linkages. The structures of Ara 3 -Hyp and Ara 4 -Hyp, which are the major constituents of Hyp-linked β-Larabinofuranosides in dicotyledons (7), are Arafβ1-2Arafβ1-2Arafβ-Hyp and Arafα1-3Arafβ1-2Arafβ1-2Arafβ-Hyp, respectively (8,9). However, despite the broad distribution of Hyp-linked β-L-arabinofuranosides in plant cells, degradative enzymes have not been identified yet.…”