Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

Abstract

The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (Mr) 15,000 which is posttranslationally converted into the authentic lectin polypeptide Of Mr 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [3HJleucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organeliar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase.Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 miliimolar Mg- Until now most of the information about the biosynthesis and processing of plant lectins has been obtained with seed lectins. In vivo and in vitro studies of lectin synthesis in soybean (Glycine max) (14), field bean (Vicia faba) (4), pea (Pisum sativum) (5, 6), french bean (Phaseolus vulgaris) (1), castor bean (Ricinus communis) (10), rice (Oryza sativa), and wheat (Triticum aestivum) (9) have shown that these lectins are all synthesized as higher mol wt precursors which are subsequently processed at the co-and/or posttranslational levels.Lectins parts of the flower, especially the ovary (E Van Damme, unpublished data). The GNA' is a tetrameric protein built up of four identical subunits of M, 13,000, which does not contain carbohydrate (12).In this paper we report the biosynthesis of the snowdrop lectin in ripening ovaries. We show evidence that this lectin is synthesized in the ER as a precursor of M, 15,000 which is posttranslationally modified. MATERIALS AND METHODSMaterial. Flowering plants of Galanthus nivalis were collected locally. Unless used immediately they were kept in water to prevent wilting. Ovaries were excised immediately before the onset of the experiments.Radioactive Labeling of the Ovaries. Ovaries were cut longitudinally and each half incubated with a small droplet (10 ,l) containing 10 ,Ci [3H]leucine for appropriate times. Five ovaries were used for each experimental condition. In chase experiments ovaries were labeled with [3H]leucine for 2 h, washed free of labeled precursor, and incubated for different times in the presence of 20 Al of a saturated solution of unlabeled leucine. During the experiments ovaries were kept on a parafilm sheet in closed Petri dishes at 20°C. To prevent dessication moistened filter paper was placed below the parafilm sheets on which the ovaries were kept.Tissue Homogenization. After labeling for the appropriate times the ovaries were rinsed with distilled water and blotted dry. Two different media were used for homogenization and isolation of the organelles. Both contained 100 mm Tris-HCl (pH 7.8) and 12% sucrose; medium A contained in addi...

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

“…In order to find the reagents that will specifically probe for glycosylation abnormalities in HeLa cells, we tested four lectins with different sugar specificity: Galanthus nivalis agglutinin (GNL; binds to glycoconjugates with terminal mannose) (Van Damme and Peumans 1988;Fouquaert et al 2007), Griffonia simplicifolia lectin-II (GS-II; binds to GlcNAc and agalactosylated N-glycans) (Lamb et al 1983;Tateno et al 2007), Lotus tetragonolobus lectin (LTL; binds to Fucα1-3GlcNAc) (Pereira and Kabat 1974;Tateno et al 2007) and wheat germ agglutinin (WGA; binds to GlcNAcβ1-4GlcNAcβ1-4GlcNAc and Neu5Ac) (Nagata and Burger 1974;Tateno et al 2007). For WGA staining, a slight decrease in lectin binding to the surface of all tested COG-deficient cells was detected (Figure 1B), indicating a defect in protein sialylation.…”

Alpha-(1-3)- and alpha-(1-6)-D-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro

“…The following lectins were used in our studies: Galanthus nivalis agglutinin (GNA) (snowdrop; family Amaryllidaceae), Hippeastrum hybrid agglutinin (HHA) (amaryllis; family Amaryllidaceae), Narcissus pseudonarcissus agglutinin (NPA) (daffodil; family Amaryllidaceae), and Listera ovata agglutinin (LOA) (twayblade; family Orchidaceae). GNA, HHA, and NPA were isolated from bulbs of snowdrop, amaryllis, and daffodil, respectively, as described by Van Damme and colleagues (27,28,30,31). LOA was isolated from leaves of twayblade essentially as described previously (29).…”

Mannose-specific lectin from the mushroom Hygrophorus russula

“…BanLec, GNA, HHA and TxLcI were isolated from banana, snowdrop, amaryllis and Tulipa by modification of previously described methods using mannose-agarose (Kaku et al 1990;Koshte et al 1990;Oda et al 1986;Shibuya et al 1988;Van Damme et al 1988;Van Damme et al 1987) Each lectin was incubated with biotin amidocaproate N-hydroxysuccinimide ester (Sigma) in 0.1 M NaHCO 3 with its haptenic sugar for 12 h at 4ºC, desalted with Sephadex G-25 (GE Healthcare UK Ltd.) and lyophilized. The degree of biotinylation was determined by using the 4-hydroxyazobenzene-2-carboxylic acid (HABA) assay (Green et al 1970).…”