Degradation of secretory immunoglobulin M in B lymphocytes occurs in a postendoplasmic reticulum compartment and is mediated by a cysteine protease.

In 38C B lymphocytes, membrane IgM is expressed on the surface, whereas secretory IgM (sIgM) is rapidly degraded. Here, we localize this degradation and characterize the proteases involved in this process. Upon treatment with brefeldin A, degradation of sIgM in 38C cells was strongly inhibited, as was secretion from the sIgM-secreting D2 hybridoma. Moreover, the brefeldin A-induced Golgi resorption resulted in galactosylation of sIgM and partial resistance to endoglycosidase H. However, sIgM avoided degradation neither due to modified terminal glycosylation nor as a consequence of the brefeldin A-induced altered milieu of the endoplasmic reticulum. When these modifications were prevented by inhibiting retrograde transport with nocodazole or by abrogating terminal glycosylation with swainsonine, sIgM was still rescued from degradation. The unaffected breakdown in the presence of nocodazole also argued against recycling of sIgM to be degraded in the endoplasmic reticulum. Furthermore, upon removal of brefeldin A, degradation of galactosylated sIgM resumed in 38C cells, as did secretion from D2 cells. These results indicate that functional export of proteins from the endoplasmic reticulum is a prerequisite for sIgM degradation. Biochemical characterization of this novel postendoplasmic reticulum/pre-trans-Golgi proteolytic pathway included application of inhibitors to a broad spectrum of proteases. Among the compounds tested, only calpain inhibitor I exerted strong inhibition. The involvement of cysteine protease(s) in the degradation of sIgM was corroborated by the inhibitory effect of diamide. We conclude that B lymphocytes avoid secretion by active and selective targeting of sIgM to a developmentally regulated postendoplasmic reticulum degradation pathway in which degradation is mediated by a cysteine protease.     

Post-translational regulation of IgM expression in B lymphocytes. Selective nonlysosomal degradation of assembled secretory IgM is temperature-dependent and occurs prior to the trans-Golgi.

B cells express on their surface the membrane form of IgM (mIgM). Upon differentiation, the resulting plasma cells synthesize and secrete large amounts of the secretory form of IgM (sIgM). Surprisingly, B lymphocytes synthesize an excess of secretory mu chain over the expressed membrane mu chain. However, the sIgM is degraded intracellularly, indicating regulation of IgM expression at the post-translational level. In the present report, we show that the assembly, maturation, and degradation of IgM in 38C B lymphocytes are highly accelerated above a certain threshold temperature. Furthermore, the degradation of sIgM is delayed and takes place by the time the maturation of mIgM in the trans-Golgi is almost completed. Neither chloroquine nor monensin has any effect on this degradation, demonstrating a nonlysosomal pre-trans-Golgi process. In addition, the degradation is of endoglycosidase H-sensitive assembled sIgM molecules. We conclude that the degradation of sIgM in 38C B lymphocytes is a postendoplasmic reticulum, pre-trans-Golgi process. We suggest that this degradation process plays a role in the post-translational regulation of expression of soluble lumenal sIgM.     

Cellular origins of human polymeric and monomeric IgA: intracellular and secreted forms of IgA

Intracellular and secreted IgA from pokeweed mitogen (PWM)-stimulated normal peripheral blood lymphocytes, from 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated peripheral blood lymphocytes of a patient with chronic lymphocytic leukemia (CLL), or from an IgA-producing human Epstein Barr virus (EBV)-transformed lymphoblastoid cell line were analyzed by molecular-sieve chromatography, electrophoresis in sodium dodecyl sulfate, and sucrose density ultracentrifugation. Fluorochrome-labeled anti-human IgA and secretory component (SC) were used as probes for the detection of polymeric IgA in individual cells. These methods demonstrated that the majority of intracellular IgA occurred in monomeric form, even when the predominant form of secreted IgA was polymeric. Sequential analyses of the IgA secreted by PWM-stimulated normal peripheral blood lymphocytes revealed that the proportion of polymeric IgA increased with the time of culture and that polymers represented the prevalent form of secreted IgA from the fifth day of culture. Although approximately one-half of TPA-stimulated CLL cells bound fluorochrome-labeled SC, only trace amounts of extracellular and intracellular polymeric IgA were detected in both culture supernatants and lysates. Culture supernatants of an IgA-secreting EBV-transformed cell line contained predominantly polymeric IgA. However, intracellular IgA was largely represented by monomers. The predominance of intracellular monomers in polymeric IgA-secreting cells suggested that the pathway of the assembly of human IgA molecules is analogous to that described for mouse IgA synthesis.     

Developmental regulation of IgM secretion: the role of the carboxy-terminal cysteine

B lymphocytes do not secrete IgM, and plasma cells only secrete IgM polymers. Here we show that both events are attributable to the tailpiece found at the carboxyl terminus of mus chains, and we specifically implicate Cys-575. Thus, if Cys-575 was mutated, IgM was secreted by B cells. Similarly, a mutant IgG containing a mus tailpiece became largely retained within the cell; secretion was restored upon mutation of the tailpiece cysteine. Removal of Cys-575 also allowed hypersecretion of monomeric IgM by plasmacytoma cells. Following further removal of Cmu1, heavy chains were secreted in the absence of light chains. Thus, in B and plasma cells, Cys-575 is involved both in the polymerization of IgM and in intracellular retention of unpolymerized intermediates.     

Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization

Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.     

Sequential expression of immunoglobulin on developing mouse B lymphocytes: a systematic survey that suggests a model for the generation of immunoglobulin isotype diversity.

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocyte acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIg or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial porportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM ans sIgD. Anti-mu antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD ig or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.     

In vitro regulation of IgA subclass production. III. Selective transformation of IgA1 producing cells by Epstein-Barr virus

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.     

Insights in progressive myoclonus epilepsy: HSP70 promotes cystatin B polymerization

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu^2 + for chaperone-dependent polymerization. Cu^2 + interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu^2 + modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu^2 +/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s).     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

Persistence of lpr/lpr-derived IgG2a secreting B cells in normal----lpr/lpr adult radiation chimeras or lpr/lpr----normal kidney capsule chimeras.

Lethally irradiated MRL/lpr mice reconstituted with bone marrow stem cells from a normal mouse strain develop a state of split hematopoietic chimerism; erythrocytes, granulocytes, and macrophages are derived from the normal stem cell inoculum while the peripheral T lymphocytes are derived from radioresistant lpr host cells. Moreover, these mice have normal levels of serum IgM and IgG2a produced by radioresistant host B cells, even though they have relatively few sIgM+ B cells. In order to better understand the differentiation and regulation of B cells present in these chimeric mice, the current study was undertaken to localize and to assess the functional capacity of the lpr B cells producing the serum antibodies. Surface IgG2a+ cells could not be found in the spleen or lymph nodes of these mice, but large lymphocytes containing cytoplasmic IgG2 of host (lpr) allotype could be readily detected, even though they constituted less than 1% of the total spleen population. The host-derived serum IgG2 and IgG2+ cells were even present in the spleens of "leaky" mice that had relatively normal numbers of donor-derived sIgM+ B cells. These lpr B cells secreted IgG2a antibody that bound ssDNA, but they could not respond to immunization with SRBC. These results indicate that the lpr-derived radioresistant B cells have a limited capacity for proliferation and are already committed to the memory lineage. The presence of similar B cells in normal mice transplanted with neonatal lpr/lpr spleen fragments suggests that lpr/lpr B cell development is inherently abnormal.     

Galactose-decorated pH-responsive nanogels for hepatoma-targeted delivery of oridonin

Nanogels based on the polymers of galactosylated chitosan-graft-poly (N-isopropylacrylamide) (Gal-CS-g-PNIPAm) were used as carriers of oridonin (ORI) for tumor targeting. Three ORI-loaded nanogels with various degrees of galactose substitution were prepared, and their characteristics were evaluated. The release behavior of ORI from these nanogels was pH-dependent, and the release could be accelerated under mildly acidic conditions. The cytotoxicity of ORI-loaded nanogels was pH-sensitive. ORI-loaded nanogels exhibited a higher antitumor activity than drug-loaded nanogels without galactosylation, and the anticancer activity increased in relation to increases in the number of galactose moieties of the nanogels in HepG2 cells. In contrast, the cytotoxicity of ORI-loaded nanogels against MCF-7 cells decreased compared with that of drug-loaded nanogels without galactosylation. Results demonstrated that these nanogels could enhance the uptake of ORI into HepG2 cells via asialoglycoprotein receptor-mediated endocytosis. These galactose-decorated pH-responsive nanogels were well-suited for targeted drug delivery to liver cancer cells.     

The infective polymerization of conformationally unstable antithrombin mutants may play a role in the clinical severity of antithrombin deficiency

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.     

Rainbow trout (Oncorhynchus mykiss) sIgM- leucocytes secrete an interleukin-2 like growth factor after mitogenic stimulation in vitro

Two secreted proteins were detected in culture supernatants of PHA or PMA stimulated, immunomagnetically separated, sIgM(-) leucocytes of rainbow trout (Oncorhynchus mykiss) with 60kDa and with 12-15kDa (multiple bands). So called conditioned media (CM), containing these proteins, induced significant activation of blood and head kidney leucocytes. Immunomagnetically separated, naive as well as PHA activated sIgM(-) T lymphocytes and LPS prestimulated sIgM(+) B lymphocytes could be identified to be responding to these secreted proteins. Using a monoclonal antibody specific for mouse IL-2 (clone JES6-1A12), one of the multiple 12-15kDa proteins could be stained in Western blots. It was also shown that the induced proliferation was due to this protein in the CM, as the same anti-IL-2 mab was able to block the CM induced proliferation. Furthermore, survival of the IL-2 dependent mouse cell line HT-2 was enhanced after addition of various concentrations of CM. The data presented show, for the first time, that mitogen stimulated trout sIgM(-) leucocytes secrete a cytokine like growth factor sharing functional and structural similarities with mammalian IL-2.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

Studies on the generation of B lymphocytes in fetal liver and bone marrow.

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative. Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.     

Involvement of a guanine-nucleotide-binding component in membrane IgM-stimulated phosphoinositide breakdown

Cross-linking of membrane immunoglobulin, the B cell receptor for antigen, activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by phospholipase C. This reaction yields two intracellular second messengers, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes an increase in cytoplasmic Ca2+. The experiments reported here demonstrate that activation of phospholipase C by membrane IgM (mIgM) involves a guanine nucleotide-dependent step. Saponin was used to permeabilize WEHI-231 B lymphoma cells and permit direct manipulation of nucleotide and Ca2+ concentrations. Very high levels of Ca2+ (greater than 100 microM) activated the phospholipase maximally without a requirement for cross-linking of mIgM. However, at much lower, physiologically relevant Ca2+ concentrations (100 to 500 nM), receptor-stimulated PtdInsP2 hydrolysis could be demonstrated. The ability of anti-IgM antibodies to activate phospholipase C in permeabilized WEHI-231 cells was greatly increased by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues (guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate), but not by guanosine diphosphate or guanosine diphosphate analogues or by a nonhydrolyzable analogue of adenosine triphosphate. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein analogous to those that couple receptors to adenylate cyclase is involved in the activation of phospholipase C by mIgM in WEHI-231 B lymphoma cells. In order to characterize this putative GTP-binding component, we examined the ability of pertussis toxin and cholera toxin to affect anti-IgM-stimulated inositol phosphate production. These bacterial toxins covalently modify and modulate the activity of various GTP-binding regulatory proteins and in some cell types can block receptor-stimulated PtdInsP2 breakdown. In WEHI-231 B lymphoma cells, neither toxin blocked signaling by mIgM. Thus mIgM appears to be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to both pertussis toxin and cholera toxin.     

Cooperative assembly of beta-barrel pore-forming toxins

Bacterial beta-barrel pore-forming toxins are secreted as water-soluble monomeric proteins and assemble into beta-barrel-shaped pores/channels through membranes of target cells, causing cell death and lysis. The pore assemblies that undergo various intermediate stages are symbolized by the association of multi-subunit structures in cells. Crystal structures of water-soluble monomers and membrane-embedded oligomeric pores, and recent studies involving biochemical detection and direct visualization of the sequential assembly of the toxin monomers have solved the mystery of how the pores are formed. Here, we review the mechanism of the cooperative assembly of several toxins of interest to explain the nature of the activities of the toxins.     

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.     

Generation of phosphatidic acid and diacylglycerols following ligation of surface immunoglobulin in human B lymphocytes: potential role in PKC activation.

We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.     

Cell surface immunoglobulin of carp lymphocytes (Cyprinus carpio L.)

Molecular size and polypeptide chain composition of cell membrane immunoglobulin (mIg) on lymphocytes of carp were studied using lactopreoxidase-catalysed surface radioiodination and SDS-polyacrylamide gel electrophoresis. Carp lymphocytes prepared from pronephros, blood and thymus carry mIgM in relatively high quantity. That means about 5-10% of the radiolabelled macromolecular cell surface material precipitates as IgM. Cell surface IgM on carp lymphocytes is present as monomeric IgM (m.w. 220000-260000) and HL subunit (m.w. 110000). There are differences among molecular weights of mIg monomers of pronephric lymphocytes (m.w. 220000) and thymocytes (m.w. 260000), whereas blood lymphocytes show both components. Following reduction and alkylation H and L chains were observed. Additional thymocytic mIg possesses two unidentified components with m.w. 35000-40000 and 110000.