Enzymic assay of C3b receptor on intact cells and solubilized cells.

The C3b receptor of human erythrocytes is known to act as a cofactor for the cleavage of the complement protein C3b by the serine proteinase C3b/C4b Ina. The same cofactor activity is shown to be present on human tonsil B-lymphocytes. The cofactor activity of the C3b receptor can be assayed, on intact cells or in solubilized extracts of cells, by determining the rate of C3b cleavage in the presence of fixed concentrations of C3b and of C3b/C4b Ina. This assay method was used to compare the characteristics and relative quantities of C3b receptors on erythrocytes and lymphocytes. The cofactor activities associated with these two cell types resemble each other, but are distinct from the serum cofactor proteins, C4bp and Factor H, in antigenicity and in pH- and ionic-strength-dependence, and are distinct from Factor H in substrate specificity. Assay of cofactor activity in intact cells indicates that there are about 80-fold more receptors per cell on the lymphocyte surface than on erythrocytes. Assays with cells made permeable by detergent show that, whereas essentially all of the receptors on erythrocytes are on the cell surface, B-lymphocytes contain a large internal receptor pool, which makes up more than 80% of the total cofactor activity of the cell.     

Antagonistic effects of phorbol esters on lymphocyte activation. Evidence that protein kinase C provides an early signal associated with lytic function

There is increasing evidence that protein kinase C plays a role in the transduction of an activation signal in lymphocytes. The bulk of this evidence is based on pharmacological experiments involving the tumor promoter phorbol myristate acetate (PMA) as a protein kinase C agonist. However, in cytotoxic T lymphocytes, PMA has been shown to both stimulate and inhibit lytic function. By examining the effects of a series of phorbol esters on protein kinase C activity in lymphocytes, we will demonstrate that these antagonistic effects of PMA on cytotoxic T lymphocyte function are related to multiple effects of PMA on protein kinase C activity.     

Receptor- and phorbol-ester-mediated redistribution of protein kinase C in human platelets. Evidence that aggregation promotes degradation of protein kinase C.

Translocation of Ca2+/phospholipid-dependent protein kinase (PKC) activity from cytosolic to membrane fractions was assessed in washed human platelet suspensions. Phorbol myristate acetate (PMA) induced a rapid loss of PKC activity from the cytosolic compartment in stirred platelets, which was not accompanied by measurable increases in membrane-associated activity, but was paralleled by a decrease in total cellular enzyme activity (cytosol plus membrane). When platelet aggregation was prevented by not stirring, (i) cytosolic activity was decreased by PMA, (ii) significant and maintained (1-15 min with PMA) increases in membrane-bound PKC were detected, and (iii) the decline in total enzyme activity was markedly slower. In stirred platelets, total and specific inhibition of PMA-induced aggregation by a fibrinogen-derived peptide (RGDS, i.e. Arg-Gly-Asp-Ser) promoted maximal increases in membrane-associated PKC in the presence of PMA and completely prevented the loss in cellular activity. Thrombin and collagen both induced a decrease in cytosolic PKC and a loss of total activity, but a significant rise in membrane activity was seen only with collagen; ADP had no detectable effect on enzyme distribution. These results demonstrate an agonist-induced redistribution of PKC and indicate that platelet aggregation may play an important role in the proteolysis, and hence persistence, of membrane-associated PKC. This observation has implications for the potency and duration of PKC-mediated responses induced by agonists and exogenous PKC activators.     

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gβ, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant‐specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C–terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub‐population of Gβ/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine‐rich extracellular C–terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine‐rich C–terminus is extracellular.     

Immune evasion of Enterococcus faecalis by an extracellular gelatinase that cleaves C3 and iC3b

Enterococcus faecalis (Ef) accounts for most cases of enterococcal bacteremia, which is one of the principal causes of nosocomial bloodstream infections (BSI). Among several virulence factors associated with the pathogenesis of Ef, an extracellular gelatinase (GelE) has been known to be the most common factor, although its virulence mechanisms, especially in association with human BSI, have yet to be demonstrated. In this study, we describe the complement resistance mechanism of Ef mediated by GelE. Using purified GelE, we determined that it cleaved the C3 occurring in human serum into a C3b-like molecule, which was inactivated rapidly via reaction with water. This C3 convertase-like activity of GelE was shown to result in a consumption of C3 and thus inhibited the activation of the complement system. Also, GelE was confirmed to degrade an iC3b that was deposited on the Ag surfaces without affecting the bound C3b. This proteolytic effect of GelE against the major complement opsonin resulted in a substantial reduction in Ef phagocytosis by human polymorphonuclear leukocytes. In addition, we verified that the action of GelE against C3, which is a central component of the complement cascade, was human specific. Taken together, it was suggested that GelE may represent a promising molecule for targeting human BSI associated with Ef.     

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.     

Oxidation product(s) in acetaldehyde reacts with NAD(P)H and interferes with assay of alcohol dehydrogenase

A decrease in absorbance at 340 nm, at rates similar to those obtained with alcohol dehydrogenases in routine assays, occurred when NADH or NADPH was mixed with acetaldehyde that had been exposed to air for various durations. NAD(P)H was apparently oxidized by interfering substance(s) present in acetaldehyde. Reagent-grade acetaldehyde from newly opened bottles as well as acetaldehyde redistilled under strictly O2-free conditions contained minimal amounts of NAD(P)H-reacting substance(s). Redistillation under poor anaerobic conditions or in air increased the amount of NAD(P)H-reacting substance(s) in redistilled acetaldehyde. NADPH reacted at a higher rate than NADH with the interfering substance(s) in Tris-Cl buffer at pH 7.5. Also, the reaction was faster in Tris buffer than in phosphate buffer at pH 7.5. The NAD(P)H-oxidizing reaction may not be apparent when the nominal concentration of acetaldehyde used was below 5 mM, but the measured ethanol dehydrogenase activity could be significantly lower with acetaldehyde containing a measurable level of interfering substance(s). This study suggests that acetaldehyde is most easily tested with NADPH for the presence of a significant level of interfering substance(s) and that redistillation, if necessary, must be performed under strictly O2-free conditions.     

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.     

Evidence that sabinene is an essential precursor of C(3)-oxygenated thujane monoterpenes

The volatile oil of immature Artemisia absinthium L. leaves contains sabinyl acetate (42%), 3-thujone (32%), sabinene (12%), and α-thujene (3%) as major constitutents, and label from the acyclic precursor [1-³H]geraniol was incorporated, under aerobic conditions, into these thujane-type monoterpenes in proportion to their natural abundance in this tissue. Light had little effect on the synthesis of these monoterpenes from exogenous geraniol; however, at reduced oxygen levels, label from geraniol accumulated in the olefin sabinene while much less sabinyl acetate and 3-thujone were formed, suggesting a route to the ester and ketone by the allylic, nonphotochemical, oxygenation of sabinene. Supporting evidence for the intermediary role of the olefin was provided by isotopic dilution studies in which sabinene, but not α-thujene, blocked formation of the oxygenated derivatives from the labeled precursor. [10-³H]Sabinene was incorporated directly as a substrate in A. absinthium leaves into both [10-³H]sabinyl acetate and 3-[10-³H]thujone. Furthermore, [³H]sabinene was specifically incorporated into 3-thujone in Tanacetum vulgare and into the diastereomeric ketone 3-isothujone in Salvia officinalis, confirming the role of this bicyclic olefin as the essential precursor of C(3)-oxygenated thujane monoterpenes.     

OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2

OKB7, an IgG2 mouse monoclonal antibody, binds to the C3d receptor (CR2) of human B lymphocytes in or near the active site of the receptor. OKB7 precipitates the same molecule as monoclonal antibodies B2 and HB-5 which have previously been reported to react with the C3d receptor at epitopes distant from the active site. The epitope recognized by OKB7 is distinct from those bound by B2 and HB-5 as shown in competitive binding experiments and is near the complement binding site since OKB7 blocks EAC3d rosetting at concentrations as low as 1 microgram/ml in the absence of cross-linking antibodies.     

Folding of intestinal brush border enzymes. Evidence that high-mannose glycosylation is an essential early event.

A polyvalent antiserum which precipitates the native, folded, but not the denatured molecular forms of pig intestinal aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) was used to determine the kinetics of polypeptide folding of the two newly synthesized brush border enzymes. In pulse-labeled mucosal explants, complete synthesis of the polypeptide chains of aminopeptidase N and sucrase-isomaltase required about 2 and 4 min, respectively, whereas maximal antiserum precipitation was acquired with half-times of 4-5 and 8 min, respectively. Fructose, which induces a defective cotranslational high-mannose glycosylation, increased the half-time of polypeptide folding to about 12 min for aminopeptidase N as well as for sucrase-isomaltase. Short-pulse experiments suggested that fructose exerts its effect by slowing the rate of glycosylation, making this partially a posttranslational process. In the presence of fructose, not only the malglycosylated forms but also the electrophoretically normal, high-mannose-glycosylated form of the brush border enzymes were retained in the endoplasmic reticulum and proteolytically degraded. The results obtained demonstrate an intimate interrelationship between glycosylation and polypeptide folding in the synthesis of membrane glycoproteins and, more specifically, indicate that the timing of these two early biosynthetic events is essential for correct polypeptide folding.     

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.     

Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein,Giα

In a previous report we shwed that glucocorticoed inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Giα levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Giα. In order to establish the nature of the coupliing between cytosolic Giα and cytosolic PLC we examined the effects of Protein activators, and inhibitors on cytosolic PLC activity from rat spenocytes and the rat lymphoma cell line Nb 2, with [³H] PI and [³H]PIP₂ as substrates. (1) Neither GTP nor its nonhydrolyzable analogue, GTPγS, at 100 μm had any effect on the calcium stimulated as well as the basal PLC activity. (2) Howevr, affinity purified antibodies to Giα1 and Giα2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP₂ as substrate, soluble PLC activity was inhibited 50–70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. (3)Administration of Giα1 antisense oligonucleotides to splenocytes for 48 h produced 25–40% decrease in cytosolic Giα1 levels compared to control. The soluble PLC activity with both PI and PIP₂ as substrates was also reduced by 25–50% compared to control conditions. This suggest that cytosolic Giα is associated with the activation of splenocyte soluble PLC. (4) Pertussis toxin administered in vivo sugnificantly reduced cytosolic Giα immunoreactivity and soluble PLC activiry when PI was used as substrate, providing additional evidence that cytosolic Giα is associated with the activation of splencyte soluble PLC. (5) Another agent that has beeen used extensively to define G-protein coupled processes is NaF/AlCl₃. NaF(4mM; with or without AlCl₃ inhibited soluble PLC activity with PIP₂ as substrate, in contrast ot the stimulatory effect that has been reported in the activation of membrane PLC. 6) because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperzine (50 μm, TFP), an inhibitor of protein phosphatase 2B; TFP (50 μm) signigicantly inhibited soluble PLC activity PI was used as substrate. These results suggest a direct involvement of cytosolic Giα in the activation of soluble PLC form splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Giα coupled PLC is addressed in the second paper.     

Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3

In the present study we examined human neutrophils for the expression of a receptor capable of binding C3dg and defined the relationship of this receptor to those that have been previously described, namely CR1, CR2, and CR3. C3dg was isolated from serum depleted of plasminogen, supplemented with 20 mM Mg++, and incubated at 37 degrees C for 6 to 8 days. The purified protein was homogeneous when analyzed by polyacrylamide gel electrophoresis and exhibited an apparent m.w. of 41,000. C3dg was polymerized by treatment with dimethyl suberimidate, and the dimer was isolated by gel filtration. Binding of both monomeric and dimeric 125I-labeled C3dg to neutrophils was saturable, and the latter ligand bound to an average of 12,400 sites/cell among nine normal individuals. At 4 degrees C, bound monomeric C3dg dissociated from neutrophils with an average t1/2 of 30 min, whereas dimeric C3dg dissociated with a t1/2 in excess of 120 min. Specific binding of multimeric C3dg was cation independent and was competitively inhibited by molar concentrations of iC3b and C3d that were equivalent to the inhibitory concentrations of unlabeled C3dg; C3b was less able to compete with C3dg for binding to these sites. The capacity of this neutrophil receptor to bind iC3b, C3dg, and C3d suggested its possible identity as CR2 or CR3. However, no specific binding to neutrophils of 125I-labeled HB-5 monoclonal anti-CR2 was detected. Furthermore, uptake of 125I-labeled C3dg was not inhibited by saturating concentrations of rabbit anti-CR1, anti-Mac-1, or OKM10. Thus, a receptor resides on neutrophils that binds the C3d region of iC3b and C3dg and is distinct from CR1, CR2, and CR3.     

C3 convertase of the alternative complement pathway. Demonstration of an active, stable C3b, Bb (Ni) complex

The purposes of this study were to demonstrate the C3 convertase complex, C3b, Bb (EC 3.4.21.47), of the alternative pathway of complement by ultracentrifugation and to determine whether the metal ion required for enzyme formation is present in the active enzyme complex. It has been shown previously that C3b,Bb formed with Ni2+ rather than Mg2+ exhibits enhanced stability. Using sucrose density gradient ultracentrifugation, an enzymatically active C3b,Bb(Ni) complex could be demonstrated which has a sedimentation coefficient of 10.7 S and which is stable in 10 mM EDTA. Upon formation of the enzyme with the radioisotope 63Ni2+, the ultracentrifugal distribution of the metal correlated with that of the enzyme complex. The molar ratio of Ni to C3b,Bb was 1:1. Displacement of Ni by Mg during formation of the enzyme indicated that both metals may bind to the same site in the enzyme. Binding of 63Ni to the catalytic site bearing fragment Bb was significantly stronger than its binding to C3b or to the zymogen, Factor B. It is proposed that there is one metal-binding site in the C3b,Bb enzyme which is not susceptible to chelation by EDTA and which is located in the Bb subunit.