Complement activation induces the expression of decay-accelerating factor on human mesangial cells.

In the present study we evaluated the effect of complement activation by immune complexes (IC) on the expression of decay-accelerating factor (DAF) on human mesangial cells (MC). MC in culture were incubated with an Ag (DNP-Gelatin) that binds to fibronectin present in the MC matrix. Subsequently, MC were incubated with anti-DNP antibodies in the presence of human serum. By immunoperoxidase staining we showed that these incubations resulted in IC formation and deposition of human C3 and terminal complement components (C5b-9) on the mesangial matrix and on the surface of MC. By immunoperoxidase staining and by RIA we showed that IC formation and complement activation significantly increased DAF expression on the MC plasma membrane. The induction of DAF expression was a consequence of deposition of terminal complement components on the MC because, zymosan-activated serum and IC formation in the presence of C5- or C8-deficient serum failed to increase MC DAF expression. Furthermore, the observed increased DAF expression was the consequence of increased DAF synthesis by MC. Thus, both cycloheximide and actinomycin D blocked the increase on MC DAF observed after incubation with IC and serum. MC DAF had biophysical and functional characteristics similar to DAF in other cells. Thus, 1) MC DAF was resistant to trypsin but was removed from the MC membrane by pronase; 2) phosphatidylinositol-specific phospholipase C removed 48 +/- 4% of MC DAF indicating that MC DAF is anchored in the cell membrane by phosphatidylinositol groups; 3) DAF isolated from MC-inhibited complement-mediated hemolysis and demonstrated a molecular mass of 83 kDa. In conclusion, deposition of terminal complement components on human MC trigger new synthesis and membrane expression of DAF. Because DAF protects cells against complement-mediated lysis, we postulate that DAF may protect glomerular cells during IC and complement-mediated glomerulonephritis.     

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.     

The influence of membrane components on regulation of alternative pathway activation by decay-accelerating factor.

Decay-accelerating factor (DAF) is a C regulatory protein which functions in membranes to inhibit autologous C activation on cell surfaces. A liposome model was used to study the mechanism of DAF action and examine the effects of membrane-bound glycophorin and LPS on the regulatory activity of DAF. Liposomes were incubated in MgEGTA-treated human serum and activation of the alternative pathway measured by C3b binding. Liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol activated the alternative pathway in proportion to their content of PE. Incorporation of 10(-7) mol/mol phospholipid of either human E or HeLa cell-derived DAF inhibited C activation by liposomes containing 40% phosphatidylethanolamine by 50%, an efficiency comparable to that observed in intact E. HeLa DAF that had been treated with phosphatidylinositol-specific phospholipase C to remove its glycolipid anchor had no effect on C activation by liposomes at concentrations as high as 10(-5) mol/mol phospholipid. Incorporation of DAF into liposomes prepared with bound C3b inhibited the deposition of additional C3b by C3bBbP. However, the incorporated DAF increased the amount of Bb generated from B in the presence of D indicating that accelerated decay of the convertase was the primary effect of DAF. Similarly, treatment of intact human E with anti-DAF decreased the amount of Bb generated by the alternative pathway convertase. To study the effects of other membrane components on DAF activity, liposomes were prepared with purified human glycophorin A or LPS. In glycophorin liposomes the presence of PE was required to activate the alternative pathway and DAF inhibited this activation. In contrast, LPS liposomes bound C3b independently of PE and the incorporation of DAF had no effect. These results demonstrate that within a membrane, DAF's inhibitory activity on the alternative pathway C3 convertase is mediated independently of other membrane proteins, that in this model the major activity of DAF is to accelerate convertase decay, and that the presence of other membrane molecules that may serve as C3 acceptors can circumvent DAF function.     

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.     

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.     

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.     

Effect of hybrid complement regulatory proteins on xenogeneic cells

To suppress C3 fragment deposition in the classical pathway complement activation on xenogeneic membranes, decay accelerating factor (DAF) was the most effective molecule among the complement regulatory proteins (CRPs) used in the present study. C3 fragment deposition was closely related to subsequent xenogeneic cell lysis. However, other molecules were also very effective in different ways and include phosphatidylinositol (PI)-anchored short consensus repeat (SCR) 2-4 of membrane cofactor protein (MCP-PI), PI-anchored C1 esterase inhibitor (C1-INH-PI), and PI-anchored SCR8-11 of complement receptor type 1 (CR1-PI). On the other hand, regarding a strategy for downregulating C4 fragment deposition, the use of only C1-INH-PI and PI-anchored SCR1-3 of the C4b-binding protein (C4bp-PI) was found to be effective.     

阵发性睡眠性血红蛋白尿症红细胞乙酰胆碱酯酶的研究

用化学方法测定了乙酰胆碱脂酶（ＡｃｈＥ）活性,阵发性睡眠性血红蛋白尿症（ＰＮＨ）红细胞远低于正常红细胞。为了进一步研究ＰＮＨＡｃｈＥ（—）的红细胞,采用Ｐｒｏｔｅｉｎ　Ａ　Ｓｅｐｈａｒｏｓｅ　６ＭＢ结合ＡｃｈＥ单抗亲和层析法分离出ＰＮＨＡｃｈＥ（—）的红细胞。用间接免疫荧光流式细胞术检测,ＰＮＨ细胞ＡｃｈＥ低于正常,而ＰＮＨＡｃｈＥ（—）红细胞未能检出ＡｃｈＥ。３Ｈ－肌醇标记实验证明,正常红细胞膜区带４．１处有较高的放射活性,而ＰＮＨ红细胞极低,ＰＮＨＡｃｈＥ（—）红细胞完全无放射活性。用ＡｃｈＥ抗体做免疫印渍实验证明了ＡｃｈＥ存在区带４．１部位。ＤＭＰＣ诱导正常和ＰＮＨ红细胞,检测二者囊泡化的程度,发现ＰＮＨ病人红细胞远比正常人红细胞易于囊泡化。     

Aurin Tricarboxylic Acid Protects against Red Blood Cell Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinemia

Objectives

Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage.

Design and Methods

We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera.

Results

We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes.

Conclusions

We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.     

Activation of the alternative complement pathway by isolated human glomerular basement membrane

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.     

Targeting of functional antibody-decay-accelerating factor fusion proteins to a cell surface

Recombinant soluble complement inhibitors hold promise for the treatment of inflammatory disease and disease states associated with transplantation. Targeting complement inhibitors to the site of complement activation and disease may enhance their efficacy and safety. Data presented show that targeting of decay-accelerating factor (DAF, an inhibitor of complement activation) to a cell surface by means of antibody fragments is feasible and that cell-targeted DAF provides significantly enhanced protection from complement deposition and lysis compared with soluble untargeted DAF. An extracellular region of DAF was joined to an antibody combining site with specificity for the hapten dansyl, at the end of either C(H)1 or C(H)3 Ig regions. The recombinant IgG-DAF chimeric proteins retained antigen specificity and bound to dansylated Chinese hamster ovary cells. Both soluble C(H)1-DAF and C(H)3-DAF were effective at inhibiting complement-mediated lysis of untargeted Chinese hamster ovary cells at molar concentrations within the range reported by others for soluble DAF. However, when targeted to a dansyl-labeled cell membrane, C(H)1-DAF was significantly more potent at inhibiting complement deposition and complement-mediated lysis. Cell-bound C(H)1-DAF also provided cells with protection from complement lysis after removal of unbound C(H)1-DAF. Of further importance, the insertion of a nonfunctional protein domain of DAF (the N-terminal short consensus repeat) between C(H)1 and the functional DAF domain increased activity of the fusion protein. In contrast to C(H)1-DAF, C(H)3-DAF was not significantly better at protecting targeted versus untargeted cells from complement, indicating that a small targeting vehicle is preferable to a large one. We have previously shown that for effective functioning of soluble complement inhibitor CD59, binding of CD59 to the cell surface close to the site of complement activation is required. Significantly, such a constraint did not apply for effective DAF function.     

Host-specific evasion of the alternative complement pathway by schistosomes correlates with the presence of a phospholipase C-sensitive surface molecule resembling human decay accelerating factor.

Schistosoma mansoni parasites recovered from the blood stream were found to be nonactivators of the alternative complement pathway (ACP) when exposed to sera of homologous but not heterologous host species. Schistosomes could be converted into activators of the homologous ACP by treatment with phospholipase C. Antibodies to either human or guinea pig decay accelerating factor (DAF), a 70-kDa glycosylphosphatidylinositol anchored membrane glycoprotein which controls ACP activation on the mammalian cell plasma membrane, bound to the surface of immature schistosomes and immunoprecipitated a molecule of similar molecular mass from detergent extracts of surface iodinated parasites. Phospholipase C treatment drastically reduced the reactivity of the worms with the anti-DAF antibodies. These data suggest that schistosomes evade the ACP by inserting functional host DAF into their surfaces, possibly through adsorption of the molecule's lipophilic diacyglycerol membrane anchor moiety into the outer lipid bilayer of the parasite.     

Characterization of the active sites in decay-accelerating factor

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.     

Homologous species restriction in lysis of human erythrocytes: a membrane-derived protein with C8-binding capacity functions as an inhibitor

An intrinsic membrane protein with a m.w. of 65,000 that can bind human C8 has been identified after separation of human erythrocyte membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransfer to nitrocellulose sheets. The protein, tentatively designated as the C8-binding protein (C8bp) could be isolated from papain-treated erythrocyte (E) membranes by phenol-water extraction and isoelectric focusing. In a functional assay, with chicken (ch) E as target cells, C8bp inhibited the lysis of ch E C5b67 intermediates by human C8 and C9, whereas the lysis by rabbit C8 and C9 was not affected. Because the decay accelerating factor (DAF) from human erythrocyte membranes also inhibits the activity of C3/C5 convertases in an homologous system, we tested whether or not a DAF activity was present in C8bp. C8bp, however, did not accelerate the decay of the classic C3 convertases. Thus, it appears that C8bp and DAF are two different factors of E membranes with a similar molecular size inhibiting different sites of the activation cascade of complement while they can function synergistically to minimize the self-inflicted damage by complement.     

Human recombinant soluble decay accelerating factor inhibits complement activation in vitro and in vivo.

Complement plays a role in activating the inflammatory response and has been implicated in the pathogenesis of some inflammatory diseases. With a view toward controlling unwanted C activation, we evaluated the C regulator, human decay accelerating factor (DAF). Three forms of recombinant DAF were purified from transfected Chinese hamster ovary cells: glycophosphatidylinositol (GPI)-linked membrane DAF (mDAF) extracted from cell membranes; spontaneously shed soluble DAF (sDAF) derived from mDAF; and a novel secreted protein (seDAF), generated by deletion of the signal for GPI attachment. We show that all three molecules inhibit both the classical and alternative pathways of C activation. The following observations indicate that mDAF extracted from Chinese hamster ovary cells reincorporates into RBC membranes via its GPI anchor: 1) cells that are preincubated with mDAF and then washed remain fully protected from C-mediated hemolysis; 2) incubation with phosphatidylinositol-specific phospholipase C abolishes this protection; and 3) sDAF and seDAF, which lack a GPI anchor, do not associate with cell membranes. mDAF is a more potent inhibitor of C-mediated hemolysis than either sDAF or seDAF, suggesting that incorporation into cell membranes greatly enhances the efficiency with which DAF inhibits C activation on the cell surface. In contrast, C activation in the fluid phase is inhibited by sDAF and seDAF, but not by mDAF, possibly due to interference by serum lipoproteins. A reversed passive Arthus reaction in guinea pigs was used to evaluate the ability of recombinant seDAF to inhibit C activation in vivo. When administered at dermal sites, seDAF reduced the severity of immune complex-mediated inflammatory reactions induced by a reversed passive Arthus reaction, as judged by both gross and histologic examination. These data indicate that seDAF may be useful as an anti-inflammatory therapeutic.     

Characterization of mouse DAF on transfectant cells using monoclonal antibodies which recognize different epitopes

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay-accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI-DAF) and a transmembrane form (TM-DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI-DAF or TM-DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI-DAF transfectant cells and established seven unique mAbs (RIKO-1-7). Western blotting analysis using RIKO-3, which reacts with both GPI-DAF and TM-DAF, and RIKO-4, which is an anti-GPI-DAF specific mAb, indicated that GPI-DAF was expressed on erythrocytes, spleen and testis, and that TM-DAF was expressed only in testis.     

Evasion of complement-mediated lysis and complement C3 deposition are regulated by Francisella tularensis lipopolysaccharide O antigen

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag.     

Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor

When whole serum C is activated by cobra venom factor complexes (CoFBb), paroxysmal nocturnal hemoglobinuria (PNH) III E (the most C-sensitive type) are hemolyzed, but normal and PNH II E (the intermediately sensitive type) are not. Previous studies have shown that after exposure to CoFBb and serum, PNH III E bind relatively large amounts of the trimolecular C complex, C5b67, whereas normal and PNH II E bind virtually none. In the studies reported herein, we have observed that when normal and PNH III E are incubated with isolated C5, C6, and 125I-C7 in the presence CoFBb, the normal E bind more C5b-7 than the PNH cells. When C7-deficient serum is included in the reaction mixture, however, the PNH E are once again observed to bind much greater amounts of C5b-7. These observations suggest that plasma and membrane factors act in concert to restrict the assembly of the trimolecular C5b-7 complex on human E. PNH III E appear to be deficient in the membrane component of this inhibitory system.     

Effect of glycoinositolphospholipid anchor lipid groups on functional properties of decay-accelerating factor protein in cells.

The inositol ring in the glycoinositolphospholipid (GPI) anchor of human decay-accelerating factor (DAF) is unmodified in nucleated cells, whereas it is fatty acid acylated in erythrocytes (Ehu). To assess the effect of this and of the glycerol sn-2-associated acyl substituent on the abilities of DAF to cell membrane incorporate and function, 1) endogenous (physiologically anchored) DAF proteins bearing three- and two-"footed" GPI anchors were purified from Ehu and HeLa cells and 2) synthetic DAF variants bearing alternative one- "footed" anchors (retaining either the sn-1 glycerol- or inositol-associated lipid) were prepared by alkaline hydroxylamine treatment and phosphatidylinositol-specific phospholipase D digestion of Ehu DAF, respectively. The different DAF species were added to antibody-sensitized sheep erythrocytes (EshA) and their abilities to insert into the plasma membranes of the cells and control subsequent complement activation on their surfaces were compared. DAF proteins bearing all four GPI anchor structures adhered to the Esh hemolytic intermediates and inhibited expression of C3 convertase (C4b2a) activity. However, mixing of DAF-treated EshA with untreated EshAC142 and stripping of cell-associated DAF proteins with vesicles showed that only the physiologically anchored proteins remained stably associated with the lipid bilayer and functioned intrinsically. Both three- and two-"footed" Ehu and HeLa DAF proteins exhibited comparable ability to incorporate and function in the intermediates as well as to accumulate to levels 1000-fold higher/cell in Schistosoma mansoni schistosomula. These findings indicate that 1) an intact inositolphospholipid-containing GPI anchor is necessary for stable membrane integration and intrinsic function, 2) endogenous GPI anchors (with either unsubstituted and acylated inositol) incorporate and function with comparable efficiency, and 3) the transfer of either endogenous DAF form can account for the previously described circumvented uptake of human C3b by blood stage schistosomula.