Chemoenzymatic synthesis and application of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination

Sialoglycopeptide (SGP) is referred as the glycopeptide in hen's egg yolk, which has an N-linked, complex-type, disialyl biantennary oligosaccharide with an alpha-(2-->6)-sialyl N-acetyllactosamine residue. The residue is known as a binding ligand of type-A human influenza virus hemagglutinin. We describe herein a simple synthesis of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination that makes use of the natural source ingredient, SGP, and the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). Its inhibitiory activity for influenza virus hemagglutination is 40 times higher than that of SGP, and its competitive inhibition is determined to be over 300 times higher than that of fetuin. These results indicate that a sialoglycopolymer having a multivalent sialo-oligosaccharide could potentially be used for the prevention of influenza virus infection.     

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.     

Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Identification and characterization of a novel antigen complex on mouse mammary tumor cells using a monoclonal antibody against platelet glycoprotein Ic

The rat monoclonal antibody GoH3 identifies a complex of glycoproteins Ic and IIa on human and mouse platelets. The GoH3 epitope is located on glycoprotein Ic. A novel glycoprotein complex is identified by GoH3 on the surface membranes of mouse mammary epithelial tumor cells. This antigen complex is composed of glycoprotein Ic noncovalently associated with a monomor or a disulfide-linked multimer of a high molecular weight glycoprotein (Ic-binding protein (IcBP]. Glycoprotein Ic is synthesized as a large precursor with asparagine N-linked high mannose oligosaccharides. Processing of this precursor involves a proteolytic cleavage of the large polypeptides into two smaller disulfide-linked polypeptide chains, Ic alpha (heavy) and Ic beta (light), and conversion of the majority of the high mannose oligosaccharides into complex-type glycans. Likewise, glycoprotein IcBP is initially glycosylated with high mannose asparagine N-linked oligosaccharides which are processed to complex units in the mature form. Association of glycoprotein Ic with IcBP occurs within the cell soon after their synthesis. The kinetics of labeling show non-coordinate processing consistent with the idea that the concentration of glycoprotein Ic limits complex formation and the subsequent processing of glycoprotein IcBP.     

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35–38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.     

Oligosaccharide-Related Epitope Specific for a Brain-Specific Glycoprotein, 1D4 Antigen

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.     

Natural killer cells discriminate between high mannose- and complex-type asparagine-linked oligosaccharides

Chinese hamster ovary cell lines with different types of N-linked oligosaccharides were tested as targets for control and lymphokine treated natural killer (NK) cells. The targets tested were parent cells, Lec1 mutants and Lec4 mutants. Due to an apparent defect in GlcNAc transferase V, Lec4 cells produce complex-type N-linked oligosaccharides devoid of GlcNAc beta(1-6) linked branches. Lec1 cells form only high mannose-type N-linked oligosaccharides because they lack GlcNAc transferase I activity. Lec1 cells are very sensitive to lysis by beta-interferon treated human NK cells, but both parent and Lec4 cells are resistant to NK lysis. The ability to discriminate between parent and Lec1 targets was demonstrated with untreated control effectors as well as those which were pretreated with either beta-interferon, gamma-interferon or interleukin-2. Both control and lymphokine-boosted NK cells exhibit much greater lytic activity against targets having only high mannose-type N-linked oligosaccharides. Five oligosaccharide structures resembling those found on N-linked glycoproteins were tested for their ability to block NK lysis of Lec1 targets. Only the high mannose-type glycopeptide from 7S soybean glycoprotein was inhibitory in the mu molar range. At the same concentration, none of the complex-type oligosaccharides had any effect on lytic activity. The results suggest that a high mannose-type N-linked oligosaccharides is recognized at some step in NK cell-mediated lysis.     

Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside

GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for (1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed. These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with ³H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes, interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation. All solvent ratios are by volume. An erratum to this article can be found at     

Molecular differentiation in pea powdery-mildew haustoria

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.Abbreviations ehm extrahaustorial membrane - ELISA enzyme-linked immunosorbent assay - HC haustorial complex - hpm haustorial plasma membrane - IIF indirect immunofluorescence - MAb monoclonal antibody - M_r apparent molecular weight - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Mr. D. Mills and Mr. P. Stanley for help with the EM immunogold techniques. This work was supported by an Agricultural and Food Research Council grant and a studentship from the Science and Engineering Research Council.     

Identification of a novel UDP-Glc:GlcNAc beta1-->4-glucosyltransferase in Lymnaea stagnalis that may be involved in the synthesis of complex-type oligosaccharide chains

Several studies suggest, that the snail Lymnaea stagnalis contains glycoproteins whose oligosaccharide side chains have structural features not commonly found in mammalian glycoproteins. In this study, prostate glands of L. stagnalis were incubated in media containing either [(3)H]-mannose, [(3)H]-glucosamine, or [(3)H]-galactose, and the metabolically radiolabeled protein-bound oligosaccharides were analyzed. The newly synthesized diantennary-like complex-type asparagine-linked chains contained a considerable amount of glucose, next to mannose, GlcNAc, fucose, galactose, and traces of GalNAc. Since glucose has not been found before as a constituent of diantennary N-linked glycans as far as we know, we assayed the prostate gland of L. stagnalis for a potential glucosyltransferase activity involved in the biosynthesis of such structures. We report here, that the prostate gland of L. stagnalis contains a beta1-->4-glucosyltransferase activity that transfers glucose from UDP-glucose to acceptor substrates carrying a terminal N-acetylglucosamine. The enzyme prefers substrates carrying a terminal GlcNAc that is beta6 linked to a Gal or a GalNAc, structures occurring in O-linked glycans, or a GlcNAc that is beta2 linked to mannose, as is present in N-linked glycans. Based on combined structural and enzymatic data, we propose that the novel beta1-->4-gluco-syltransferase present in the prostate gland may be involved in the biosynthesis of Glcbeta1-->4GlcNAc units in complex-type glycans, in particular in N-linked diantennary glycans.     

Bovine Reissner’s fiber (RF) and the central canal of the spinal cord: an immunocytochemical study using a set of monoclonal antibodies against the RF-glycoproteins

The subcommissural organ secretes N-linked complex-type glycoproteins into the cerebrospinal fluid. These glycoproteins condense to form Reissner’s fiber (RF), which extends along the fourth ventricle and central canal of the spinal cord. A set of three monoclonal antibodies (Mabs 3E6, 3B1, and 2A5) has been obtained using these glycoproteins as immunogens. Competitive and sandwich enzyme-linked immunoassay methods have demonstrated that the three monoclonal antibodies are directed against different epitopes, and that there is no competition among them for their binding to glycoproteins of RF. Mab 3E6 displays immunoblotting properties that are similar to those of a polyclonal antibody against the pool of glycoproteins from RF, but that are different from those of Mabs 3B1 and 2A5. All three antibodies immunostain the bovine subcommissural organ and RF. A population of ependymal cells is stained by the polyclonal antibody, and Mabs 2A5 and 3E6, but not by Mab 3B1. The material present in a population of ependymal cells of the central canal, and the glycoproteins secreted by the subcommissural organ thus probably have partial chemical identity. Some evidence suggests that the immunoreactive ependymal cells are secretory cells. The luminal surface of the central canal is coated by a thin layer of material with immunocytochemical characteristics different from those of the ependymal cells; such a coat may correspond to material released from RF. Received: 19 December 1995 / Accepted: 30 April 1996     

Demonstration of glucuronic acid on brain glycoproteins which react with HNK-1 antibody

Ependymins, a family of extracellular glycoproteins of goldfish and mammalian brain, were shown to contain N-linked complex glycan chains. These glycoproteins reacted with a monoclonal antibody, HNK-1 which recognizes a membrane antigen on a subset of human lymphocytes, myelin-associated glycoprotein glycoprotein epitope reacting with HNK-1 antibody was previously shown to include a terminal 3-sulfoglucuronosyl residue present in certain glycolipids of the nervous tissue (Chou et al., Biochem. Biophys. Res. Commun. 1985, 128, 383-388). In this report, the presence of glucuronic acid in ependymins was demonstrated by gas-liquid chromatography and mass spectrometry. We suggest that a 3-sulfoglucuronosyl residue may be the common epitope on HNK-1-reactive glycoproteins.     

Enhancement of phorbol ester induced cell aggregation after alterations in asparagine-linked oligosaccharides

Summary— Human erythroleukemia (K-562) cells grown in the presence of phorbol 12,13-dibutyrate formed aggregates of cells not seen in untreated control cultures. Furthermore, the proportion of cells in aggregates and the size of the aggregates both increased dramatically in cultures treated with both phorbol ester and kifunensine, an inhibitor of asparagine-linked oligosaccharide processing. Relative to control cells, phorbol ester treated cells exhibited a greater proportion of N-linked oligosaccharides of the complex-type. Kifunensine prevented this change and caused an accumulation of Man₉GlcNAc₂. The enhanced aggregation of cells treated with phorbol ester plus kifunensine depended on phorbol ester concentration and was blocked by inhibitors of protein kinase C (H7, sphinganine and sangivamycin). In flow cytometry analysis, phorbol ester treated K-562 cells showed an increase in CD44, a glycoprotein involved in cell adhesion. Moreover, monoclonal antibody to CD44 augmented reaggregation of phorbol ester treated cells. The results implicate phorbol ester induction of CD44 in aggregation of K-562 cells and demonstrate that the presence of high mannose-type asparagine-linked oligosaccharides on cell glycoproteins correlates with increased aggregation of phorbol ester treated cells.     

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants

We have investigated the structure of glycans N-linked to the proteins of the moss Physcomitrella patens. The structural elucidation was carried out by western blotting using antibodies specific for N-glycan epitopes and by analysis of N-linked glycans enzymatically released from a total protein extract by combination of MALDI–TOF and MALDI–PSD mass spectrometry analysis. Nineteen N-linked oligosaccharides were characterised ranging from high-mannose-type and truncated paucimannosidic-type to complex-type N-glycans harbouring core-xylose, core-(1,3)-fucose and Lewis^a, as previously described for proteins from higher plants. This demonstrates that the processing of N-linked glycans, as well as the specificity of glycosidases and glycosyltransferases involved in this processing, are highly conserved between P. patens and higher plants. As a consequence, P. patens appears to be a new promising model organism for the investigation of the biological significance of protein N-glycosylation in the plant kingdom, taking advantage of the potential for gene targeting in this moss.Abbreviations Asn asparagine - CID collision-induced dissociation - Glc glucose - GlcNAc N-acetylglucosamine - Man mannose - MALDI–TOF MS matrix-assisted laser desorption ionization–time of flight mass spectrometry - PNGase A peptide N-glycosidase A - PSD post-source decay     

The Lex Carbohydrate Sequence Is Recognized by Antibody to L5, a Functional Antigen in Early Neural Development

Abstract: The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N -glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5-positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N -glycans containing a 3-fucosyl N -acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N -acetyllactosamine sequence [Lewis^x (Le^x) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the Le^x sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

N-linked oligosaccharides of cobra venom factor contain novel alpha(1-3)galactosylated Le(x) structures

Cobra venom factor (CVF), a nontoxic, complement-activating glycoprotein in cobra venom, is a functional analog of mammalian complement component C3b. The carbohydrate moiety of CVF consists exclusively of N-linked oligosaccharides with terminal alpha1-3-linked galactosyl residues, which are antigenic in human. CVF has potential for several medical applications, including targeted cell killing and complement depletion. Here, we report a detailed structural analysis of the oligosaccharides of CVF. The structures of the oligosaccharides were determined by lectin affinity chromatography, antibody affinity blotting, compositional and methylation analyses, and high-resolution (1)H-NMR spectroscopy. Approximately 80% of the oligosaccharides are diantennary complex-type, approximately 12% are tri- and tetra-antennary complex-type, and approximately 8% are oligomannose type structures. The majority of the complex-type oligosaccharides terminate in Galalpha1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1, a unique carbohydrate structural feature abundantly present in the glycoproteins of cobra venom.     

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.     

Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase

Previously, we developed a transgenic tobacco BY2 cell line (GT6) in which glycosylation was modified by expressing human beta(1,4)-galactosyltransferase (betaGalT). In this study, we produced a mouse monoclonal antibody in GT6 cells, and determined the sugar chain structures of plant-produced antibodies. Galactose-extended N-linked glycans comprised 16.7%, and high-mannose-type and complex-type glycans comprised 38.5% and 35.0% of the total number of glycans, respectively. N-linked glycans with the plant-specific sugars beta(1,2)-xylose and alpha(1,3)-fucose comprised 9.8%. The introduction of human betaGalT into suspension cultured tobacco cells resulted in the production of recombinant proteins with galactose-extended glycans and decreased contents of beta(1,2)-xylose and alpha(1,3)-fucose.     

The Golgi apparatus ofPhytophthora cinnamomi makes three types of secretory or storage vesicles concurrently

Summary The formation of three types of vesicles in the oomycetePhytophthora cinnamomi was investigated using ultrastructural and immunocytochemical techniques. All three vesicles are synthesised at the same time; one type serves a storage role; the others undergo regulated secretion. A monoclonal antibody Lpv-1 that is specific for glycoproteins contained in the storage vesicles labelled the endoplasmic reticulum (ER), elements in the transition region between ER and Golgi stack, and cis, medial and trans Golgi cisternae. Cpa2, a monoclonal antibody specific for glycoproteins contained within secretory dorsal vesicles labelled the transition region, cis cisternae and a trans-Golgi network. Vesicles possessing a structure characteristic of mature secretory ventral vesicles were observed in close association with the trans face of Golgi stacks. The results suggest that all three vesicles are formed by the Golgi apparatus. Double immunogold labelling with Lpv-1 and Cpa-2 showed that these two sets of glycoproteins occurred within the same Golgi cisternae, indicating that both products pass through and are sorted concurrently within a single Golgi stack.