Streptavidin-induced lysis of homologous biotinylated erythrocytes. Evidence against the key role of the avidin charge in complement activation via the alternative pathway

It is shown that non-covalent attachment of streptavidin, as well as of avidin, to biotinylated human erythrocytes induces homologous hemolysis by complement. Rabbit antiserum against human C3 is found to inhibit the lysis specifically as compared with non-immune rabbit serum. Efficiency of lysis inhibition is greater for avidin- and streptavidin-induced lysis of biotinylated human erythrocytes than for antibody-sensitized sheep erythrocytes. In contrast to positively charged avidin (pI 11), streptavidin is a neutral protein. Hence, hemolysis of streptavidin-carrying erythrocytes is inconsistent with the suggestion on the crucial role of avidin charge in lysis. Membrane alterations (cross-linking and clusterization of biotinylated components) induced by avidin (streptavidin) seem to be a more plausible explanation for the lysis.     

Reduced immunoglobulin G activates complement system with decreased cooperativity

Sheep erythrocytes sensitized with intact antibody or reduced and alkylated antibody were lysed by guinea-pig serum indicating that reduced and alkylated antibody bound and activated complement. Reduction of antibody caused erythrocyte lysis to exhibit pseudo-first-order kinetics, while the lysis kinetics of erythrocytes sensitized with intact antibody was sigmoidal. Analysis of erythrocyte lysis by complement according to the von Krogh equation showed that reduction of antibody diminished the von Krogh exponent $\text{n}$ from 2.8 to 1.3, while the value of $\text{K}$ remained unchanged at 0.17 (complement dilution). These observations suggested that the sole effect of the reduction of antibody inter-heavy-chain and heavy-light chain disulfide bonds was to diminish the cooperativity of antibody-complement interaction.     

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.     

MEMBRANE LESIONS IN IMMUNE LYSIS : Surface Rings, Globule Aggregates, and Transient Openings

It is known that there are 100 Å-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 Å-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K⁺, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter

Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.     

Regulation of the alternative pathway of complement by pH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia. The abnormal PNH erythrocytes are highly susceptible to complement-mediated lysis in vitro, especially at pH 6.4. Lysis has been shown to be due to alternative pathway activation. The purpose of this study was to determine why lysis of PNH erythrocytes is increased at acidic pH. The results presented demonstrate that at pH 6.4: binding of C5 and Factor B to C3b deposited on human erythrocytes is markedly enhanced; generation of the two C3 convertases, C3(H2O), Bb and C3b,Bb is increased; and control of C3b on human erythrocytes by CR1 and Factor I is diminished. In addition, it was found that rabbit erythrocytes, which activate the human alternative pathway, are also lysed much better at pH 6.4 than at pH 7.4. These results indicate that the optimal pH for the initiation and amplification of the alternative complement pathway, and probably also for the activation of the membrane attack complex, is 6.4.     

The indiction by complement of a change in KSCN-dissociable red cell membrane lipids.

During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56 degrees C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins,and hexoses. Membranes from erythocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than a membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN inidcate that the membrane pertubations caused caused by complement may be specific. We conclude that complement-lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propragated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.     

In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.     

Haemolytic activity in the serum of brown trout, Salmo trutta

Brown trout serum contains a natural, spontaneous, antibody-independent lytic activity and a haemolysin antibody complement-mediated lytic activity against unsensitized and trout antibody-sensitized sheep erythrocytes, respectively. The use of various activators and inactivators of the mammalian complement system demonstrated that trout serum possesses complement or complement-like components similar in activity to those present in the classical and alternative pathways found in mammals. A single injection of trout with sheep erythrocytes stimulated the production of antibody-secreting cells in lymphoid organs and increased the levels of natural haemolysins. A second injection of sheep erythrocytes further raised the haemolysin values and antibody-secreting cell counts. Serum complement from homologous or closely related fish species was more effective for use in the haemolysin and antibody-secreting cell assays than that from heterologous sources, except guinea pig. Based on physico-chemical properties, gel filtration and immunoelectrophoretic studies, natural and induced anti-sheep erythrocyte haemolysins were found to be similar molecules and are possibly high molecular weight IgM antibodies.     

Species specificity of recognition by the alternative pathway of complement

The recognition function of the alternative complement pathway was studied with isolated human and rabbit components. Zymosan and homologous and heterologous erythrocytes were used as representative activators or nonactivators. The binding affinity of Factor B and Factor H for particle-bound C3b was measured. In both species, the average affinity of Factor H for bound C3b on homologous cells (nonactivators) was eight to 10 times higher than on zymosan particles (activators). The interaction between Factor H and C3b on rabbit erythrocytes was species-specific: rabbit Factor H bound strongly to rabbit C3b on rabbit erythrocytes and also on human erythrocytes, which are nonactivators for the rabbit alternative pathway. Human Factor H bound strongly to human C3b on human erythrocytes but seven times weaker on rabbit erythrocytes, which are activators of the human alternative pathway. No substantial differences were found in the binding of Factor B to bound C3b regardless of the nature of the particle to which C3b was bound. The results indicate that in the two species studied, the molecular mechanism of recognition is analogous and that recognition is species-specific.     

Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.     

Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry

The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.     

Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain

Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.     

Membrane factors responsible for homologous species restriction of complement-mediated lysis: evidence for a factor other than DAF operating at the stage of C8 and C9

Species-restricted lysis of complement refers to the relative inefficiency of complement to lyse cells from the homologous species. Restriction occurs at least at the steps involving C3/C5 convertase formation and the C9 insertion phase of the complement cascade, and is presumed to be mediated by inhibitory factors in the target cell membrane. In this study, we have examined whether decay accelerating factor (DAF), a membrane protein known to modulate C3/C5 convertase activities on cell surfaces, acts as a regulatory protein in species-restricted lysis of human erythrocyte (E). The role of DAF was assessed in homologous lysis by the classic pathway, in reactive lysis, and in lytic steps requiring C8 and C9. The results indicated that DAF participated in regulating C3/C5 deposition on the surface of homologous E, but had no effect on homologous restriction in reactive lysis and in the reaction of C8 and C9 with antibody-sensitized E C1-7. Treatment of E with pronase or with dithiothreitol (DTT) abolished the restricting effect of homologous C8/C9, indicating that species-restricted lysis by C5b-9 involves membrane factor(s) sensitive to pronase and DTT.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

The interaction of cells persistently infected with canine distemper virus with antiviral antibody and complement

Cells persistently infected with canine distemper virus can be lysed by antibody and complement. This reaction is dependent upon the alternative complement pathway. In the absence of antiviral antibody, the virus-infected cells will activate the alternative complement pathway, although this will not produce cell lysis.     

Binding of avidin to bacteria and to the outer membrane porin of Escherichia coli

Abstract The binding of avidin to different bacteria was studied using tetramethylrhodamine isothiocyanate (TRITC)-conjugated avidin in fluorescence microscopy, enzyme immunoassay and [¹⁴C]biotin-avidin complex. We observed binding of avidin to all Gram-negative bacteria tested and to some Gram-positive bacteria. The binding was dose-dependent, reached the maximum in 10 min, was optimal at physiological pH and occurred at 4°C, 22°C, and 37°C. This binding of avidin was independent of the saturation of its biotin-binding sites. The avidin receptor of the cell envelope of Escherichia coli K-12 was shown to be the major porin protein (OmpF/OmpC) of the outer membrane.     

Dual-affinity avidin molecules

A recently reported dual-chain avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (K(d) approximately 10(-15) M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate avidins in which the neighboring biotin-binding sites of dual-chain avidin exhibit two different affinities for biotin. In these novel avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These "dual-affinity" avidin molecules open up wholly new possibilities in avidin-biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters.     

Cytotoxic effect of poly-dispersed single walled carbon nanotubes on erythrocytes in vitro and in vivo

Single wall Carbon Nanotubes (SWCNTs) are hydrophobic and do not disperse in aqueous solvents. Acid functionalization of SWCNTs results in attachment of carboxy and sulfonate groups to carbon atoms and the resulting acid functionalized product (AF-SWCNTs) is negatively charged and disperses easily in water and buffers. In the present study, effect of AF-SWCNTs on blood erythrocytes was examined. Incubation of mouse erythrocytes with AF-SWCNTs and not with control SWCNTs, resulted in a dose and time dependent lysis of erythrocyte. Using fluorescence tagged AF-SWCNTs, binding of AF-SWCNTs with erythrocytes could be demonstrated. Confocal microscopy results indicated that AF-SWCNTs could enter the erythrocytes. Treatment with AF-SWCNTs resulted in exposure of hydrophobic patches on erythrocyte membrane that is indicative of membrane damage. A time and dose dependent increase in externalization of phosphatidylserine on erythrocyte membrane bilayer was also found. Administration of AF-SWCNTs through intravenous route resulted in a transient anemia as seen by a sharp decline in blood erythrocyte count accompanied with a significant drop in blood haemoglobin level. Administration of AF-SWCNTs through intratracheal administration also showed significant decline in RBC count while administration through other routes (gavage and intra-peritoneal) was not effective. By using a recently developed technique of a two step in vivo biotinylation of erythrocytes that enables simultaneous enumeration of young (age <10 days) and old (age>40 days) erythrocytes in mouse blood, it was found that the in vivo toxic effect of AF-SWCNTs was more pronounced on older subpopulation of erythrocytes. Subpopulation of old erythrocytes fell after treatment with AF-SWCNTs but recovered by third day after the intravenous administration of AF-SWCNTs. Taken together our results indicate that treatment with AF-SWCNTs results in acute membrane damage and eventual lysis of erythrocytes. Intravenous administration of AF-SWCNTs resulted in a transient anemia in which older erythrocytes are preferably lysed.