A novel protein that participates in nonself discrimination of malignant cells by homologous complement.

The human complement (C) system protects an individual against substances of nonself origin, including xenografts and microbial pathogens. Human cells express C-regulatory proteins, CD46 and CD55, thereby circumventing attack by C3, a major effector of C. Nevertheless, certain malignant cells, particularly those undergoing apoptotic stress, can activate homologous C, overcoming the regulatory actions of CD46 and/or CD55. The molecular mechanisms whereby malignant cells are tagged by homologous C3 remain largely unknown. We identified a novel gene product that converts human cells into targets for homologous complement. Only malignant cells and cell lines exposed to Fas or X-irradiation stimuli produced this protein, designated M161Ag, which was an unglycosylated 43-kDa protein. Analysis of cloned cDNAs indicated that this molecule was a secretory protein containing five amino acids encoded by TGA codons. Its functions were unique in that once secreted from the tumor cells, it bound back to the surface of these cells and activated homologous complement (C3) via the alternative pathway, allowing for C3 deposition on the membrane. This molecule may offer new insight into innate immunity; surveillance of tumor cells by complement is a common feature in the human immune system.

Purification and characterization of a membrane protein (gp45-70) that is a cofactor for cleavage of C3b and C4b.

Based on preliminary evidence indicating that a cell-associated protein of U937 (a human monocyte-like cell line) possessed cofactor activity and was not the C3b/C4b receptor, we sought to further characterize this protein. A sequential four-column purification procedure was devised that includes C3(H2O) affinity chromatography to isolate in reasonable yields and purity a cell-associated protein of U937 and several other human cell lines. Based on its pattern and Mr on SDS-PAGE, acidic pI, and ligand specificity, it is identical to a recently described C3(H2O) or C3b-binding membrane glycoprotein of human PBL and cell lines; having no presently identified function, it was termed gp45-70. After purifying this protein, we determined its functional capabilities and compared them to those of the other complement proteins with regulatory activity directed at components comprising the C3 convertases. This protein was the most efficient (50 times that of H) yet-described cofactor for the I-mediated first cleavage of C3b. It also was a cofactor for the first cleavage of C4b, but was not as efficient as C4bp. The second cleavage of C3b and C4b was not efficiently mediated. It had no ability to accelerate decay in the classical or alternative pathway C3 convertases. Based on this unique activity profile and ability to be surface labeled, we have renamed this molecule membrane cofactor protein (MCP). We suggest that this protein plays a major role in preventing autologous complement activation.

Purification and characterization of a human membrane protein that activates the alternative complement pathway and allows the deposition of homologous complement C3.

A human myeloid cell subline, P39+, is found to be a target for human complement (C) via the alternative pathway and to allow the deposition of multiple C3 fragments on its membranes, though expressing the complement regulatory proteins decay-accelerating factor and membrane cofactor protein. The parent cell line, P39-, which is phenotypically similar to the P39+ subline, does not allow the deposition of homologous C3 fragments. In this study, we established a monoclonal antibody, M161 Ab, which reacted with P39+ but not P39- cells. This Ab recognized a 43-kD protein in P39+ cell lysate transblotted onto nitrocellulose. Using this Ab as a probe, we purified the 43-kD protein, namely, M161 antigen (Ag). M161 Ag had a basic isoelectric point (pI), 9.3-9.4 by chromatofocusing, and was precipitated as an insoluble material at the pI point. The purified M161 Ag was a single-chain protein and did not possess N- or O-linked carbohydrates. When the purified M161 Ag was transblotted onto nitrocellulose and incubated with Mg(2+)-EGTA serum, human C3 fragments were efficiently deposited on M161 Ag. The major species of the deposited C3 fragments was C3b. Furthermore, the C3 fragments bound to the M161 Ag were detached by 1 M hydroxylamine, suggesting that a covalent ester linkage sustains M161 Ag-C3b interaction. NH2-terminal amino acid analysis revealed that M161Ag is a novel membrane protein. Hence, it appeared that M161 Ag is a potent activator of human alternative complement pathway on human cells that activates homologous C3 and allows the deposition of C3b on itself. Thus, under some conditions, homeostasis of complement is maintained even on human cells, not only by the complement regulatory proteins, but also by membrane C3-activating molecules on which C3b is deposited.

Complement-mediated tumor cell damage induced by antibodies against membrane cofactor protein (MCP, CD46).

We have developed polyclonal and monoclonal antibodies against human membrane cofactor protein (MCP) to use as tools to investigate the functions of MCP on intact nucleated cells. Two human T cell lines, CEM and TALL, are CR1- and DAF-. Pretreatment of these cell lines with M177 and polyclonal anti-MCP, which inhibit cofactor activity almost completely, resulted in effective C3 deposition immediately following addition of these cells to Mg2+/EGTA/human sera. The deposited C3 remained expressed partly on the cell surface and most of them were gradually converted to C3bi. Some of the deposited C3 were complexed with membrane proteins, since 140- and 250-kD bands became significantly accumulated on SDS-PAGE by treatment with the antibodies. We next tested whether these C3-coated cells were damaged by complement-mediated cytolysis. p18, an inhibitor of membrane attack complex (MAC) formation, was negative in TALL but positive in CEM. TALL was lysed efficiently only by treatment with the polyclonal anti-MCP, while CEM showed only slight lysis with the same treatment. Monoclonal antibodies to MCP, including M177, caused only minimal cell destruction. Based on these results, together with the fact that decay-accelerating factor (DAF) serves as a factor for preventing C3 attack on human cells, we conclude that MCP and DAF cooperatively protect host cells from C3 targeting and, in these T cell lines, MCP is sufficient for preventing C3 deposition even without DAF. After all, human cells undergo almost no autologous complement-mediated cytolysis if they express at least one of the functionally active inhibitors, MCP, DAF, or p18.

Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein.

Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

Molecular cloning and chromosomal localization of human membrane cofactor protein (MCP). Evidence for inclusion in the multigene family of complement-regulatory proteins.

Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.

Toll-like receptor 3 in nasal CD103+ dendritic cells is involved in immunoglobulin A production.

Intranasal inoculation with influenza hemagglutinin subunit with polyinosine-polycytidylic (polyI:C), a synthetic analog for double-stranded RNA, enhances production of vaccine-specific immunoglobulin (Ig) A, which is superior to IgG in prophylactic immunity. The mechanism whereby polyI:C skews to IgA production in the nasal-associated lymph tissue (NALT) was investigated in mouse models. Nasally instilled polyI:C was endocytosed into CD103+ dendritic cells (DCs) and induced T-cell activation, including interferon (IFN)-γ production. According to knockout mouse studies, polyI:C activated the Toll-like receptor 3 signal via the adapter TICAM-1 (also called TRIF), that mainly caused T-cell-dependent IgA production. Nasal CD103+ DCs activated transforming growth factor-ß signaling and activation-induced cytidine deaminase upon polyI:C stimulation. IgA rather than IgG production was impaired in Batf3-/- mice, where CD103+ DCs are defective. Genomic recombination occurred in IgA-producing cells in association with polyI:C-stimulated DCs and nasal microenvironment. PolyI:C induced B-cell-activating factor expression and weakly triggered T-cell-independent IgA production. PolyI:C simultaneously activated mitochondrial antiviral signaling and then type I IFN receptor pathways, which only minimally participated in IgA production. Taken together, CD103+ DCs in NALT are indispensable for the adjuvant activity of polyI:C in enhancing vaccine-specific IgA induction and protective immunity against influenza viruses.

Membrane cofactor protein of complement is present on human fibroblast, epithelial, and endothelial cells.

Membrane cofactor protein (MCP) of the complement system is a iC3/C3b binding molecule with cofactor activity that has been identified on all human peripheral blood cells except erythrocytes. Human mononuclear and platelet MCP is dimeric with molecular weights of 68,000 and 63,000 and is expressed in three phenotypic patterns. To further determine its tissue distribution, surface-labeled human fibroblast, epithelial, and endothelial cells and cell lines were assessed for the presence of MCP by iC3 affinity chromatography and by immunoprecipitation with a monospecific anti-MCP rabbit polyclonal antibody. All sources of adult and fetal fibroblast and epithelial cells and cell lines examined and umbilical vein endothelial cells expressed MCP. The molecular weight and phenotypic patterns of MCP were similar to those of peripheral blood cells. MCP was synthesized by fibroblast and epithelial cell lines. Solubilized extracts of these cell lines expressed factor I-dependent cofactor activity for the first cleavage of iC3/C3b which was abrogated by removal of MCP. Expression of MCP was modulated by SV40 transformation of two fetal fibroblast lines. There was a 5- to 10-fold increase in expression of MCP and a preferential expression of the lower species such that the phenotypic designation was changed. The wide tissue distribution and activity profile of MCP suggest that it is likely to play an important role in the regulation of the complement cascade.

Identification of a third component of complement-binding glycoprotein of human platelets.

Utilizing affinity chromatography, a C3-specific binding protein was isolated from 125I surface-labeled human platelets. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated two bands with mean Mr of 64,000 and 53,000, characteristic variability in the relative density of the two bands in a given individual, and the presence of N-linked complex oligosaccharides as well as sialic acid residues not associated with N-linked sugars. These characteristics are similar to those of a human leukocyte iC3- and C3b-binding glycoprotein, termed gp45-70. Further analysis showed that leukocyte gp45-70 and the platelet C3-binding glycoprotein have identical Mr and other similar structural features. Functional characterization of solubilized platelet preparations indicated that gp45-70 has cofactor activity. This membrane glycoprotein is structurally and antigenically distinct from decay accelerating factor (DAF), a complement regulatory protein previously identified on human platelet membranes. DAF and gp45-70 have complementary activity profiles inasmuch as DAF can prevent assembly of and dissociate the C3 convertases but has no cofactor activity, whereas gp45-70 has cofactor activity but no decay accelerating activity. We suggest that these two proteins function conjointly to prevent autologous complement activation.

The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.

Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Regulatory system of guinea-pig complement C3b: tests for compatibility of guinea-pig factors H and I with human factors.

Two proteins that are involved in cleavage of methylamine-treated C3 of guinea-pig origin (C3(MA)gp) have been isolated from guinea-pig serum. One of them functioned as a cofactor of human factor I (Ihu) for cleavage of C3(MA)gp and its molecular size was 150 kDa. The other was functionally pure and able to cleave C3(MA)gp together with human factor H (Hhu). They appear to be analogous to human factors H and I in the guinea-pig and will be referred to as Hgp and Igp. Methylamine-treated human C3 [C3(MA)hu] was not a compatible substrate for Hgp or Igp: little cleavage of C3(MA)hu was observed if human factor H (Hhu) or I was substituted with the guinea-pig counterpart. C3(MA)gp, on the other hand, served as a substrate, though less efficiently, for Hhu and Ihu. Human C4b-binding protein (C4bp) and membrane cofactor protein (MCP) as well as Hhu could participate in cleavage of C3(MA)gp by Igp or Ihu. In these assays, C3(MA)gp was degraded again less efficiently than C3(MA)hu. Interestingly, human C3b/C4b receptor (CR1) mediated factor I-dependent cleavage of C3(MA)hu and C3(MA)gp to a similar extent regardless the sources of factor I. These results suggest that factor I-dependent C3b regulatory system is species-specific except in the case of CR1, which may function as a cofactor irrespective of species.

Acceleration of site-to-site transfer of C1- by a monoclonal antibody to C1-s.

A monoclonal antibody to human C1-s (a subcomponent of C1-), M365 blocked the complement-mediated lysis of C1(-)-coupled IgM-sensitized sheep erythrocytes (EAIgMC1). However, M365, via its binding to C1-s, did not inhibit C2 activation and only partially inhibited C4 activation, both attributable to the proteolytic action of C1s. Therefore, the inhibition of lysis of EAIgMC1 is not due to the direct effect of antibody binding to C1-s in the C1- molecule. M365, when added to the pre-formed EAIgMC1, deprived the whole C1 molecule from the cells. It was also found that M365-bound C1 could not bind to EAIgM. These phenomena were induced only when M365, one of seven monoclonal antibodies to C1-s, was employed and when IgM antibody-sensitized E was used. The observed C1- liberation and C1 binding inhibition by M365 were found to be less effective when the C4b bearing cells, EAIgMC4, were used. But the addition of M365-bound C1- to EAIgMC4 promoted the formation of the C3 convertase, C4b2a, which caused an incremental lysis of the C4b bearing cells. The results are interpreted to mean that C1-, once complexed with M365, still remains active and acquires the ability to transfer from one C1 binding site on IgM to another to activate the convertases. C4b must be a prerequisite on the cells to induce the effective C1- transfer and resulting increase of C3 convertase sites. We hypothesize that the M365-C1-s association alters the conformation of C1q and thereby leads to the dissociation of whole C1- from IgM. The M365-C1- complex, therefore, can move from site to another.

Involvement of the pertussis toxin-sensitive GTP-binding protein in regulation of expression and function of granulocyte complement receptor type 1 and type 3.

Human polymorphonuclear leukocytes (PMN) express receptors for complement (C) C3b and C3bi termed CR1 and CR3, respectively. The addition of PMA or fMLP to PMN enhances the capacity of these receptors to promote binding of C3b- and C3bi-coated erythrocytes. fMLP-dependent increase of the binding of these ligand-coated erythrocytes was completely abolished by prior exposure of the PMN to pertussis toxin (IAP). GTP-binding protein (Gi alpha) was ADP-ribosylated and dysfunctional by this treatment. On the other hand, PMA-dependent binding of these ligands, as well as control binding, was inhibited only slightly, if at all, by the IAP treatment. The levels of C receptor expression on cell surface were determined by flow cytometry using monoclonal antibody against CR1 and those against the alpha and beta chains of CR3 (CR3 is composed of alpha and beta chain). Upon exposure of PMN to the chemotactic factor or PMA, or upon incubation of the cells at 37 degrees C, the surface expression of CR1 and CR3 alpha was increased. IAP also blocked an fMLP-induced increase of CR1 and CR3 alpha, but did not block the temperature- or PMA-dependent increase of these receptors. Opsonized zymosan (SOZ), another ligand for CR3, also led to an increase of both CR1 and CR3 alpha. Neither PMA nor SOZ brought about an increase of the surface expression of CR3 beta, but fMLP caused a slight increase of CR3 beta in an IAP-sensitive manner. Based on the IAP-sensitivity of the receptor expression, therefore, it appears that at least two separate mechanisms are operative in the control of C receptors. In addition, the alpha and beta chains of CR3 are regulated independently. The present data offer evidence suggesting that C receptor functions are in part regulated through a GTP-binding protein via modulation of their surface expression.

Analysis of C3b/C4b receptor (CR1) polymorphic variants by tryptic peptide mapping.

The human C3b/C4b receptor (CR1) binds the major activation and opsonic fragments of the third (C3) and fourth (C4) components of complement. CR1 is a single chain integral membrane glycoprotein widely distributed on peripheral blood cells. Four codominantly inherited allelic variants with Mrs of 160,000, 190,000, 220,000 and 250,000 have been described. To address the structural basis for this unusual polymorphism, CR1 from donors expressing three of the four allelic variants was purified from surface labeled (125I) erythrocytes by iC3-Sepharose affinity chromatography and the variants compared by tryptic peptide mapping (TPM). The TPMs of each variant contained the same major peaks and minor peak areas and were nearly identical to one another. Tryptic peptide mappings of the 190,000 Mr erythrocyte CR1, which was purified prior to iodination, were similar to those derived from surface iodinated CR1. The TPMs of erythrocyte and granulocyte CR1 from the same donor differed by a single peak of increased prominence in the granulocyte map. These results indicate a conservation in amino acid sequence for those peptides detected. In view of these data and those of other studies of the structure and genetics of CR1 and related proteins, it is suggested in this paper that the allelic variation relates to CR1, being composed of repeating amino acid sequences.

Covalently-bound human C4b dimers consisting of C4B isotype show higher hemolytic activity than those of C4A in the C3-bypass complement pathway.

The ability to form a covalent dimer of human C4b was investigated with purified isotypes C4A and C4B, and antibody-sensitized liposomes supplemented with C1. In this system, no C4A or C4B formed a complex with the antibody or C1. Whereas both C4A and C4B isotypes formed dimers to a similar extent, C4B formed an ester-linked dimer and C4A an amide-linked dimer. Both of these dimers served as a subunit for the C3-bypass pathway C5 convertase, since liposomes bearing Ab, C1 and a dimer of C4A or C4B, allowed the formation of C5 convertase by the addition of C2. The degree of complement-mediated liposome lysis however, was observed to be 2-3 times higher in the C4B-bearing particles than in those bearing C4A. These results indicate that the second C4b-binding site on the first C4b is different between C4A and C4B, and that in the C3-bypass pathway, C4B has a higher degree of hemolytic activity than C4A, as in the conventional classical complement pathway.

Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP).

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.

Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b.

Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and Factor I can cleave methylamine-treated C4(C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0 and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase.

Analysis of C5b-8 binding sites in the C9 molecule using monoclonal antibodies: participation of two separate epitopes of C9 in C5b-8 binding.

C5b-8 binding sites in C9 were examined using mAbs raised against C9. Among 16 mAbs, two, designated P40 and X197, blocked C9-mediated EAC1-8 lysis. C9 pretreated with the mAbs failed to bind to EAC1-8 at 4 degrees C. In addition, the mAbs became inaccessible to the C9 that had been incorporated into EAC1-8 at 4 degrees C. These findings suggest that C9 binding to EAC1-8, but not its membrane spanning or polymerization, is blocked by mAbs. By immunoblotting analysis using alpha-thrombin proteolytic fragments derived from C9 [a N-terminal fragment of mol. wt 25,000 (C9a) and a C-terminal one of mol. wt 37,000 (C9b)] and tryptic fragments of C9 (mol. wts 53,000 (C9a') and 20,000 (C9b')), the epitopes of P40 and X197 were mapped to the N-terminal and C-terminal regions of C9b, respectively. Both P40 and X197 bound to the C9 polymerized with Zn2+ in the fluid phase, whereas X197 but not P40 reacted with the membrane attack complex (MAC) formed on membranes. The results suggest that two distinct epitopes are involved in C9 binding to EAC1-8, and behave in a different manner for globular C9 bound to EAC1-8 at 4 degrees C, C9 assembled in MAC, or poly-C9 induced by Zn2+. These mAbs may be useful in clarifying the conformational states of C9 and in analyzing the molecular interaction between C9 and its inhibitors.

Proteolytic elimination of decay-accelerating factor (DAF): lytic abnormality coincides with removal of DAF in papain-treated human erythrocytes.

Erythrocytes (E) from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack decay-accelerating factor (DAF) and this partly causes increasing susceptibility of the E to complement. Several reagents have been used to convert normal E to the complement-sensitive (PNH-like) cells. The relationship between DAF amounts and complement susceptibility of these PNH-like cels has been examined. Of the reported reagents for preparation of PNH-like cells, 2-amino-ethylisothiouronium bromide (AET), papain, and periodate efficiently converted normal E to the complement-sensitive cells, but only papain reduced the quantity of DAF on the cells. Further, of the proteases we tested only papain cleaved DAF to liberate its major fragment from the cells. The papain-treated cells lysed in a similar fashion to PNH cells as the serum concentration increased. The major papain-digested product of DAF had Mr, 55,000, lacked hydrophobicity, and retained the ability to inhibit the C3 convertases. These findings suggest that papain allows liberation from cells of functional domains as well as most of the antigenic epitopes of DAF to generate a PNH-like cell.