The fate of IgE bound to rat basophilic leukemia cells. II. Endocytosis of IgE oligomers and effect on receptor turnover

We have assessed the internalization of variously sized oligomers of IgE bound to rat basophilic leukemia (RBL) cells by measuring their accessibility to the extracellular environment, and by direct visualization of the radiolabeled ligands. We also followed the fate of the internalized ligands and their receptors, as well as the fate of the free receptor on cells internalizing oligomers. In contrast to monomeric IgE, surface-bound oligomeric IgE was internalized. Notably, dimers provided an effective signal for internalization, although larger oligomers seem to be internalized more efficiently. In our experiments, 48% of the cell-bound dimers and 67% of the trimers were eliminated from the cell surface in 180 min. One-half of the maximal internalization observed with dimers and trimers occurred in 25 and 11 min, respectively. Release of radioactivity into the supernatant followed internalization; the released radioactivity did not bind to fresh cells and was only partially TCA-precipitable. Radioactive ligands remaining associated with the cells were unchanged as judged by m.w; they also were shown to remain receptor-bound. During either internalization or release of substantial amounts of the originally cell-bound oligomers, there was no increase in IgE-binding activity. In contrast, there was a transient drop (25%) in the number of free surface receptors suggesting internalization of the free receptors together with the oligomer-occupied receptor. Cells that failed to release histamine (RBL-I) processed dimeric and trimeric IgE similarly to histamine-releasing (RBL-2H3) cells. We conclude that dimeric and trimeric IgE are internalized by RBL cells and later are released to the medium in a partially degraded form. The ligand-bound receptor seems to be internalized with the ligand, along with some free receptor, and does not appear to be reusable or to recycle rapidly to the cell surface.     

Characteristics of human basophil sulfidopeptide leukotriene release: releasability defined as the ability of the basophil to respond to dimeric cross-links

Human basophils release approximately 90 pmol of LTC4/micrograms histamine when challenged with anti-IgE antibody, but donor to donor variation produces a 1000-fold range of response. There is little conversion to LTC4 to LTE4 in purified preparations of basophils, but conversion to LTE4 does occur if cell densities are high during incubation. Like histamine release, leukotriene release is calcium and temperature dependent and is complete in 20 min, with a t1/2 of approximately 8 min. The process of desensitization also ablates leukotriene release, but there is a distinct two phase process where leukotriene release is enhanced after 5 min of desensitization, whereas histamine release is inhibited and total ablation of leukotriene release occurs only after 45 min of desensitization. Human basophils respond well to stimulation with covalently cross-linked trimeric IgE myeloma but respond poorly to dimeric IgE. This differential sensitivity to the two forms of cross-linked IgE is most exaggerated in the context of leukotriene release, where dimer is 30-fold less efficacious and 100- to 1000-fold less potent than trimer on some donors' basophils. This dichotomy of response is also observed in antigen-challenged cells, where the bivalent hapten, BPO2, also poorly induces leukotriene release in accord with the fact that it predominantly induces dimeric cross-links of penicillin-specific IgE. Anti-IgE dose-response curves reveal a region of dimeric cross-link dominance that may explain the peculiar differences observed in pharmacologic studies of basophil release induced with antigen vs anti-IgE. In addition, there is a continuum of "releasability," where some donors' basophils display no response (histamine or leukotriene release) to dimeric IgE, and others' basophils are essentially equally responsive to both dimeric and trimeric IgE. This releasability difference manifests itself by conferring increased sensitivity to antigenic challenge in those donors' basophils capable of responding to dimeric cross-links such that these donors' basophils are capable of releasing histamine upon antigen challenge while possessing only 50 molecules of cell surface antigen-specific IgE; other dimer-insensitive donors' basophils require 6 to 10-fold greater IgE densities for equal histamine release.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

Demonstration of Fcgamma receptors on human basophil granulocytes.

Fcgamma receptors were detected on human basophil granulocytes. The mononuclear cell fraction of human peripheral blood was incubated with heat-aggregated human IgG (HGG) followed by 125I-anti-HGG. Autoradiography of the cells showed that the majority of basophil granulocytes gave a significant number of grains. Basophils were not labeled by preincubation of the same cells with monomeric HGG followed by 125I-anti-HGG. However, the binding of aggregated HGG to basophils was inhibited by the presence of a high concentration of monomeric HGG or its Fc fragment but not by the Fab fragment. Evidence was obtained that Fcgamma receptors are distinct from IgE receptors on the same cells: i) Saturation of basophils with IgE did not affect the binding of aggregated HGG to the cells. ii) Preincubation with and the presence of aggregated HGG failed to affect the binding of 125I-IgE to basophils, or to block passive sensitization of the cells with IgE antibodies. iii) The Fcgamma receptors did not co-cap with IgE receptors. Aggregated HGG failed to induce histamine release from basophils even in the presence of D2O. It was also found that the presence of aggregated HGG on basophils did not modulate IgE-mediated histamine release from the cells.     

Cytophilic binding of IgE to the macrophage. III. Involvement of cyclic GMP and calcium in macrophage activation by dimeric or aggregated rat myeloma IgE

Discharge of lysosomal enzymes, measured by release of β-glucuronidase, was studied in uninduced rat macrophages stimulated in vitro with rat monoclonal IgE (IR 162) in different states of aggregation. Monomeric IgE showed negligible activity, while dimeric and aggregated IgE were shown to induce a rapid and selective release of β-glucuronidase as well as new synthesis of the enzyme, without change in the cytoplasmic marker, leucine aminopeptidase. Lysosomal enzyme release is related to the dose of dimeric IgE, becoming maximal above 2.5 μg/ml. β-Glucuronidase release from macrophages by dimers is competitively inhibited by monomeric IgE but only at high ratios, approximately 100-fold greater than those needed to block mast cell release of the same enzyme. The difference in inhibitability is consistent with the difference in binding affinity of macrophages and mast cells for monomeric IgE. This observation rules out the participation of the few remaining mast cells contained in the macrophage monolayer in β-glucuronidase release. Dimeric or aggregated IgE produced a rise in cyclic GMP coincident with the peak fixation of IgE by macrophages. Elevation of cyclic GMP by pharmacological means also stimulated β-glucuronidase release and new synthesis, as well as enhancing the effect on these of aggregated IgE. Enzyme release by IgE did not occur in the absence of extracellular calcium. We conclude that IgE, which has been cross-linked to form dimers before binding to specific macrophage receptors, triggers the cell and that cyclic GMP (and perhaps calcium) modulates the early step of macrophage activation.     

Transient Dimers of Allergens

Background

Allergen-mediated cross-linking of IgE antibodies bound to the FcεRI receptors on the mast cell surface is the key feature of the type I allergy. If an allergen is a homodimer, its allergenicity is enhanced because it would only need one type of antibody, instead of two, for cross-linking.

Methodology/Principal Findings

An analysis of 55 crystal structures of allergens showed that 80% of them exist in symmetric dimers or oligomers in crystals. The majority are transient dimers that are formed at high protein concentrations that are reached in cells by colocalization. Native mass spectrometric analysis showed that native allergens do indeed form transient dimers in solution, while hypoallergenic variants of them exist almost solely in the monomeric form. We created a monomeric Bos d 5 allergen and show that it has a reduced capability to induce histamine release.

Conclusions/Significance

The results suggest that dimerization would be a very common and essential feature for allergens. Thus, the preparation of purely monomeric variants of allergens could open up novel possibilities for specific immunotherapy.     

Identification of histamine releasing factor(s) in the late phase of cutaneous IgE-mediated reactions

We have shown that fluids collected from antigen-challenged skin blisters during the late phase reaction cause the release of substantial amounts of histamine (means = 42%, n = 14) from human basophils in vitro. Control fluids collected either during the immediate phase or from an unchallenged blister released less than or equal to 10% histamine from both basophils and lung mast cells. Late phase blister fluids induced low levels of histamine release from human lung cells (means = 11%, n = 4) that were slightly but not significantly greater than levels induced by control blister fluids. The characteristics of basophil release were similar to IgE-mediated stimuli in dose dependence, calcium and temperature requirements, and kinetics. The IgE dependence of the late phase blister fluid was demonstrated by desensitization of the basophils to anti-IgE, which obviated the response to anti-IgE and blister fluid but did not affect a non-IgE-mediated stimulus. Removal of the cell surface IgE with lactic acid also abolished the response to both anti-IgE and late phase blister fluid. Incubation of the "stripped" cells with serum containing IgE myeloma restored the response to anti-IgE but failed to affect response to late phase blister fluid. The characteristics of release obtained with this factor closely resemble those of an IgE-dependent histamine releasing factor from cultured macrophages previously described by our group.     

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.     

Processing, stability, and receptor binding properties of oligomeric envelope glycoprotein from a primary HIV-1 isolate

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is thought to exist on the virion surface as a trimer of non-covalently associated gp120/gp41 molecules. We expressed trimeric envelope glycoprotein from three primary, macrophage tropic HIV-1 isolates in baby hamster kidney cells and analyzed the furin-mediated cleavage, stability, and receptor binding properties of the oligomers. The envelope glycoprotein was secreted in a soluble form deleted of its transmembrane anchor and the intracytoplasmic domain (gp140). A mixture of trimers, dimers, and monomers of gp140 as well as monomeric gp120 was detected on polyacrylamide gels. Analysis by sucrose gradient centrifugation revealed that trimers and dimers were essentially composed of uncleaved gp140, whereas most of the gp120 was found in the monomeric fraction. To analyze the effect of the cleavage of gp140 to gp120/Delta41 on trimerization, we co-expressed the furin protease along with gp140. Surprisingly, furin expression changed the subcellular localization of the envelope glycoprotein, which became in majority sequestered in the major furin compartment, the trans-Golgi network, as judged by confocal laser microscopy. The envelope glycoprotein secreted from furin-co-expressing cells was almost completely cleaved to gp120 and Deltagp41, but gp120 was found exclusively in the monomeric fraction, with a few residual oligomers being composed of uncleaved gp140. Secreted uncleaved gp140 trimers were purified to homogeneity and analyzed for their capacity to interact with cellular receptors CD4 and CC chemokine receptor 5 (CCR5). Receptor binding was analyzed on CD4- and CCR5-expressing cells as well as on peripheral blood mononuclear cells. Trimers showed greatly reduced binding to CD4 as compared with monomers. Neither monomers nor trimers bound directly to CCR5. In conclusion, our results show that the cleaved form of the envelope glycoprotein does not form stable trimers, suggesting that gp120/gp41 oligomers on the virion surface might be stabilized by a yet to be identified mechanism and that the virion might attach to CD4 via a monomeric form of gp120. These results are relevant to the development of an envelope-based vaccine against AIDS.     

The mechanism of basophil histamine release induced by antigen and by the calcium ionophore A23187.

The mechasism of human basophil histamine release by the calcium ionophore A23187 has been compared to that induced by the interaction of antigen with cell bound IgE antibody. Ionophore induced histamine release (Ion. H.R.) occurs with the leukocytes of both normal and allergic donors. It is completely calcium dependent; LaCl3 inhibits both Ion. H.R. and antigen induced histamine release (Ag. H.R.) at about 10-minus 7 M. The kinetics of Ion. H.R. suggest that this process has no "desensitization" phase as does Ag. H.R. and the ionophore is fully active on antigen-desensitized cells. Pharmacologic studies indicate that dibutyryl cyclic AMP and agents which increase endogenous cyclic AMP levels do not inhibit Ion. H.R. as they inhibit the early stages of Ag. H.R. Of the agents which affect microtubules, colchicine inhibits and D2O enhances Ion. H.R. in a manner which is qualitatively similar but quantitatively less marked than their effects on Ag. H.R. The metabolic antagonist 2-deoxyglucose inhibits both Ion. H.R. and Ag. H.R. in a similar fashion. Based on these data and the observation that cells pretreated with ionophore show a marked (synergistic) enhancement of Ag. H.R. we conclude that Ion. H.R. has a similar or identical mechanism to the later stages if Ag. H.R. but "short circuits" the cyclic AMP-associated events of Ag. H.R.     

Basic characteristics of human lung mast cell desensitization

Human lung parenchymal mast cells displayed both specific and nonspecific desensitization. The kinetics of both release and desensitization were approximately equal to 3 times faster than human basophils, but a similar relationship between release and desensitization suggests similar biochemistries in basophils and mast cells. Arachidonic acid metabolite (PGD2 and LTC4) release was slower to desensitize (t1/2 of 8 min) than histamine release (t1/2 of 3 min), the ratio of which is similar to the ratio observed in basophils. Ionophore A23187-induced release was unaffected by desensitization to anti-IgE antibody, and calcium-45 uptake was inhibited by desensitization, suggesting that desensitization inhibits the early post-cross-linking "influx" of calcium that is necessary for mediator release in mast cells. In contrast to the above similarities in basophil and mast cell desensitization, mast cell desensitization, unlike that of basophils was not inhibited by diisopropylfluorophosphate.     

Biochemical analysis of desensitization of mouse mast cells

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.     

Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain

When the low density lipoprotein (LDL) receptor was solubilized from bovine adrenal cortex membranes and subjected to electrophoresis in the absence of reducing agents, a disulfide-bonded dimeric species was demonstrated. Formation of these covalent bonds was blocked when the tissue was homogenized in the presence of sulfhydryl alkylating agents, indicating that the native receptor was self-associated noncovalently and that the disulfide bond formation occurred only after homogenization. The disulfide-linked dimers were disrupted and the receptor was restored to a monomeric form when inside-out adrenal vesicles were treated with trypsin, suggesting that the disulfide bond formation involved the 50-amino acid cytoplasmic domain of the receptor. When the receptor was solubilized from bovine adrenal cortex membranes and then purified by ion exchange and affinity chromatography, it could be covalently coupled into dimers and trimers in the presence of bivalent cross-linking agents. Receptor dimers could also be demonstrated by chemical cross-linking of intact cells that were transfected with an expressible cDNA encoding the normal human LDL receptor. Dimer formation was markedly reduced in transfected cells expressing mutated cDNAs that had premature termination codons at positions 792, 807, and 812, which produced shortened receptors that retained 2, 17, and 22 of the original 50 amino acids of the cytoplasmic domain, respectively. The first two mutant receptors, which did not form oligomers, did not enter coated pits and were not rapidly internalized by cells. However, the mutant receptor that terminates at position 812 was internalized normally even though oligomer formation was greatly reduced. Moreover, a mutant receptor with a cysteine substituted for a tyrosine at position 807, which internalized very slowly, showed a normal susceptibility to chemical cross-linking. Deletion of external domains of the LDL receptor, including the epidermal growth factor homology region and the O-linked sugar domain, did not alter susceptibility to chemical cross-linking. We conclude that the cytoplasmic domain of the LDL receptor is responsible both for self-association into oligomers and for clustering in coated pits, but the available data do not establish a causal connection between these two events.     

Mouse mast cell activation and desensitization for immune aggregate-induced histamine release

Immune aggregate-induced histamine release and desensitization were studied in mouse mast cells. Maximal histamine release was rapid, occurred at 37 degrees C, and required the addition of alpha-L-phosphatidyl-L-serine and Ca2+. The amount of histamine released varied with the composition of the immune aggregates and was dependent on the antibody concentration. Saturation of mast cell Fc epsilon receptors with rat or mouse IgE had no effect on subsequent immune aggregate-induced release. The incubation of mouse mast cells with immune aggregates in the absence of cations of alpha-L-phosphatidyl-L-serine did not stimulate the release of histamine but resulted in desensitization of the cells for release with the addition of the same or unrelated immune aggregates. Such cells are capable, however, of IgE-mediated histamine release. Mast cells desensitized for IgE-mediated histamine release by incubation with anti-IgE were capable of immune aggregate-induced release. These data suggest that IgE-mediated and immune aggregate-induced triggering of mouse mast cells occurs through separate receptors.     

Human lung macrophage-derived histamine-releasing activity is due to IgE-dependent factors

Human lung macrophages obtained from surgical specimens spontaneously secreted a factor(s) (which we term macrophage factor) during 24-hr culture that induced calcium-dependent histamine release from human basophils and lung mast cells. Macrophage factor induced noncytotoxic histamine release from purified (85%) basophils. The kinetics of release were relatively slow and similar to that of anti-IgE. We performed a series of experiments to test the IgE dependence of macrophage factor-induced release. Preincubation of basophils with anti-IgE in calcium-free medium resulted in complete desensitization to macrophage factor-induced histamine release (i.e., when calcium and macrophage factor were added to the basophils, no histamine release occurred), and preincubation with macrophage factor in calcium-free medium resulted in partial desensitization to anti-IgE-induced histamine release. Pretreatment of basophils with pH 3.9 lactic acid buffer, which dissociates basophil IgE from its receptors, markedly reduced the capacity of basophils to release histamine in response to macrophage factor. Basophils that were incubated with IgE myeloma (but not with IgG) after lactic acid treatment partially or completely regained their capacity to release histamine in response to macrophage factor. Fluid-phase IgE myeloma (15 micrograms/ml) (but not IgG) inhibited basophil histamine release induced by two macrophage-derived supernatants, whereas IgE myeloma (200 micrograms/ml) did not inhibit release due to other supernatants. IgE-affinity columns removed the histamine-releasing activity of five macrophage-derived supernatants, and IgG-affinity columns had similar effects. However, neither affinity column removed the histamine-releasing activity of three other macrophage-derived supernatants. On Sephadex G-75 chromatography, nearly all of the histamine-releasing activity migrated as single peak with an apparent m.w. of 18,000. These results suggest that, although macrophage factor are heterogeneous, they are related, as they are a IgE-dependent factors that induce histamine release by interacting with cell surface IgE. These macrophage factors may be responsible for stimulation of basophil/mast cell mediator release in chronic allergic reactions.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Biologically active decorin is a monomer in solution

It has been reported that decorin and its protein core can have molecular masses nearly double the size of those previously published, suggesting a dimeric structure. In this study we tested whether biologically active decorin and its glycoprotein core would form dimers in solution. We used homo- and hetero-bifunctional chemical cross-linking reagents, BS3 and sulfo-SMPB, respectively, as well as glutaraldehyde and found no preferential dimer formation, whether chemical cross-linking was performed in the presence or absence of live cells. Under the same experimental conditions, we easily detected dimers of epidermal growth factor receptor and basic fibroblast growth factor, two glycoproteins known to dimerize. Only at very high cross-linker to decorin molar ratios (2000:1) were trimers and multimers observed, but performing the chemical cross-linking in the presence of a reducing agent abolished these. The elution of decorin protein core in Superose 6 gel chromatography gave masses compatible with monomeric proteins, both before and after denaturation with 2.5 M guanidine HCl. Matrix-assisted laser desorption ionization gave a mass of 44,077 Da for decorin protein core, without any evidence of dimers or oligomers. Extensive oligomerization of the decorin protein core was observed only after dialysis against water and freeze-drying. These oligomers were considered artifacts because they were independent of chemical cross-linking and were resistant to heat denaturation and disulfide-bond reduction. Oligomeric preparations showed markedly reduced biological activity in both phosphorylation and collagen fibrillogenesis assays. Thus, biologically active decorin is a monomer in solution and, as such, is a monovalent ligand for various extracellular matrix proteins, growth factors, and cell surface receptors.     

Photosystem I: a search for green plant trimers.

Recent blue-native gel electrophoresis studies gave evidence for the existence of dimeric and trimeric PSI complexes in green plants. We used single particle electron microscopy to investigate all the larger particles from the thylakoid membrane of pea (Pisum sativum var. Charmette). Peak fractions with monomeric, dimeric and trimeric Photosystem I were obtained after solubilization with digitonin and size-exclusion chromatography. The analysis showed that only a few percent of dimers and trimers were present. In the best resolved trimers some of the monomers were oriented upside down. Many classes were fuzzy, indicating a non-specific or flexible orientation. From these results we conclude that the green plant PSI is monomeric within the green plant membrane.     

Release of histamine from human leukocytes stimulated with the tumor-promoting phorbol diesters. II. Interaction with other stimuli

The tumor promoter and irritant, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), previously shown to be a potent histamine releaser, has been used to further probe the mechanism of histamine release from human basophils. TPA and the calcium ionophore, A23187, produced a synergistic response in which subeffective concentrations of each stimulus (which alone produced less than 3% release) together produced over 70% histamine release. TPA also synergized with the IgE cross-linking stimulus anti-IgE. Desensitization of cells by incubation with anti-IgE in the absence of calcium rendered the cells unresponsive to anti-IgE and super-responsive to TPA. This marked increase in the TPA response was the result of an increase in the rate of TPA-induced histamine release, and occurred in the absence of extracellular calcium. The ability of various concentrations of anti-IgE to "sensitize" cells to TPA paralleled their ability to produce histamine release in untreated cells rather than their ability to desensitize the cells. These results suggest that in the absence of calcium, anti-IgE induces desensitization of some activation of other elements of the histamine-release process. The anti-IgE dose-response pattern of this activation event further suggests that it is an integral part of the anti-IgE-induced release process itself.     

Crystal structures of saposins A and C

Saposins A and C are sphingolipid activator proteins required for the lysosomal breakdown of galactosylceramide and glucosylceramide, respectively. The saposins interact with lipids, leading to an enhanced accessibility of the lipid headgroups to their cognate hydrolases. We have determined the crystal structures of human saposins A and C to 2.0 Angstroms and 2.4 Angstroms, respectively, and both reveal the compact, monomeric saposin fold. We confirmed that these two proteins were monomeric in solution at pH 7.0 by analytical centrifugation. However, at pH 4.8, in the presence of the detergent C(8)E(5), saposin A assembled into dimers, while saposin C formed trimers. Saposin B was dimeric under all conditions tested. The self-association of the saposins is likely to be relevant to how these small proteins interact with lipids, membranes, and hydrolase enzymes.