Characterization and localization of laccase forms in stem and cell cultures of sycamore

A laccase-type polyphenoloxidase (EC 1.10.3.2.), abundantly secreted by suspension-cultured sycamore (Acer pseudoplatanus) cells was purified to homogeneity. This laccase form is a glycoprotein (molecular weight 110000) with high mannose and complex glycans. The polypeptide moiety has a molecular weight of 66 000, indicating that the glycoprotein is 40% carbohydrate. Laccase is abundantly present in both the cell wall and the culture medium of suspension-cultured sycamore cells, but it is not detected in the cytoplasm, indicating that this large protein is efficiently secreted by the cells. Polyclonal rabbit antiserum was raised against the deglycosylated protein and was used to probe extracts of sycamore stem tissues. A second laccase form (molecular weight 56 000), antigenically related to laccase from cell cultures, is abundant in the epidermis of sycamore stems. In addition, this 56 kDa laccase form co-localizes with lignin precursors on tissue prints from sycamore stems. A polypeptide (molecular weight 50 000-56 000), antigenically related to sycamore laccase, was also immunodetected in most plant organs previously described in the literature as polyphenoloxidase-rich.     

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and β-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.     

Inhibition of disulfide bonding of von Willebrand protein by monensin results in small, functionally defective multimers

The biosynthesis of von Willebrand protein by human endothelial cells was impaired by the presence of the carboxylic ionophore monensin. Several processing steps that have been localized to the Golgi apparatus were affected in a dose-dependent manner, including carbohydrate processing, dimer multimerization, and precursor cleavage. Since multimerization was more susceptible to the ionophore than was precursor cleavage, it appears that these processing steps are separate events. As expected, dimer formation, which occurs in the rough endoplasmic reticulum, was unaffected by monensin. Thus, at high concentrations of monensin, only dimer molecules were produced and secreted. The observed inhibition of multimer formation and precursor cleavage were not likely the result of incomplete carbohydrate processing, since inhibition of complex carbohydrate formation by swainsonine did not interfere with the other processing steps. Monensin also affected the capacity of endothelial cells to store von Willebrand protein, as the ratio of secreted to cell-associated protein increased dramatically in the presence of monensin, and the processed forms could not be found in the treated cells. The low molecular weight multimers produced in the presence of monensin did not incorporate in the endothelial cells' extracellular matrix nor did they bind to the matrix of human foreskin fibroblasts. In summary, the presence of monensin in human endothelial cell culture produced experimental conditions that mimic Type IIA von Willebrand disease, in that the cells synthesized and secreted only low molecular weight von Willebrand protein multimers, which were functionally defective.     

N-linked oligosaccharide processing is not necessary for glycoprotein secretion in plants

The role of N-glycans in the secretion of glycoproteins by suspension-cultured sycamore cells was studied. The transport of glycoproteins to the extracellular compartment was investigated in the presence of a glycan-processing inhibitor, castanospermine. Castanospermine has been selected because it inhibits homogeneously glycan maturation in sycamore cells and leads to the accumulation of a single immature N-glycan. The structure of this glycan has been identified as Glc₃Man₇GlcNAc₂ by labeling experiments, affinity chromatography on concanavalin A-Sepharose and proton NMR. In contrast with previous results showing that N-glycosylation is a pre-requisite for secretion of N-linked glycoproteins, this secretion is not affected by the presence of castanospermine. As a consequence, the presence of this unprocessed glycan is sufficient for an efficient secretion of glycoproteins in the extracellular compartment of suspension-cultured sycamore cells.     

Targeting and post‐translational processing of human α1‐antichymotrypsin in BY‐2 tobacco cultured cells†

The post‐translational processing of human α₁‐antichymotrypsin (AACT) in Bright Yellow‐2 (BY‐2) tobacco cells was assessed in relation to the cellular compartment targeted for accumulation. As determined by pulse‐chase labelling experiments and immunofluorescence microscopy, AACT sent to the vacuole or the endoplasmic reticulum (ER) was found mainly in the culture medium, similar to a secreted form targeted to the apoplast. Unexpectedly, AACT expressed in the cytosol was found in the nucleus under a stable, non‐glycosylated form, in contrast with secreted variants undergoing multiple post‐translational modifications during their transit through the secretory pathway. All secreted forms of AACT were N‐glycosylated, with the presence of complex glycans as observed naturally on human AACT. Proteolytic trimming was also observed for all secreted variants, both during their intracellular transit and after their secretion in the culture medium. Overall, the targeting of human AACT to different compartments of BY‐2 tobacco cells led to the production of two protein products: (i) a stable, non‐glycosylated protein accumulated in the nucleus; and (ii) a heterogeneous mixture of secreted variants resulting from post‐translational N‐glycosylation and proteolytic processing. Overall, these data suggest that AACT is sensitive to resident proteases in the ER, the Golgi and/or the apoplast, and that the production of intact AACT in the plant secretory pathway will require innovative approaches to protect its structural integrity in vivo. Studies are now needed to assess the activity of the different AACT variants, and to identify the molecular determinants for the nuclear localization of AACT expressed in the cytosol.     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Xylose-specific antibodies as markers of subcompartmentation of terminal glycosylation in the Golgi apparatus of sycamore cells.

Antibodies specific for xylose-containing plant complex N-linked glycans are used for indirect immunolocalization of xylosyltransferase in sycamore cells. The use of high pressure freezing and freeze substitution for sample preparation resulted in very good morphological preservation of the different Golgi cisternae. Xylosyltransferase shows a diffuse distribution all over the Golgi stacks and xylosylation appears to be an early processing event that is initiated in the cis Golgi compartment.     

Proteinase-inhibitor synthesis in tomato plants: Evidence for extracellular deposition in roots through the secretory pathway

The cellular and subcellular localization of proteinase Inhibitors I and II proteins, synthesized in transgenic tomato (Lycopersicon esculentum L.) plants from chimeric genes regulated by the 35S promoter, was investigated by immunocytochemical techniques. Newly synthesized inhibitor proteins were deposited in the cell vacuoles as in wild-type plants, but were also secreted into the cell walls of outer epidermal and secretory cells of the root cap. The Na ionophore monensin increased the levels of proteinase inhibitors found in rough endoplasmic reticulum, Golgi cisternae and in the cell walls of transgenic plants, supporting a role for the secretory pathway in the sorting and targeting of Inhibitor I and II proteins. The two inhibitor proteins were detected by Western-blot analysis in water-washes obtained from roots of transgenic tomato seedlings, confirming their extracellular presence. Wild-type tomato plants exhibited the presence of Inhibitor I and II proteins in the external cell walls, using silver-enhanced immunogold labelling, but not by Western-blot analysis. The extracellular Inhibitor I from transgenic plant roots migrated in electrophoretic gels with a slightly different apparent mass than the Inhibitor I isolated from tomato leaf vacuoles, indicating that specific structural features of this inhibitor protein have been altered during or after extracellular deposition. The presence of extracellular inhibitors in roots may help provide protection for the growing meristems against insects or microorganisms present in the soil.Abbreviations CaMV cauliflower mosaic virus - TEM transmission electron microscope Transmission electron microscopy was performed at the Electron Microscopy Center (EMC) of Washington State University. The authors thank the EMC staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Greg Pearce, Scott Johnson, and Martha L. Orozco for their advice and technical help. The work was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants Nos. DCB-8702538 and DCB-8608594.     

The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein

A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins.     

Subcellular sites of processing of precursors to neurosecretory peptides in the bag cells of Aplysia: Inferences from the effects of monensin,FCCP, and chloroquine

The effects of the sodium ionophore monensin were examined in the bag cells of Aplysia californica in order to identify the subcellular sites of processing of precursors to their neurosecretory products. Incubation of bag cells in media containing 10 μM monensin led to a marked disruption of the morphology of the Golgi apparatus without affecting that of other organelles. Exposure of bag cells to monensin led to a significant impairment of processing of the largest precursor and of an intermediate protein which gives rise to the immediate precursors to the final secreted products, the egg-laying hormone (ELH) and the acidic peptide (AP). Furthermore, ELH and AP were never produced in the presence of monensin during the time course of these experiments. When axonal transport was allowed to proceed, the contents of bag-cell terminals indicated that the intermediate protein is the first to be packaged in Golgi-derived vesicles, and in monensin-treated cells may be transported without being processed further. In contrast to these results, the protonophore FCCP-impaired precursor and intermediate cleavage equally, indicating that monensin and FCCP have different effects on intracellular transport and precursor processing. These data are interpreted to indicate that the largest ELH-AP precursor is normally processed within the Golgi apparatus, and that the disruption of this organelle induced by monensin produces the impairment seen in its processing. The impairment of cleavage of the intermediate species, and the blockade of production of AP and ELH, are probably the result of monensin-induced impairment of production of proteolytically competent secretory granules by the Golgi apparatus.     

Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information

Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.     

Identification of the protein storage vacuole and protein targeting to the vacuole in leaf cells of three plant species

Protein storage vacuoles (PSVs) are specialized vacuoles devoted to the accumulation of large amounts of protein in the storage tissues of plants. In this study, we investigated the presence of the storage vacuole and protein trafficking to the compartment in cells of tobacco (Nicotiana tabacum), common bean (Phaseolus vulgaris), and Arabidopsis leaf tissue. When we expressed phaseolin, the major storage protein of common bean, or an epitope-tagged version of alpha-tonoplast intrinsic protein (alpha-TIP, a tonoplast aquaporin of PSV), in protoplasts derived from leaf tissues, these proteins were targeted to a compartment ranging in size from 2 to 5 microm in all three plant species. Most Arabidopsis leaf cells have one of these organelles. In contrast, from one to five these organelles occurred in bean and tobacco leaf cells. Also, endogenous alpha-TIP is localized in a similar compartment in untransformed leaf cells of common bean and is colocalized with transiently expressed epitope-tagged alpha-TIP. In Arabidopsis, phaseolin contained N-glycans modified by Golgi enzymes and its traffic was sensitive to brefeldin A. However, trafficking of alpha-TIP was insensitive to brefeldin A treatment and was not affected by the dominant-negative mutant of AtRab1. In addition, a modified alpha-TIP with an insertion of an N-glycosylation site has the endoplasmic reticulum-type glycans. Finally, the early step of phaseolin traffic, from the endoplasmic reticulum to the Golgi complex, required the activity of the small GTPase Sar1p, a key component of coat protein complex II-coated vesicles, independent of the presence of the vacuolar sorting signal in phaseolin. Based on these results, we propose that the proteins we analyzed are targeted to the PSV or equivalent organelle in leaf cells and that proteins can be transported to the PSV by two different pathways, the Golgi-dependent and Golgi-independent pathways, depending on the individual cargo proteins.     

Distribution of xylosylation and fucosylation in the plant Golgi apparatus

Antibodies have been immunopurified which are specific for carbohydrate epitopes containing the β1→2 xylose or α1→3 fucose residues found on complex N-linked glycans in plants. The antibody specificity was determined by taking advantage of an Arabidopsis thaliana N-glycosylation mutant which lacks N-acetyl-glucosaminyltransferase I and is unable to synthesize complex glycans. These antibodies were used to immunolocalize xylose- and fucose-containing glycoproteins in suspension-cultured sycamore cells (Acer pseudoplatanus). By mapping the enzymatic reaction products within the Golgi apparatus, the fucosyl- and xylosyltransferase subcellular localization was made possible using immunocytochemistry on thin sections of high-pressure frozen and freeze-substituted sycamore cells. This procedure allows a much better preservation of organelles, and particularly of the Golgi stack morphology, than that obtained with conventionally fixed samples. Glycoproteins containing β→2 xylose and α1→3 fucose residues were immunodetected in the cell wall, the vacuole, and the Golgi cisternae. The extent of immunolabeling over the different cisternae of 50 Golgi stacks was quantified after treatment with anti-xylose or anti-fucose antibodies. Labeling for xylose-containing glycoproteins was predominent in the medial cisternae, while fucose-containing glycoproteins were mainly detected in the trans compartment. Therefore, in plants, complex N-linked glycan xylosylation probably occurs mostly at the medial Golgi level and α1→3 fucose is mainly incorporated in the trans cisternae. Finally, fucose- and xylose-containing glycoproteins were also immunolocalized, albeit to a lesser extent, in earlier Golgi compartments. This indicates that the glycosylation events are a continuous process with some maxima in given compartments, rather than a succession of discrete and compartment-dependent steps.     

Dissecting cardosin B trafficking pathways in heterologous systems

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins’ high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.     

Sorting of proteins to the vacuoles of plant cells.

The secretory system of plant cells sorts a large number of soluble proteins that either are secreted or accumulate in vacuoles. Secretion is a bulk-flow process that requires no information beyond the presence of a signal peptide necessary to enter the endoplasmic reticulum. Many vacuolar proteins are glycoproteins and the glycans are often modified as the proteins pass through the Golgi complex. Vacuolar targeting information is not contained in glycans as it is in animal cells; rather, targeting information is in polypeptide domains as it is in yeast cells. Several such domains have now been identified, but these show little or no amino acid sequence homology. We discuss the possibilities that targeting of protein to plant vacuoles may involve receptors as well as aggregation of protein at low pH.     

Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.     

Inhibition of pollen-tube growth and protein secretion by the monovalent ionophore monensin

Summary Apple pollen tubes grown in vitro in the presence of up to 1 M monensin, which is known to slow down or stop vesicular traffic originating from the Golgi complex, were severely inhibited by the carboxylic ionophore. Monensin at concentrations as low as 5 nM significantly decreased the secretory release of protein into the extracellular space. During 2 h of germination in the presence of monensin, ³H-leucine incorporation into the membrane versus the soluble fraction increased with respect to the controls. These data seem to be consistent with an effective action of monensin on the secretory routes which, in pollen tubes, deliver products from the dictyosomes to the extending tip. The importance of monovalent cation homeostasis in the functioning of secretory paths, also in unique cells such as pollen tubes, emerges clearly from the present data.     

The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone

The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [³⁵S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium dodecyl-sulfate polyacrylamide-gel electrophoresis     

Glycodelin A, not glycodelin S, is apoptotically active. Relevance of sialic acid modification

Glycodelin, previously known as PP14 (placental protein-14), is a kernel lipocalin secreted by the glandular epithelium of the endometrium upon progesterone stimulation and by the seminal vesicles. The isoform of the protein present in female reproductive tissue, glycodelin A (GdA), and the male counterpart, glycodelin S (GdS), have identical amino acid sequences, but strikingly different N-linked glycans. It is well documented in literature that GdA is an immunosuppressive protein, and we have shown that this activity is due to its ability to induce apoptosis in activated T cells. The precise role of GdS in seminal plasma is not known. In this study, we report that GdS is not apoptotically active. We observe that the apoptotic activity requires the presence of sialic acid residues on the complex glycans, as in the case of GdA; however, complex glycans of GdS are non-sialylated. We have expressed the wild-type protein in Pichia pastoris, which does not add sialic acid to the secreted proteins, and confirmed our observations that the protein is apoptotically inactive in the non-sialylated form. Our results indicate that differential glycosylation modulates the function of the different glycodelin isoforms.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.