Precursors of ricin and Ricinus communis agglutinin Glycosylation and processing during synthesis and intracellular transport

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1 Eur. J. Biochem. 146, (1985) 0 FEBS 1985 Precursors of ricin and Ricinus communis agglutinin Glycosylation and processing during synthesis and intracellular transport J. Michael LORD Department of Biological Sciences, University of Warwick, Coventry (Received June 12/September 24, 1984) - EJB During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass Pretreatment of intact castor bean endospenn tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the M, form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies. Recent studies into the synthesis of the castor bean lectins have shown that their constituent A and B polypeptide chains are not the products of distinct transcripts. Both A and B chain sequences are encoded by a single mrna species [l]. The precursor polypeptide is synthesized on bound polysomes and is cotranslationally segregated into the lumen of the endoplasmic reticulum with concomitant removal of an N-terminal signal sequence and core glycosylation [2]. The A and B chains of both ricin and Ricinus communis agglutinin are N-glycosylated [3]. The assembly and processing of carbohydrate moieties linked to aspargine residues has been extensively studied [for review, see 41. The initial glycosylation is achieved by the transfer en bloc of an oligosaccharide [Gl~~Man~(GlcNAc)~ -1 from its dolichol carrier to a nascent polypeptide chain in the rough endoplasmic reticulum [5, 61. Core glycosylated proteins within the endoplasmic reticulum are susceptible to the action of endoglucosaminidase H [4]. In siru, the core glycosylated proteins undergo a variety of oligosaccharide modification steps [7, 81. Some of these occur in the Golgi apparatus during intracellular transport and render the modified oligosaccharide chain resistant to the action of endoglucosaminidase H [9]. In the case of animal and viral glycoproteins, acquired endoglucosaminidase H resistance is regarded as evidence that the glycoproteins have passed through, and been modified in, the Golgi apparatus [4, lo]. The present paper describes the application of this type of analysis during the intracellular transport of the glycosylated castor bean lectin precursors. The precursors acquired partial resistance to endoglucosaminidase H suggesting that they are Abbreviations. RAC,, Ricinus communis agglutinin, type I ; PhMeS04F, phenylmethylsulphonyl fluoride; SDS, sodium dodecyl sulphate. Enzyme. UDP-N-acetylglucosamine: glycolipid N-acetylglucosaminyltransferase (EC ). transported and modified by the Golgi apparatus. The addition of fucose to the oligosaccharide side chain has been identified as a potential Golgi modification. MATERIALS AND METHODS ~-[~%]Methionine, ~-[5,6-~H]fucose, N-acetyl-~-[l-~H]- glucosamine, GDP-~-[U-'~C]fucose, and UDP-N-acetyl-D- [U-14C]glucosamine were purchased from Amersham International (Amersham, Bucks, UK); Sepharose 6B and protein- A-Sepharose 4 B were from Pharmacia (Uppsala, Sweden); endo-n-acetylglucosaminidase H was from Miles Laboratories (Elkhart, IN, USA); proteinase K was from Boehringer (Mannheim, FRG); and rabbit antiserum against RCAI was from Vector Laboratories (Burlingame, CA, USA). Tunicamycin was a generous gift from Dr R. Hamill (Eli Lilly and Co., Indianapolis, IN, USA). Labeling in vivo and tissue fractionation Conditions described in the preceding paper [l 11 were used for the growth of plant tissue, for labeling excised endosperm slices with [35S]methionine ( kbq per slice), [3H]fucose ( kbq) and N-a~etyl-[~H]glucosamine ( kbq), and for fractionating tissue homogenates by sucrose density gradient centrifugation. When it was necessary to maintain the attachment of ribosomes to the rough endoplasmic reticulum membrane, the tissue was homogenized and fractionated in the presence of excess MgZf as described previously [ 121. Tunicamycin treatmenl Endosperm slices were each overlaid with 15 p1 of tunicamycin solution (1 mg/ml in 1 mm NaOH) and were

2 41 2 incubated at room temperature for 90 min, before the addition of radioactive label. Control slices were overlaid with a corresponding volume of 1 mm NaOH solution. Endoglucosarninidase H digestion of imrnunopvecipitates Immunoprecipitation was performed as described [2] except that 0.1 M lactose replaced galactose in the 1% Nonidet P40 buffer and 0.1 M lactose was added to each of the washing buffers. The antigen-antibody complexes were recovered using protein-a-sepharose and were released by resuspending the washed beads in 50 pl of 0.1 M glycine, ph 2.5, containing 0.1 M lactose. Protein was precipitated from the supernatant using cold trichloroacetic acid (10% final volume), recovered by centrifugation and washed with acetone. After drying, 10 p1 of 1% (w/v) SDS, 0.1 M citrate buffer ph 5.6, 1 mm PhMeS04F were added and the mixture was heated to 100 C for 2 min. When cool, 60 pl of 0.1 M citrate buffer, ph 5.6, 1 mm PhMeS04F were added and the mixture was then divided into two equivalent aliquots. 2.5 pl of endo-n-acetylglucosaminidase H (1 mu/pl) was added to one aliquot and the control aliquot received 2.5 p1 of water. Digestion was allowed to occur for 3 h at 37T. Glycosyltransferase assays After centrifugation, sucrose density gradients were collected as 1.0-ml fractions. Each fraction was halved and one half was assayed for GDP-fucose: glycoprotein fucosyl transferase by incubation with 1.85 kbq of GDP-[14C]fucose and 10 mm MgC12. After incubation at 30 C for 1 h, the reaction was stopped by adding an equivalent volume of cold 20% (w/v) trichloroacetic acid. The precipitated material was collected by filtration onto a Whatman GF/A filter disc, washed in 10% (w/v) trichloroacetic acid and chloroform/ methanol (2: 1) and its radioactivity was determined by liquid scintillation counting. The other half was assayed for UDP- N-acetylglucosamine : glycolipid N-acetyl-glucosaminyltransferase by incubation with 740 Bq of UDP-N-acetyl- ['4C]glucosamine and 10 mm MgC12. After incubation at 30 C for 1 h radioactivity incorporated into dolichyl monophosphate-n-acetylglucosamine was determined after chloroform/methanol(2 : 1) extraction as described previously ~31. Gel dectrophoresis Electrophoresis in the presence of SDS and fluorography were performed as described previously [2]. RESULTS Endosperm slices were treated with tunicamycin prior to labeling with [35S]methionine. The tissue was homogenized and fractionated and the total proteins present in the endoplasmic-reticulum-vesicle-containing fraction and in the denser (1.21 g/ml; see preceeding paper) vesicle fraction were separated electrophoretically (Fig. 1). The presence of tunicamycin did not significantly inhibit protein synthesis in comparison with untreated tissue nor did it affect the electrophoretic mobility of most of the labelled bands. One obvious exception was the appearance of a band of approximate M, in both fractions isolated from tunicamycintreated tissue (indicated by arrow, Fig. 1). This band was Fig. 1. EJfect of tunicamycin on protein synthesis in vivo. Maturing castor bean endosperm tissues was treated with tunicamycin prior to pulse labelling with [35S]methionine. The tissue was homogenized and fractionated by sucrose density gradient centrifugation. Polypeptides present in the endoplasmic-reticulum-derived vesicles and the denser vesicles were separated by dodecylsulphate/polyacrylamide gel electrophoresis and visualized by fluorography. Lane 1, molecular mass markers; lane 2, endoplasmic reticulum without tunicamycin treatment; lane 3, denser vesicles without tunicamycin treatment; lane 4, endoplasmic reticulum from tunicamycin-treated tissue; lane 5, denser vesicles from tunicamycin-treated tissue identified by immunoprecipitation as the unglycosylated lectin precursor (see below). It is slightly smaller than the unglycosylated lectin precursor (M, ) synthesized in vitro [l], the difference resulting from the removal of an N-terminal signal sequence [2]. The effect of tunicamycin on the synthesis of the lectin precursor was confirmed by immunoprecipitation using antibodies against RCAI. The results (Fig. 2) showed that in control tissue labeled with [35S]-methionine, glycosylated lectin precursor was initially present in both the endoplasmic reticulum and denser vesicle fractions (Fig. 2, lanes 2 and 3) while authentic-sized lectin subunits ultimately accumulated in the soluble (protein body matrix) fraction (Fig. 2, lane 5). In the presence of tunicamycin, although inhibition of glycosylation was by no means complete, a major non-glycosylated polypeptide band was immunoprecipitated from the two vesicle fractions (Fig. 2, lanes 6 and 7) which eventually gave rise to unglycosylated lectin subunits in the protein body fraction (Fig. 2, lane 9). The glycosylated precursor present in the endoplasmic-reticulum-containing fraction was completely sensitive to endoglucosaminidase H digestion after different chase periods with unlabelled methionine (compare Fig. 2, lanes 10, 11 and 12 with the untreated lanes 15, 16 and 17). The enzymically deglycosylated precursor had the approximately same electrophoretic mobility as the unglycosylated precursor synthesized in the presence of tunicamycin. The enzymically deglycosylated bands had a slightly lower electrophoretic mobility than

3 413 Fig. 2. N-Glycosylution of lectin precursors and subunits. Control castor bean endosperm tissue or tissue which had been treated with tunicamycin was pulse-labelled with [35S]methionine for 1 h and was subsequently chased with excess unlabelled methionine for various timcs. Tissue was homogenized and fractionated to give endoplasmic-reticulum-containing vesicles (E), denser vesicles (V) and a soluble fraction containing protein body matrix components (S). Polypeptides immunopreeipitated from the various fractions by antiserum raised against RCA, were separated by dodecylsulphate/polyaerylamide gel electrophoresis visualized by fluorography. In some instances (lanes 10-14) immunoprecipitates were digested with endoglucosaminidase H prior to electrophoresis. Lane 1, molecular mass markers; lane 2, endoplasmic reticulum, 90 min chase; lane 3, denser vesicles, 90 min chase; lane 4, soluble fraction, 90 min chase; lane 5, soluble fraction, 17 h chase; lanes 6-9, as lanes 2-5 respectively but from tunicamycin-treated tissue; lane 15, endoplasmic reticulum, no chase; lane 16, endoplasmic reticulum, 30 mi, chase; lane 17, endoplasmic reticulum, 90 min chase; lane 18, denser vesicles, 90 min chase; lane 19, soluble fraction, 17 h chase; lanes 10-14, as lanes respectively except that immunoprecipitates were treated with endoglucosaminidase H before electrophoresis the non-glycosylated bands, possibly due to the residual asparagine-lin ked N-acetylglucosamine residues. In contrast the glycosylated precursor present in the denser vesicle fraction was only partially susceptible to endoglucosaminidase H digestion and was not completely deglycosylated (compare Fig. 2, lanes 13 and 18). Endoglucosaminidase H also had different effects on the authentic lectin subunits which accumulate in the protein body matrix (Fig. 2, lane 19). The largest-mr polypeptide band (RCAI B chain) was completely sensitive to endoglucosaminidase H, while the intermediate band (a mixture of ricin B chain and heavy A chain [l, 141) was partially sensitive, and the smallest-mr band (a mixture of ricin and RCAI A chains) appeared to be completely resistant to endoglucosaminidase H (Fig. 2, lane 14). The B chains of both ricin and RCAI have previously been shown to be sensitive to endoglucosaminidase H [I]. The deglycosylated RCA, B chain has the same electrophoretic mobility as the endoglucosaminidase-h-insensitive A chains, while the deglycosylated ricin B chain accounts for the faint band below the A chains (Fig. 2, lane 14). The faint uppermost band (Fig. 2, lane 14) is either endoglucosaminidase-h-insensitive heavy A chain or partially deglycosylated RCAI B chain. Collectively these results confirmed that the lectin precursor was N- glycosylated and showed that the multiple bands observed (for example, Fig. 2, lane 2) were due to oligosaccharide heterogeneity. Further, the precursor in the denser vesicle fraction, in contrast to that present in the endoplasmic reticulum vesicles, had acquired endoglucosaminidase H resistance. This resistance was not complete, however, suggesting that the precursor present in the denser vesicle fraction contained a mixture of endoglucosaminidase-h-sensitive and endoglucos- aminidase-h-resistant oligosaccharide chains. This resistance to enzymic digestion may have resulted from the addition of further sugar molecules to the oligosaccharides. Although the original analysis of the sugars attached to purified ricin only identified glucosamine and mannose [15,16], a recent analysis has shown that purified ricin A chain additionally contains one molecule each of fucose and xylose (B. Foxwell, personal communication). Accordingly, [3H]fucose was utilized in pulse-chase experiments. After a 1 -h pulse with [3H]fucose followed by a 90-min chase with unlabeled fucose, both the fraction containing endoplasmic reticulum vesicles and the denser vesicles contained radioactive protein (Fig. 3 A). After an 18-h chase, fucosylated protein was largely absent from these particulate fractions and had accumulated in the soluble phase (Fig. 3B). Fig. 4 summarizes these experiments and clearly indicates that fucose was initially added in the fraction containing endoplasmic reticulum vesicles, after which the fucosylated protein was transported via the denser vesicle fraction to the protein bodies. Electrophoretic analysis showed that the lectins were the only major cellular glycoproteins which became labeled when intact endosperm tissue was supplied with either [3H]fucose or N-a~etyl-[~H]glucosamine (Fig. 5). Radioactivity from both sugars was initially present in the precursor (Fig. 5, lanes 4 and 6). Longer exposure of the gel to X-ray film confirmed that the precursors in the fraction containing endoplasmic reticulum vesicles (Fig. 5, lane 3) also contained [3H]fucose. Ultimately this radioactivity accumulated in authentic lectin subunits (Fig. 5, lanes 9 and 10). Radioactive lectins recovered from the soluble (protein body matrix) phase of [3H]fucoselabeled endosperm homogenates were hydrolyzed in trifluoro-

4 414 + o 8- + x 4-1 'I Chase period (h Fig. 4. Intracellular transport of fucosylated protein. Maturing castor bean endosperm tissue was pulse-labelled with [3H]fucose for 1 h and was subsequently chased with excess unlabelled fucose for various time periods. During the chase period tissue was homogenized and fractionated. At each stage the proportion of the total radiolabelled protein in the endoplasmic reticulum (O), denser vesicle (0) and soluble fraction (0) was determined - Fraction Fig. 3. Fucosylated protein initially present in cellular organelles accumulates in the soluble fraction. Maturing castor bean endosperm tissue was pulse-labelled with [3]fucose for 1 h and was subsequently chased with excess unlabelled fucose for 90 rnin (A) or for 18 h (B). The tissue was fractionated by sucrose density gradient centrifugation and collected in 1.O-ml fractions. The protein present in each fraction was precipitated with cold trichloroacetic acid, counted and its radioactivity was expressed as a percentage of the total incorporated acetic acid. The released radioactivity ran as a single spot during descending paper chromatography which comigrated with authentic fucose (data not shown). The addition of fucose to the oligosaccharide chain indicated that the precursor had passed through the Golgi apparatus [17]. Since fucose addition occurred in the fraction containing endoplasmic reticulum vesicles, this fraction was probably heterogeneous. To examine this possibility further, an endosperm homogenate was fractionated on a sucrose density gradient and collected as 1.O-ml fractions. Aliquots of each fraction were assayed for the enzyme catalyzing synthesis of dolichyl monophosphate-n-acetylglucosamine, an endoplasmic reticulum marker, and for glycoprotein fucosyltransferase. The distribution of these two ezymes confirmed that the endoplasmic reticulum vesicle fraction also contained the fucosyltransferase activity, although a slight difference in their relative distribution suggested that they might occur in distinct membrane fractions (Fig. 6A). The gradient used contained EDTA which stripped the rough endoplasmic reticulum of ribosomes [12]. The inclusion of excess Mg2+ in the homogenization medium and gradient solutions maintained the attachment of ribosomes to the endoplasmic reticulum Fig. 5. Lectin precursors and subunits are the major cellular glycoproteins. Maturing castor bean endosperm tissue was pulse-labelled with [35S]methionine,[3H]fucose or N-a~etyl[~H]glucosamine for 1 h and was subsequently chased with the appropriate unlabelled compound for various time periods. The tissue was homogenized and fractionated, and polypeptides from the endoplasmic-reticulum-containing vesicles (E), denser vesicles (V) or soluble (S) fractions were separated by dodecylsulphate/polyacrylamide gel electrophoresis and visualized by fluorography. Lanes 1 and 14, molecular mass markers; lane 2, methionine-labelled endoplasmic reticulum, 60 rnin chase; lane 3, fucose-labelled endoplasmic reticulum, 90 rnin chase; lane 4, N-acetylglucosamine-labelled endoplasmic reticulum, 90 rnin chase; lanes 5-7, as lanes 2-4 except for the denser vesicle fraction; lane 8, methionine-labelled soluble fraction, 17 h chase; lane 9, fucoselabelled soluble fraction, 18 h chase; lane 10, N-acetylglucosaminelabelled soluble fraction, 18 h chase; lanes 11-13, as lanes 8-10 except the chase period was 90 rnin

5 t ( Fraction Fig. 6. Intracellular distribution of glycosyltrmsferases. Maturing castor bean endosperm tissue was homogenized and fractionated by sucrose density gradient centrifugation in the absence (A) or presence (B) of excess Mg2+. Gradients were collected as 1.0-ml fractions which were divided into equal aliquots. One aliquot was assayed for glycoprotein fucosyltransferase (0) while the other was assayed for glycolipid-n-acetylglucosamin yltransferase ( 0) membrane. This increased the mean buoyant density of the glycolipid-n-acetylglucosaminyltransferase associated with the rough microsomes (Fig. 6B). In contrast, the presence of Mg2+ did not affect the gradient distribution of the bulk of the fucosyltransferase activity. Although the assay method employed here depends on the presence of endogenous acceptors, it is clear that a proportion at least of the fucosyltransferase was associated with a smooth-surfaced membrane fraction distinct from the endoplasmic reticulum. While it is tempting to assume that such a fraction was Golgi-derived, this has not been rigorously established at present. DISCUSSION The results presented in this paper establish that the castor bean lectin subunits are the major glycoproteins synthesized by maturing endosperm cells. The oligosaccharide chains are added cotranslationally to the lectin precursor in the endoplasmic reticulum. The precursor appers as a group of glycosylated polypeptides which are converted to a single-sized polypeptide band if N-glycosylation is prevented by tunicamycin or if the oligosaccharides are removed enzymatically using endoglucosaminidase H. The precursor is transported from the endoplasmic reticulum to the protein bodies via a distinct vesicle population of mean buoyant density 1.21 g/ ml. During transport from the endoplasmic reticulum to this vesicle fraction, the oligosaccharide moieties undergo modification. This modification results in the appearance of a smaller 41 5 number of glycosylated polypeptides of slightly lower molecular mass than the core glycosylated endoplasmic reticulum forms (compare lanes 2 and 3, Fig. 2). More significantly modification renders the oligosaccharide chains insensitive to endoglucosaminidase. The modification steps include the addition of fucose which, by analogy with animal and viral glycoproteins, indicates that the precursor is transported from the endoplasmic reticulum to the denser transporting vesicles via the Golgi apparatus [17]. The intracellular transport route proposed in the present paper for the castor bean lectins is identical to that recently proposed for phytohaemagglutinin in developing Phaeseolus cotyledons [ 181. Phytohaemagglutinin is also fucosylated during transport, a step that was attributed to a Golgi apparatus fucosyltransferase [19]. Treatment of intact endosperm tissue with tunicamycin inhibits glycosylation of the lectin precursor. Inhibition was not complete and both glycosylated and unglycosylated precursor chains were synthesized. Glycosylation is not required for intracellular transport and unglycosylated lectin subunits accumulate in the protein bodies (Fig. 2, lane 9). This also demonstrates that glycosylation is not required to give the precursor a conformation which allows cleavage by the protein body endoproteinase which generates the lectin subunits. The attachment of fucose is likely to be only one of a range of carbohydrate modification steps which occur after the initial core-glycosylation of the nascent lectin precursor. In the absence of information concerning the structure of the oligosaccharide chains during the various stages of intracellular transport, these additional processing steps remain unknown. I thank Croda Premier Oils (Hull, UK) for generously providing the castor bean seeds. This work was supported by the Science and Engineering Research Council (UK) through grants GR/B43753 and GRlC REFERENCES 1. Butterworth, A. G. & Lord, J. M. (1983) Eur. J. Biochem. 137, Roberts, L. M. & Lord, J. M. (1981) Eur. J. Biochem. 119, Olsnes, S. & Pihl, A. (1976) in The Specijkity of Animal, Bacterial andplant Toxins (Cuatrecasas, P., ed.) pp , Chapman and Hall, London. 4. Hubbard, S. C. & Ivatt, R. J. (1981) Annu. Rev. Biochem. 50, Snider, M. & Robbins, P. (1981) Methods Cell. Biol. 23, Hanover, J. A. & Lennarz, W. J. (1981) Arch. Biochem. Biophys. 211, Elting, J. J., Chen, W. W. & Lennarz, W. J. (1980) J. Biol. Chem. 255, Goldberg, D. E. & Kornfeld, S. (1983) J. Biol. Chem. 258, Tarentino, A. L. & Maley, F. (1975) Biochem. Biophys. Res. Commun. 67, Strous, G. J. A. M. & Lodish, H. F. (1980) Cell22, Lord, J. M. (1985) Eur. J. Biochem. 144, Lord, J. M., Kagawa, T., Moore, T. S. & Beevers, H. (1973) J. Cell. Biol. 57, Mellor, R. B. &Lord, J. M. (1979) Planta (Berl.) 144, Cawley, D. B., Hedblom, M. L. & Houston, L. L. (1978) Arch. Biochem. Biophys. 190, Funatsu, G., Yoshitake, S. & Funatsu, M. (1978) Agr. Biol. Chem. 42,

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