Theileria parva: bovine helper T cell clones specific for both infected lymphocytes and schizont membrane antigens

Two Theileria parva-specific bovine helper T cell clones were used to identify T. parva-derived antigens expressed on the surface of schizont infected lymphoblastoid cells. Although the clones proliferated in response to both the immunizing (Muguga) and heterologous stocks of T. parva, the patterns of the responses differed, showing that the two clones recognized different antigenic epitopes. Both clones were stimulated by autologous infected cells, without an additional source of antigen-presenting cells, as well as by purified schizonts and by a subcellular membrane fraction prepared from infected lymphoblastoid cells, when antigen-presenting cells were present. The membrane fraction was shown to be enriched for schizont membranes as indicated by the presence of a schizont surface antigen detected by immunoblotting using a schizont-specific monoclonal antibody. Elimination of schizonts with the anti-theilerial drug, parvaquone, resulted in reduced antigenicity of the membrane fraction as detected by both the T cell clones and the schizont-specific monoclonal antibody. We conclude that the T. parva-infected cell surface antigens recognized by the T cell clones are of schizont membrane origin. Although the antigens have not yet been characterized biochemically, the monoclonal antibody-specific epitope appears to be distinguishable from the T cell epitopes.     

Cell surface expression of 4β-galactosyltransferase accompanies rat parotid gland acinar cell transition to growth

Rat parotid gland acinar cells stimulated to divide by a chronic regimen of isoproterenol demonstrate a dramatic increase in the synthesis of the glycosyltransferase 4β-galactosyltransferase. A plasma membrane localization for much of the increase in 4β-galactosyltransferase was determined by density gradient membrane fractionation. Golgi-enriched fractions showed no increase in specific activity, while plasma membrane activity increased 40-fold. This selective increase at the cell surface was confirmed by immunofluorescence of intact, nonpermeabilized cells from treated glands, using a monospecific antibody prepared against the purified bovine milk transferase. In detergent-permeabilized cells staining of nontreated cells was seen only as groups of perinuclear vesicles, presumed to be Golgi apparatus. In isoproterenol-treated and permcabilized cells both presumptive Golgi and cell surface staining was apparent. Enzyme assays performed on intact cells established that the enzyme's active site was oriented to the exterior of the cells. The transferase could be detected as early as 3 hr after the primary challenge with isoproterenol. Pretrcatment of rats with cycloheximide prevented its appearance.     

Crystal structure of botulinum neurotoxin type A in complex with the cell surface co-receptor GT1b-insight into the toxin-neuron interaction

Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.     

Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.

Extracts of isolated microvascular endothelial cells (MEC) and cultured bovine aortic endothelial cells (BAEC) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfer and incubation with albumin either radioiodinated or adsorbed to 5-nm gold particles. Both ligands reacted exclusively with two peptides of 18 and 31 kDa. To the 18 kDa peptide (excised from preparative SDS-PAGE), an antibody was raised in rabbits and purified by affinity on 18 kDa obtained from two-dimensional gel electrophoresis and immobilized on nitrocellulose paper. The specificity of the anti-18 kDa was assessed by immunoblotting and immunoprecipitation of endothelial cell extracts. To check whether the 18 kDa peptide is exposed on the endothelial cell surface and/or its components (uncoated pits, open plasmalemmal vesicles), the apical membrane of BAEC was radioiodinated, the solubilized proteins incubated with the anti-18 kDa, and the immune complexes formed were precipitated with protein A-Sepharose CL-4B. The ensuing SDS-PAGE and autoradiography revealed that from all radioiodinatable surface proteins, the 18 kDa was the only polypeptide immunoprecipitated by the anti-18 kDa antibody. To localize the 18 kDa peptide, we applied indirect immunofluorescence technique on cultured MEC and BAEC and immunoelectron microscopy (EM) on ultrathin cryosections of mouse heart. Nonpermeabilized whole MEC and BAEC incubated with anti-18 kDa followed by rhodamine-conjugated second antibody showed a relatively intense surface fluorescence often appearing as small dots. At the EM level, heart ultrathin cryosections exposed anti-18 kDa followed by gold-conjugated second antibody revealed that 18 kDa was primarily associated with the membrane of plasmalemmal vesicles of capillary endothelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.     

Ultracryotomy for the study of unfixed membranes

Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K2Cr2O7 to produce positive staining of the membrane proteins. Chromaffin cell membranes formed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface.     

Ganglioside GT1b in rat brain binds to p58, a brain-specific sodium-dependent inorganic phosphate cotransporter: expression cloning with a specific monoclonal antibody to ganglioside GT1b-binding protein

To evidence the notion that gangliosides involve neuronal cell interactions in the brain, we surveyed the presence of ganglioside-binding proteins in membrane lysates of adult rat cerebellum. Three proteins (p58, p90, and p160) were identified as GT1b-binding proteins by incubation of the blot of the membrane lysate with GT1b micelles. We generated a monoclonal antibody (mAb) specific to the polypeptide portion of the GT1b-binding proteins (YAK-2). The YAK-2 mAb specifically reacted with all three proteins on blots of proteins pretreated under nonreducing conditions for SDS-PAGE, but reacted mainly with p58 under reducing conditions, showing that p90 and p160 are oligomeric forms of p58. The binding activity of the YAK-2 mAb was completely inhibited by the presence of GT1b micelles, indicating the specificity of YAK-2 mAb for p58 and its oligomers. Immunohistochemical investigations revealed that both p58 and GT1b colocalize within the granular layer of adult rat cerebellum. Expression cloning of p58 cDNA was performed using YAK-2 mAb, and five putative clones were obtained. Among them, the nucleotide sequence of one cDNA completely matched that of rat brain-specific sodium-dependent inorganic phosphate cotransporter (rBNPI), a 61 kDa membrane protein. COS7 cells were transfected with a Flag-chimeric construct containing the rBNPI/p58 cDNA, and the membrane lysate was subjected to immunoprecipitation with anti-Flag antibody. One protein (64 kDa) was detected only with YAK-2 mAb, and the membrane lysate specifically bound to GT1b micelles. Taking together, we propose that rBNPI/p58 functions as a GT1b-binding protein in neuronal cells.     

Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein

A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane leaflet. In addition, transport could be reversibly inhibited with the sulfhydryl-specific inhibitor pyridyldithioethylamine (PDA). PDA-sensitive PS transport was also observed in nucleated human and murine cell lines. Analysis of isolated plasma membranes from 125I-PDA-labeled cells revealed marked labeling of a 32,000-Da component. Attempts to inhibit PS transport by treating the cells with proteases, lectins, or antibody suggested that the 32-kDa polypeptide is an integral membrane protein that does not contain sites critical to its function at the cell surface.     

Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.     

Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.     

An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.     

Monoclonal antibody A2B5, which detects cell surface antigens, binds to ganglioside GT3 (II3 (NeuAc)3LacCer) and to its 9-O-acetylated derivative

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides. In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study. One is the ganglioside NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1ceramide (GT3) and the other is a 9-O-acetylated derivative of GT3). This derivative was purified from 10-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-O-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5. These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18B8 in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalcohol chain. Antibody 18B8 does not bind to 9-O-acetylated GT3 or GT3 oxidized by sodium periodate.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Proteolytic activation of protein kinase Calpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein

We sought to determine the roles of PKCalpha and G(i)alpha in regulating cPLA(2) activity in bovine pulmonary artery endothelial cell membrane under peroxynitrite (ONOO(-)) stimulation. Treatment of bovine pulmonary artery endothelial cells with ONOO(-) markedly stimulates the cell membrane associated protease activity, protein kinase C (PKC) activity, phospholipase A(2) (PLA(2)) activity, and arachidonic acid (AA) release from the cells. ONOO(-) significantly increases (Ca(2+))(i) in the cells, and pretreatment with the intracellular Ca(2+) chelator BAPTA-AM prevents the increase in (Ca(2+))(i), protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment of the cells with arachidonyl trifluoromethyl ketone (AACOCF(3)) (a cPLA(2) inhibitor) prevents ONOO(-)-stimulated cPLA(2) activity and AA release without producing a significant alteration of the protease activity. Pretreatment with vitamin E and aprotinin prevents ONOO(-)-induced increase in the protease activity, PKC activity, and cPLA(2) activity in the cell membrane and AA release from the cells. Pretreatment with the PKC inhibitor calphostin C prevents ONOO(-)-caused increase in PKC activity and cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot study of the cell membrane isolated from the ONOO(-)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. An immunoblot study with anti-nitrotyrosine antibody revealed that ONOO(-) induces nitration of tyrosine residues in PKCalpha. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. An immunoblot study of the endothelial cell membrane with polyclonal cPLA(2) antibody revealed that treatment of the cells with ONOO(-) markedly increases the cPLA(2) immunoreactive protein profile in the membrane. Pretreatment of the endothelial cells with Go6976, a PKCalpha inhibitor, prevents the increase in PKC activity and cPLA(2) activity in the cell membrane under ONOO(-)-triggered condition. It, therefore, appears from the present study that treatment of the cells with ONOO(-) causes an increase in the protease activity, and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA(2) activity in the cell membrane and AA release from the cells. An immunoblot assay with polyclonal G(i)alpha antibody elicited an immunoreactive band having a molecular mass of 41 kDa. Pretreatment of the cells with pertussis toxin markedly inhibits ONOO(-)-induced increase in cPLA(2) activity and AA release without significantly altering (Ca(2+))(i), protease activity, and PKC activity in the cell membrane. Treatment of the cells with ONOO(-) causes phosphorylation of G(i)alpha in the cell membrane, and pretreatment with Go6976 prevents its phosphorylation. We suggest the existence of a pertusssis toxin sensitive G protein-mediated mechanism for activation of cPLA(2) by ONOO(-) in bovine pulmonary artery endothelial cell membrane, which is regulated by PKCalpha-dependent phosphorylation and sensitive to aprotinin for its inhibition.     

In vivo detection of membrane protein expression using surface plasmon enhanced fluorescence spectroscopy (SPFS)

Surface plasmon enhanced fluorescence spectroscopy (SPFS) was applied for the detection of expression and functional incorporation of integral membrane proteins into plasma membranes of living cells in real time. A vesicular stomatitis virus (VSV) tagged mutant of photoreceptor bovine rhodopsin was generated for high level expression with the semliki forest virus (SFV) system. Adherent baby hamster kidney (BHK-21) cells were cultivated on fibronectin-coated gold surfaces and infected with genetically engineered virus driving the expression of rhodopsin. Using premixed fluorescently (Alexa Fluor 647) labeled anti-mouse secondary antibody and monoclonal anti-VSV primary antibody, expression of rhodopsin in BHK-21 cells was monitored by SPFS. Fluorescence enhancement by surface plasmons occurs exclusively in the close vicinity of the gold surface. Thus, only the Alexa Fluor 647 labeled antibodies binding to the VSV-tag at rhodopsin molecules exposed on the cell surface experienced fluorescence enhancement, whereas, unbound antibody molecules in the bulk solution were negligibly excited. With this novel technique, we successfully recorded an increase of fluorescence with proceeding rhodopsin expression. Thus, we were able to observe the incorporation of heterologously expressed rhodopsin in the plasma membrane of living cells in real time using a relatively simple and rapid method. We confirmed our results by comparison with conventional wide field fluorescence microscopy.     

Ultracrytomy for the study of unfixed membranes

Summary Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K₂Cr₂O₇ to produce positive staining of the membrane proteins. Chromaffin cell membranes fromed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface.Dedicated to Prof. Georg Maneke on the occasion of his 70th birthday     

Synthesis of alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on human tumor cells by recombinant alpha1,3galactosyltransferase produced in Pichia pastoris.

This study describes the processing of human tumor cells or cell membranes to express alpha-gal epitopes (Galalpha1-3Gal-beta1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant alpha1,3galactosyltransferase (ralpha1,3GT), produced in the yeast Pichia pastoris. Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody. This ralpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent. It is effectively produced in P. pastoris under constitutive expression of the P(GAP) promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)(6) tag. This tag enables the simple affinity purification of ralpha1,3GT on a nickel-Sepharose column and elution with imidazole. The purified enzyme appears in SDS-PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme. ralpha1,3GT is very effective in synthesizing alpha-gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound alpha-gal epitopes/min/mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9 )cells) with neuraminidase, ralpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6 )alpha-gal epitopes per cell. Effective synthesis of alpha-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma. These data imply that ralpha1,3GT produced in P. pastoris is suitable for the synthesis of alpha-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.     

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.     

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.     

Identification of a cell surface component of Swiss 3T3 cells associated with an inhibition of cell division

Polyclonal IgG, prepared to a purified bovine cell surface sialoglycopeptide (SGP) inhibitor of cell division, was used to identify an antigenically related molecule on the surface of Swiss 3T3 cells. SDS-PAGE and Western analyses showed that the anti-SGP antibody was monospecific and primarily recognized a 66-kDA protein of 3T3 cell membranes. Treatment of intact 3T3 cells or 3T3 cell membranes with either broad and phosphatidylinositol-specific phospholipase C enzymes suggested that the antigenic material most likely existed as an integral membrane molecule, or associated as a multimeric complex, and was not anchored at the cell surface by a phospholipid. The addition of anti-SGP IgG to 3T3 cell monolayer cultures was shown to promote cell division, suggesting a regulatory function for the membrane-associated molecule.