Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with beta-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.     

Zymolyase Treatment Exposes p55 Antigen of Pneumocystis carinii

Rats exposed to Pneumocystis carinii mount antibody responses to a broad band migrating on western blot with an apparent molecular weight of 45-55 kDa. One antigen within this band, designated p55, is uniformly recognized by P. carinii exposed rats. Although the gene encoding the p55 antigen had been previously cloned, the location of this antigen within the organism was unknown. Prior attempts to localize the protein were unsuccessful. A monospecific polyclonal antiserum raised against a carboxyl-terminai 15-oligomer peptide yielded specific reactivity with a single 55 kDa band on a western blot of P. carinii. Using this antiserum, little to no reactivity could be detected with P. carinii organisms by immunofluorescence assay (IIFA). However, zymolyase treatment of P. carinii dramatically increased the intensity and proportion of organisms reactive by IFA. Zymolyase, an enzyme with β-1,3 glucanase activity, has previously been shown to remove the electron dense outer layer of the P. carinii cell wall, exposing an electron lucent layer. Immunoelectron microscopy performed on zymolyase treated organisms showed the majority of labeling occurs within the cell wall.     

Sed1p Is a Major Cell Wall Protein of Saccharomyces cerevisiae in the Stationary Phase and Is Involved in Lytic Enzyme Resistance

A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Amino acid sequence analysis revealed that this protein is the product of the SED1 gene. SED1 was formerly identified as a multicopy suppressor of erd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway. Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor. However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins. Epitope-tagged Sed1p was detected in a β-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein. The expression of Sed1p mRNA increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls. Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase. These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance.     

COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

Candida albicans mycelial wall structure: supramolecular complexes released by Zymolyase,chitinase and β-mercaptoethanol

Different techniques released from the wall of Candida albicans mycelial cells high molecular weight mannoprotein materials with different levels of complexity. SDS solubilized among others one protein of 180 kDa which reacted with a monoclonal antibody (MAb) specific of a O-glycosylated protein secreted by regenerating mycelial protoplasts [Elorza et al. (1989) Biochem Biophys Res Commun 162:1118–1125]. Zymolyase, chitinase and -mercaptoethanol, released different types of high molecular highly polydisperse mannoprotein materials (>180 kDa) that also reacted with the same MAb. These materials had N-glycosidically linked sugar chains, in addition to the O-glycosidically bonded sugars, as their molecular masses were significantly reduced by Endo H digestion. Besides, the specific materials released by either zymolyase or chitinase seemed to be the same throughout the process of germ tube formation. Transmission electron microscopy of thin sections of cells and walls showed that mannoproteins and chitin are evenly distributed throughout the entire cell wall structure.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Subcellular localization of the Streptococcus mutans P1 protein C terminus.

To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.     

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains.

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.     

Nucleotide sequences of a Korean isolate of apple stem grooving virus associated with black necrotic leaf spot disease on pear (Pyrus pyrifolia)

Pear black necrotic leaf spot (PBNLS) is a disease of pears caused by capillovirus-like particles, which can be observed under the electron microscope. The disease was analyzed by Western blot analysis with antisera raised against apple stem grooving virus (ASGV) coat protein. cDNAs covering the entire genome were synthesized by RT-PCR and RACE using RNA isolated from Chenopodium quinoa infected with sap extracted from pear leaves carrying black necrotic spot disease. The complete genome sequence of the putative pear virus, 6497 nucleotides in length excluding the poly (A) tail, was determined and analyzed. It contains two overlapping open reading frames (ORFs). ORF1, spans from nucleotide position 37 to 6354, producing a putative protein of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at nucleotide 4788 and terminates at 5750, and produces a putative protein of 36 kDa. The 241 kDa protein contains sequences related to the NTP-binding motifs of helicases and RNA-dependent RNA polymerases. The 36-kDa protein contains the consensus sequence GDSG found in the active sites of several cellular and viral serine proteases. Morphological and serological analysis, and sequence comparison between the putative pear virus, ASGV, citrus tatter leaf virus and cherry virus A of the capillovirus suggest that PBNLS may be caused by a Korean isolate of ASGV.     

Li+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy

The effect of Li⁺ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm^?1 range, of Na⁺ and Li⁺ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li⁺ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li⁺ ion treatment.     

A Protease that Participates in Yeast Cell Wall Lysis during Zymolyase Digestion

Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these results, it is hypothesized that Zymolyase B binds to the cell wall mannans and changes their conformation, making the attached proteins susceptible to proteolysis, and then releases peptide-mannan from the cell walls.     

Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae.

Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their N-terminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a beta 1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2 delta. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.     

Abolishment of N-glycan mannosylphosphorylation in glyco-engineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p.

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine- and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall. Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation. On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.     

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.     

Antigenic cell wall mannoproteins in Candida albicans isolates and in other Candida species.

Polyclonal antibodies (pAbs) and monoclonal antibodies (mAbs), raised against mannoprotein components from Candida albicans ATCC 26555 (serotype A) blastoconidia and mycelial cell walls, were used to investigate antigenic similarities among wall mannoproteins from other C. albicans serotype A and B strains, and from C. tropicalis and C. guilliermondii. Radioactively labelled walls isolated from cells grown at either 28 degrees C or 37 degrees C were digested with a beta-glucanase complex (Zymolyase 20T) to release cell-wall-bound mannoproteins. Numerous molecular species with different electrophoretic mobilities were released from the various isolates. Differences appeared to be related to both the organism and the growth temperature. Among the major protein components solubilized were mannoproteins larger than 100 kDa (high molecular mass mannoproteins), heterogeneous in size in most cases. Antigenic homology was detected among the cell wall high molecular mass mannoproteins of the two C. albicans serotype A isolates, whereas significant qualitative and quantitative differences were detected between serotype A and serotype B cell-wall-bound antigenic profiles. Moreover, C. tropicalis and C. guilliermondii wall antigenic determinants were not recognized by the preparations of pAbs and mAbs raised against C. albicans walls. A mannoprotein with a molecular mass of 33-34 kDa was present in the enzymic wall digests of all the organisms studied. When probed with pAbs raised against the protein moiety of the 33 kDa cell wall mannoprotein of Saccharomyces cerevisiae, antigenic cross-reactivity was observed in all cases except C. tropicalis. There appear to be significant antigenic differences between the mannoproteins of different isolates of C. albicans, and between those of C. albicans and other Candida species.     

The Identification of an Anther Specific Antigen inBrassicaSpecies using a Heterologous Monoclonal Antibody

Monoclonal antibody, MAb 69-139-19, raised against sheep sperm,cross-reacted with anther proteins from fourBrassicaspeciestested. Early in anther development the MAb weakly detecteda polypeptide of42 kDa. At the mid-maturation stage the MAbstill bound weakly to this band but it also detected a polydisperseband of Mr 160–230 kDa. In mature pollen MAb 69-139-19labelled the polydisperse region of 160–230 kDa very strongly,as well as a faint, but distinct, band of116 kDa. Immunogoldlabelling ofB. napusL. anthers and pollen grains also showeda differential pattern of labelling. At the early vacuolatestage the MAb recognized an antigen within the tapetum and themicrospore cytoplasm and nucleus. During the late vacuolatestage the MAb bound to the tapetal material during transferto the pollen grain wall, leading to strong labelling of thepollen wall at maturity. In sheep MAb 69-139-19 binds to thepostacrosomal region of the sperm (Hou, 1989). The polypeptidepresent in plants, which contains the epitope recognized bythe MAb is likely to be a different protein to that in sheep,but we suggest that it plays a role in sexual reproduction inBrassica.It is possible that as the polypeptide is located in the pollencoat it may be involved in pollen/stigma interactions duringpollination leading to successful adhesion and pollen tube germination. Brassica napusL; incongruity; pollen-stigma recognition; pollination; rapeseed; heterologous antibody