Biosynthesis of the Snowdrop ( Galanthus nivalis ) Lectin in Ripening Ovaries 1

Content Provider	Semantic Scholar
Author	Damme, E. Van
Copyright Year	2005
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 OfMr 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-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M, 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing. 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 coand/or posttranslational levels. Lectins occur also in different types of vegetative tissues of many dicotyledonous as well as monocotyledonous species where they can represent a significant part of the total protein content. Recently we isolated a mannose-specific lectin from bulbs of snowdrop (Galanthus nivalis) (12). Further studies have shown that the lectin is not confined to the underground parts of the plant but also occurs in leaves and stems as well as in certain ' Supported in part by grants of the National Fund for Scientific Research (Belgium) of which W. P. is a Senior Research Associate. E. V. D. acknowledges the receipt of a Fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw. 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 METHODS Material. 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 addition 1 mM EDTA whereas medium B contained in addition 2 mM Mg-acetate. The ovaries were homogenized in a cold mortar with medium A or B and the homogenate was centrifuged at 3000g for 1 min to remove nuclei and cell wall debris. Small aliquots of the extracts were withdrawn for SDS-PAGE, protein estimation, and determination of [3H]leucine uptake and incorporation in TCA-insoluble material. The remainder of the extract was used for further analyses. Isolation of Organelles. The organelles were separated from the soluble proteins and small molecules on Sepharose 4B columns (1.7 x 12 cm) (Pharmacia, Uppsala, Sweden) as described by Van der Wilden et al. (13). Briefly, homogenates made in homogenization medium A or B were applied to Sepharose 4B columns equilibrated with medium A or B, respectively. The columns were eluted with the homogenization medium and fractions of 1 ml each collected. Fractions containing the light-scattering material (organelles) in the first peak and those containing the soluble proteins (which eluted in the second peak) were pooled and used for further analyses. 2 Abbreviation: GNA, Galanthus nivalis agglutinin. 922 www.plantphysiol.org on July 21, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. BIOSYNTHESIS OF THE SNOWDROP LECTIN Fractionation of ER on Sucrose Gradients. Linear 16 to 48% (w/w) sucrose gradients were formed in medium A or B with a Beckman density gradient former on top of a 1 ml cushion of 70% sucrose. Gradients were loaded with 2 ml of organelle fraction (from the Sepharose 4B column) and centrifuged for 2.5 h at 38,000 rpm in a Beckman SW 40 rotor. Fractions of 0.5 ml were collected with an ISCO (Palo Alto, CA) gradient fractionator and used for determination of total [3H]leucine incorporation (into TCA-insoluble material), [3H]leucine incorporation in the lectin, NADH Cyt c reductase and Cyt c oxidase activity. Sucrose concentrations were determined with a refractometer. Isolation of the Lectin by Affinity Chromatography. Fractions from Sepharose 4B or sucrose gradients were brought at 0.5% Triton X-100 and 0.5 M (NH4)2SO4, and applied to small (0.2 ml bed volume) columns of immobilized mannose (Selectin 10 from Pierce Chemical Co., Rockford, IL) equilibrated with 0.5 M (NH4)2SO4. Unbound proteins were washed off with 5 ml of 0.5 M (NH4)2SO4. Then the lectin was desorbed with 4 ml of unbuffered 2,3 diaminopropane, frozen at 80°C and lyophilized. These lyophilized fractions were dissolved in 1 ml of distilled water and the lectin precipitated with TCA (10% final concentration). Finally the precipitate was dissolved in sample buffer for subsequent SDS-PAGE and fluorography (and for determination of [3H]leucine incorporation in the lectin). SDS-PAGE and Fluorography. SDS-PAGE was done on 12.5 to 25% acrylamide gradient gels using a discontinuous system as described by Laemmli (7). After fixing and destaining gels were immersed in amplify (Amersham, U.K.) for 30 min, dried under partial vacuum, and exposed to preflashed x-ray films (FUJI RX, Japan). Analytical Methods. NADH Cyt c reductase and Cyt c oxidase were assayed as described by Bowles and Kauss (2) and Sottocasa et al. (11), respectively. Agglutination assays using trypsin-treated rabbit erythrocytes were carried out as described previously (12). Protein contents of extracts were determined by the method of Bradford (3) using BSA as a standard. RESULTS Choice of Material. Ripening ovaries of snowdrop represent a favorable system for a study of the lectin synthesis for several reasons. First, the plant material is easy to prepare and to handle. We preferred ovaries over bulbs since extracts from bulbs are very viscous and rapidly turn brown. In addition, the lectin concentration within the ovaries is very high and the lectin can readily be purified. The reliability of the affinity purification procedure as described in the "Materials and Methods" section is quite high. As shown in Figure 1 the lectin fraction obtained after affinity chromatography is pure (lane 3). In addition, the same figure shows that the lectin polypeptides (which are the major labeled bands on the fluorogram of the crude extract [lane 1]) are completely removed by affinity chromatography (lane 2). GNA is Synthesized in Vivo as a Precursor. Snowdrop ovaries were labeled with [3H]leucine for different times and the (total) lectin was isolated by affinity chromatography on immobilized mannose. Subsequent analyses by SDS-PAGE and fluorography revealed that the first product to be synthesized is a polypeptide with an apparent Mr or 15,000 (Fig. 2). After longer labeling times radioactivity also appears in the lectin polypeptide with a Mr of 13,000. This sequence of appearance of radioactivity in different polypeptides suggests that GNA is synthesized as a precursor of Mr 15,000 which is subsequently converted into the 13,000 Mr lectin polypeptide. Kinetics of the Conversion of the Lectin Precursor into the Authentic Lectin. To follow the fate of the precursor and determine the kinetics of its processing pulse-chase experiments were carried out. Snowdrop ovaries were labeled for 2 h with [3H]leucine and further incubated in excess of unlabeled leucine for different Po
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