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.