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Engineering human complement component C9 reveals the significance of covalent modification for biological activity
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^ monoclonal antibody(uAbSl2) to c e l l surface antigen on neuraminidase-treated rat erythroeytes renders thel sensitive to homologous cozplelent.
ENGINEERING HUMAN COMPLEMENT C O M P O N E N T C9 R E V E A L S T H E S I G N I F I C A N C E O F COVALENT MODIFICATION FOR BIOLOGICAL ACTIVITY.
11.Takizawa~, N. Okada2, S. Matsuo3, H. Senoua and ll. OkadaI Dept of Molecular Biology, Nagoya City University School of Medicine 1, Dept of Microbiology, Fukuoka University School of Nedicinez, The Third Dept of Medcine, Nagoya University School of Medcine3
Kathryn M. Taylor, Alex Davies, B. Paul Morgan,*J. Paul Luzio, and Anthony K. Campbell. Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K. *Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QR, U.K.
Complement action on homologous cell membranes is r e s t r i c t e d by membrane inhibitors such as OAF, cR-I, MCP, IIRF and ItRF20(CD59). These complement regulatory proteins have been isolated and characterized for humans, but much less is known about their rodent counterparts and correspond -ing encoded genes. In order to understand rat complement regulatory proteins, we attempted to obtain a monoclonal antibody which was capable of rendering rat erythrocytes sensitive to homologous complement. Using neuraminidasetreated rat erythrocytes(Neu RE) as an immunogen, we established a monoclonal antibody, mAbSl2, which allows for haemolysis of Neu-RE by rat serum. We assumed that mAbS12, an IgGl isotype, blocks a complement regulatory protein, mAbSl2 detected a rat erythrocyte membrane protein with a molecular mass of 65Kda and 55Kda by western blotting analysis. 512Ag was expressed on PBMC, endothelial cells, mesangial c e l l s and several established cell lines. Furthermore mAbS12 induced C3b deposition on rat myeloma cells following treatment with rat serum. These findings show that 512Ag has important complement regulatory a c t i v i t i e s in rats, and i t s use should f a c i l i t a t e the further characterization of this membrane protein, especally with respect to physiological function.
Evidence for a Shared Binding Site in Human Complement Component C3 for Factor B and for Complement Receptor Type 3 (CR3, CDIIb/CDI8) Aiko Taniguchi-Sidleand David E. Isenman Dept.of Biochemistry,University of Toronto, Toronto, Canada M5S 1A8 Based on previous work, factor B binding to C3b was expected to involve two points of contact, one each for Ba and Bb. Studies using synthetic peptides (Fishelson, Mol. Immunol. (1991) 28:545) and the "C3o" proteolytic fragment of C3 (O'Keefe et al., JBC (1988) 263:12690) identified short sequences in the ct'-chain (752-761 and 955-964, pro-C3 numbering) which might mediate factor B binding. To further examine the role of these proposed sequencesin the context of intact C3b, we have altered their charged residues by site-directed mutagenesis. Resultant mutant recombinant C3 proteins were deposited onto sheep erythrocytes, and the ability of the cello associated C3b molecules to bind factor B was assessed by measuring the susceptibility of bound B to cleavage by D. Recombinant C3b in which airs of neg,,~ively charged amino acids near the N-terminus of ct'-chain, 52DE and~58EE, were replaced by NQ and QQ respectively,had much less ability to support activation of B by D. Mutagenesis of each of K959, E960 and D961 individuallyto alanine caused little impairmentof factor B binding, andeven the 959AAA triple mutation showed only moderate impairment. Although an RGD triplet in C3 had been implicated in the binding of iC3b to CR3, we have previously shown that its replacement by AAA has no effect on this interaction ~ (1992) 267:635). Because the "L domain" of CD1 lb has sequence similarity to a region in Bb, it seemed possible that CR3 and factor B may recognize the same or overlapping sites in C3. To lest this hypothesis, cell-bound recombinant C3b molecules were converted to iC3b and these iC3b-coated cells were assayed for their ability to form CR3-dependentrosettes. Mutagenesis of 752DE and 758EE greatly reduced the iC3b-CR3 interaction, whereas binding of the 959AAA mutant iC3b was indistinguishablefrom that of wild-type. These studies thus demonstratethat a segment near the N-terminusof or'chain, encompassing the sequence 752DEDIIAEE, first identified in the Fishelson peptide study, contains an important contact site for the binding of both factor B by C3b, and iC3b by CR3. The 959KED triplet of the C3ospecific site makes at best a minor contribution to the C3b-B interaction and has no role in binding of iC3b to CR3. (Supported by MRC Canada )
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Aggregation and polymerisation of C9 plays a key role in the permeability changes induced by the MAC. The aim of the work described here was to use protein engineering to manipulate glycosylation and disulphide-bond induced polymerisation. Their role in the permeability thresholds induced by C9 within the MAC, 'ogcther with its removal from the membrane, was then investigated. Unglycosylated C9 was generated in vitro by P e R coupled with transcription and translation. C9 without N-linked glycosyl residues, and fully glycosylated C9 missing the first 23 amino acids, a putative calcium binding site, and two cysteines from a cysteine-rich region were generated using the baculovirus expression system. Fully active g|ycosylated recombinant C9 was produced by sub-cloning P e R products into a plasmid (pVL941) colrtainin.g compatible regions to a linear baculovirus expression vector with a lethal deletion (Baculogold) enabling co-transfection into Spodoptera frugiperda (Sf9) insect cells. Increased secretion of recombinant C9 was obtained by replacing the native signal peptide with the honeybee melittin signal peptide and by use of Trichoplusia ni 5BI-4 (High Five) cells instead of Sf9 insect cells. Unglycosylatcd recombinant C9 polymerised more readily in solution than the glycosylatcd C9 secreted by insect ceils. Several of the mutations affected polymerisation and biological activity.
HETEROPOLYMER MEDIATED BINDING OF TARGET ANTIGENS TO PRIMATE ERYTHROCYTES (E) V I A C R 1 . IN V I T R O A N A L Y S E S R.P. Taylor a, C.J. Reist a, H-Y. Liangb, V. Muzykantov °, M. Zachary a, W.M. Scheld a, J. Powers a, B. Bustera aUniversity of Virginia, Charlottesville, VA, USA; bChina-Japan Friendship Hospital, Peking, China; Clnstitute of Experimental Cardiology, Moscow, Russia We have used the streptavidin/biotin system to construct soluble, bispecific heteropolymers made up of monoclonal antibodies to primate E CR1 cross-linked with monoclonal antibodies to a target antigen. These heteropolymers facilitate rapid (ca. 2 min at 37°C), specific and high avidity binding to human or monkey E of soluble and particulate antigens, including human IgM, IgE, LDL, herpes simplex virus capsid, and the bacterium Haemophilus influenzae. Specific heteropolymer mediated binding of these antigens to primate E occurs in the complete absence of complement, and quite similar levels of antigen binding are observed in the presence of complement as well. No antigen binding is demonstrable in control studies with sheep E (which lack CR1). FACS analyses and magnetic separation assays indicate the majority of the primate E contain bound antigen or heteropolymer. At saturation the absolute number of heteropolymers or protein antigens bound per E (1002000) is consistent with the number of CR1 per E, the mw of the antigens, and amplification by the streptavidin/biotin system. These heteropolymer constructs, prepared using streptavidin/biotin or other cross-linking methodologies, may find therapeutic use by immobilizing pathogens on primate E. The E-bound pathogens may then be neutralized and/or cleared from the circulation through a mechanism similar to that which occurs during Emediated processing or clearance of complement-opsonized particles and immune complexes.
^ monoclonal antibody(uAbSl2) to c e l l surface antigen on neuraminidase-treated rat erythroeytes renders thel sensitive to homologous cozplelent.
ENGINEERING HUMAN COMPLEMENT C O M P O N E N T C9 R E V E A L S T H E S I G N I F I C A N C E O F COVALENT MODIFICATION FOR BIOLOGICAL ACTIVITY.
11.Takizawa~, N. Okada2, S. Matsuo3, H. Senoua and ll. OkadaI Dept of Molecular Biology, Nagoya City University School of Medicine 1, Dept of Microbiology, Fukuoka University School of Nedicinez, The Third Dept of Medcine, Nagoya University School of Medcine3
Kathryn M. Taylor, Alex Davies, B. Paul Morgan,*J. Paul Luzio, and Anthony K. Campbell. Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K. *Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QR, U.K.
Complement action on homologous cell membranes is r e s t r i c t e d by membrane inhibitors such as OAF, cR-I, MCP, IIRF and ItRF20(CD59). These complement regulatory proteins have been isolated and characterized for humans, but much less is known about their rodent counterparts and correspond -ing encoded genes. In order to understand rat complement regulatory proteins, we attempted to obtain a monoclonal antibody which was capable of rendering rat erythrocytes sensitive to homologous complement. Using neuraminidasetreated rat erythrocytes(Neu RE) as an immunogen, we established a monoclonal antibody, mAbSl2, which allows for haemolysis of Neu-RE by rat serum. We assumed that mAbS12, an IgGl isotype, blocks a complement regulatory protein, mAbSl2 detected a rat erythrocyte membrane protein with a molecular mass of 65Kda and 55Kda by western blotting analysis. 512Ag was expressed on PBMC, endothelial cells, mesangial c e l l s and several established cell lines. Furthermore mAbS12 induced C3b deposition on rat myeloma cells following treatment with rat serum. These findings show that 512Ag has important complement regulatory a c t i v i t i e s in rats, and i t s use should f a c i l i t a t e the further characterization of this membrane protein, especally with respect to physiological function.
Evidence for a Shared Binding Site in Human Complement Component C3 for Factor B and for Complement Receptor Type 3 (CR3, CDIIb/CDI8) Aiko Taniguchi-Sidleand David E. Isenman Dept.of Biochemistry,University of Toronto, Toronto, Canada M5S 1A8 Based on previous work, factor B binding to C3b was expected to involve two points of contact, one each for Ba and Bb. Studies using synthetic peptides (Fishelson, Mol. Immunol. (1991) 28:545) and the "C3o" proteolytic fragment of C3 (O'Keefe et al., JBC (1988) 263:12690) identified short sequences in the ct'-chain (752-761 and 955-964, pro-C3 numbering) which might mediate factor B binding. To further examine the role of these proposed sequencesin the context of intact C3b, we have altered their charged residues by site-directed mutagenesis. Resultant mutant recombinant C3 proteins were deposited onto sheep erythrocytes, and the ability of the cello associated C3b molecules to bind factor B was assessed by measuring the susceptibility of bound B to cleavage by D. Recombinant C3b in which airs of neg,,~ively charged amino acids near the N-terminus of ct'-chain, 52DE and~58EE, were replaced by NQ and QQ respectively,had much less ability to support activation of B by D. Mutagenesis of each of K959, E960 and D961 individuallyto alanine caused little impairmentof factor B binding, andeven the 959AAA triple mutation showed only moderate impairment. Although an RGD triplet in C3 had been implicated in the binding of iC3b to CR3, we have previously shown that its replacement by AAA has no effect on this interaction ~ (1992) 267:635). Because the "L domain" of CD1 lb has sequence similarity to a region in Bb, it seemed possible that CR3 and factor B may recognize the same or overlapping sites in C3. To lest this hypothesis, cell-bound recombinant C3b molecules were converted to iC3b and these iC3b-coated cells were assayed for their ability to form CR3-dependentrosettes. Mutagenesis of 752DE and 758EE greatly reduced the iC3b-CR3 interaction, whereas binding of the 959AAA mutant iC3b was indistinguishablefrom that of wild-type. These studies thus demonstratethat a segment near the N-terminusof or'chain, encompassing the sequence 752DEDIIAEE, first identified in the Fishelson peptide study, contains an important contact site for the binding of both factor B by C3b, and iC3b by CR3. The 959KED triplet of the C3ospecific site makes at best a minor contribution to the C3b-B interaction and has no role in binding of iC3b to CR3. (Supported by MRC Canada )
~
Aggregation and polymerisation of C9 plays a key role in the permeability changes induced by the MAC. The aim of the work described here was to use protein engineering to manipulate glycosylation and disulphide-bond induced polymerisation. Their role in the permeability thresholds induced by C9 within the MAC, 'ogcther with its removal from the membrane, was then investigated. Unglycosylated C9 was generated in vitro by P e R coupled with transcription and translation. C9 without N-linked glycosyl residues, and fully glycosylated C9 missing the first 23 amino acids, a putative calcium binding site, and two cysteines from a cysteine-rich region were generated using the baculovirus expression system. Fully active g|ycosylated recombinant C9 was produced by sub-cloning P e R products into a plasmid (pVL941) colrtainin.g compatible regions to a linear baculovirus expression vector with a lethal deletion (Baculogold) enabling co-transfection into Spodoptera frugiperda (Sf9) insect cells. Increased secretion of recombinant C9 was obtained by replacing the native signal peptide with the honeybee melittin signal peptide and by use of Trichoplusia ni 5BI-4 (High Five) cells instead of Sf9 insect cells. Unglycosylatcd recombinant C9 polymerised more readily in solution than the glycosylatcd C9 secreted by insect ceils. Several of the mutations affected polymerisation and biological activity.
HETEROPOLYMER MEDIATED BINDING OF TARGET ANTIGENS TO PRIMATE ERYTHROCYTES (E) V I A C R 1 . IN V I T R O A N A L Y S E S R.P. Taylor a, C.J. Reist a, H-Y. Liangb, V. Muzykantov °, M. Zachary a, W.M. Scheld a, J. Powers a, B. Bustera aUniversity of Virginia, Charlottesville, VA, USA; bChina-Japan Friendship Hospital, Peking, China; Clnstitute of Experimental Cardiology, Moscow, Russia We have used the streptavidin/biotin system to construct soluble, bispecific heteropolymers made up of monoclonal antibodies to primate E CR1 cross-linked with monoclonal antibodies to a target antigen. These heteropolymers facilitate rapid (ca. 2 min at 37°C), specific and high avidity binding to human or monkey E of soluble and particulate antigens, including human IgM, IgE, LDL, herpes simplex virus capsid, and the bacterium Haemophilus influenzae. Specific heteropolymer mediated binding of these antigens to primate E occurs in the complete absence of complement, and quite similar levels of antigen binding are observed in the presence of complement as well. No antigen binding is demonstrable in control studies with sheep E (which lack CR1). FACS analyses and magnetic separation assays indicate the majority of the primate E contain bound antigen or heteropolymer. At saturation the absolute number of heteropolymers or protein antigens bound per E (1002000) is consistent with the number of CR1 per E, the mw of the antigens, and amplification by the streptavidin/biotin system. These heteropolymer constructs, prepared using streptavidin/biotin or other cross-linking methodologies, may find therapeutic use by immobilizing pathogens on primate E. The E-bound pathogens may then be neutralized and/or cleared from the circulation through a mechanism similar to that which occurs during Emediated processing or clearance of complement-opsonized particles and immune complexes.