Identification of Residues within the 727–767 Segment of Human Complement Component C3 Important for Its Interaction with Factor H and with Complement Receptor 1 (CR1, CD35)

Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727–767 segment at the NH2 terminus of C3b α′-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738–767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545–552) which implicated residues within the 744–754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of α-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727–767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O). Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727–767 segment at the NH2 terminus of C3b α′-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738–767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545–552) which implicated residues within the 744–754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of α-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727–767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O). Stringent regulation of the alternative complement pathway C3 convertase is essential for the prevention of C3 hypercatabolism and for the protection of host tissue from the deleterious effects of alternative pathway propagation. is important to alternative pathway that as a C3b molecules that have to host this regulation the soluble factor H and factor as as the factor membrane cofactor protein cofactor with and the complement of complement cofactor with and the complement of complement and complement in The in and the of the factor the molecules are of a of of a to as a complement protein this is found in the the complement molecules are on the of at the for of complement of and have that form of two of Mol. Mol. that the of which are of are for interaction with that to C3b effect two as and factor the binding of H, and CR1 to the alternative pathway C3 to an of of the from the C3b of the I cofactor to the of the protein in the cleavage of C3b terminus to residues and C3 the and a the with factor B, this the C3 with to a for alternative pathway C3 convertase this factor cleavage only the C3b is with of the soluble protein factor H and the host CR1 and there is in the that binding of H to C3b a in the Immunol. the for factor there is that factor I to H and the interaction to cleavage factor I to CR1 MCP is as only CR1 as an I cofactor for the cleavage of to residue and Immunol. and have in within the complement that are required for and I cofactor Immunol. Immunol. Immunol. are required for functional in the case of factor H, are required for the of its I cofactor Immunol. H to and although not contribute to the I cofactor of the The two of CR1 are and and are of I cofactor study on CR1 that the first in case for of the functional and residues within important have identified to the in the complement molecules the an for functional the of any within C3 the have a of approaches to within C3 that with its and soluble protein The approaches have functional blocking antibodies with the of the to which and the of and synthetic peptides from C3 as functional of the Immunol. Z. Mol. Immunol. Additionally, a with a of C3b interact with the for the of a C3 segment in a binding interaction Immunol. and Z. Immunol. have in this to and the binding of C3 molecules in which a segment of C3 with the segment of the in to a binding mutagenesis to the of binding to important and in to the of the side chains that are required for a binding Immunol. Mol. Immunol. of the approaches have implicated the NH2-terminal segment of C3 α′-chain as contributing at to the interaction of C3b with factor B, factor H, and CR1. an this segment a in C3b and the interaction with factor B, factor H, and CR1 the 727–767 peptide with factor H and CR1 for binding to C3b and in study but not the of the interaction between C3b and factor Finally, a in which the 727–767 segment the ability to interact with all Z. Immunol. Z. Mol. Immunol. had a of and peptides the 727–767 for ability to factor B and factor H. The that the to factor B the segment to the binding of factor H. mutagenesis on C3 the of the side chains within the segment and identified of residues and as important not only for the interaction of C3b with factor B but for the ability of C3b and to to CR1 and on Immunol. with the of the mutations not the interaction with factor H. the have extended previous mutagenesis study on C3 to cover the remainder of the charged residues in the with the of residues for the interaction with factor H. factor H, CR1 and MCP as for the factor cleavages of the of mutants for ability to interact with soluble of CR1 (sCR1) and MCP We have identified Glu-744 and Glu-747 within the segment as residues side chains contribute to the interaction with factor H. We have found that the charged residue contacts with CR1, although with factor H, extend over a portion of the none of the mutations had any effect on the interaction with in the in this study have identified residues within the segment at the NH2 terminus of C3b α′-chain that contribute to the binding interaction with factor H and CR1. to any in the of MCP with any of mutant to a of of with two antibodies and the mutant molecules be to be with to the of which the to which the C3 convertase cleavage from to of is important to that there between the of the and not an I cofactor binding of cofactor binding of mutant C3 molecules in this to on of in the cofactor-dependent factor I cleavage assay using the with to I cofactor for the C3(H2O) to but cleavage I of to and a with to I cofactor for the C3(H2O) to but cleavage I of to and a with to I cofactor for the C3(H2O) to but cleavage I of to and a to on of in the cofactor-dependent factor I cleavage assay using the with to I cofactor for the C3(H2O) to but cleavage I of to and a in a the factor H and CR1 of interaction within the 727–767 segment overlap, the charged residue contacts required CR1 extend over a wider portion of the segment than is the case for factor H. the negatively charged side chains of Glu-744 and Glu-747 appear to be important for the interaction with factor H, for CR1 there of charged Glu-747, and Glu-754/Asp-755 that had effects on the cleavage of C3(H2O) to of the a contacts with Glu-744 and the Lys-757/Glu-758 to be required for CR1 to as a cofactor for the third factor is of previous using factor H as the I cofactor for the third H is not a cofactor for this cleavage at at Immunol. a for contacts in to the of to the factor I to the third cleavage of C3 α-chain. The that H and I interact with be with the that residues Glu-744 and Glu-747 are for the binding of factor H to C3(H2O) is with the of the Z. Mol. Immunol. study which that C3 residues 744–754 contribute to the binding of factor H. The only mutant in which the interaction with H to be the mutant the of is the as that with that the of the and Glu-736/Glu-737 charged on to the binding of factor H to be in assay that there is the of over a of to have had an effect on the segment which now two approaches to a to H that all of the mutant for which the I cofactor assay a interaction with factor H CR1 had at none of the of the mutations any The only the mutant which to only the third factor I cleavage with CR1 as the is that is not at all in the interaction with CR1 that at only to the cofactor binding required to the third factor I The dominance of negatively charged residues as contacts on the C3 side of the interactions with CR1 and factor H, with the of interactions suggests that to charged residues on the cofactor side of the interaction form an essential of the binding interface. with to the and within CR1, that all loss-of-function mutations from the of a the of a These are in with the that the is between charged residues of CR1 and negatively charged residues C3b and mutagenesis of factor H have to be of is that there is a to the interactions of C3b with CR1 and factor H within the peptide segment the study Z. Mol. Immunol. to a binding for factor H, there is a of of which contribute a to the binding for factor H and Z. Immunol. have the of the NH2-terminal α′-chain segment of C3 to the interactions with factor H, factor B, and all of of the segment with the from and C3 and from the factor in molecules in to to the Immunol. although not that the NH2-terminal α′-chain segment on its the The in which the 727–767 segment the ability to interact with H, B, and sCR1 but not Z. Immunol. the the of loss-of-function from a a there is general agreement between the from the study and from with to the with the mutants as the only deleterious effects on the third factor cleavage with sCR1 as the all the molecules a of a of the peptide from the with a of mutants in study to the interaction with H and is in the of effect of the interaction with factor H of the residues Glu-744 and Glu-747 are in the case of the and of the two acidic residues is its in a charged residue in and on have to the of cleavage in the assay. this not is that the assay and Z. Immunol. not have of We have cleavages in the case of the and that not to the third factor I cleavage of the of at residue in the and the of Glu-754/Asp-755 a in the in the case of the the of an at residues and for the of at residues and is not that of the Glu-736/Glu-737 in the molecules effect in the of C3(H2O) to in case a of two side chains and side in is a and have and with to factor B binding have that the be the effect Immunol. is now a of from synthetic blocking and protein approaches that the acidic segment at the terminus of C3 α′-chain a for factor H, CR1, and factor B and the The protein implicated this in contributing a binding for Immunol. there is that this is not the only binding in the C3 for as as binding and with and synthetic peptides all the as contributing to binding the functional homology the I with the that CR1, factor H, and MCP are of C3b that a at binding in The of and now as a for a mutagenic that the interactions for the protein of Stringent regulation of the alternative complement pathway C3 convertase is essential for the prevention of C3 hypercatabolism and for the protection of host tissue from the deleterious effects of alternative pathway propagation. is important to alternative pathway that as a C3b molecules that have to host this regulation the soluble factor H and factor as as the factor membrane cofactor protein cofactor with and the complement of complement cofactor with and the complement of complement and complement in The in and the of the factor the molecules are of a of of a to as a complement protein this is found in the the complement molecules are on the of at the for of complement of and have that form of two of Mol. Mol. that the of which are of are for interaction with membrane cofactor protein complement protein complement factor with and the complement assay C3 of complement soluble CR1 soluble MCP membrane cofactor protein complement protein complement factor with and the complement assay C3 of complement soluble CR1 soluble MCP that to C3b effect two as and factor the binding of H, and CR1 to the alternative pathway C3 to an of of the from the C3b of the I cofactor to the of the protein in the cleavage of C3b terminus to residues and C3 the and a the with factor B, this the C3 with to a for alternative pathway C3 convertase this factor cleavage only the C3b is with of the soluble protein factor H and the host CR1 and there is in the that binding of H to C3b a in the Immunol. the for factor there is that factor I to H and the interaction to cleavage factor I to CR1 MCP is as only CR1 as an I cofactor for the cleavage of to residue and Immunol. and have in within the complement that are required for and I cofactor Immunol. Immunol. Immunol. are required for functional in the case of factor H, are required for the of its I cofactor Immunol. H to and although not contribute to the I cofactor of the The two of CR1 are and and are of I cofactor study on CR1 that the first in case for of the functional and residues within important have identified to the in the complement molecules the an for functional the of any within C3 the have a of approaches to within C3 that with its and soluble protein The approaches have functional blocking antibodies with the of the to which and the of and synthetic peptides from C3 as functional of the Immunol. Z. Mol. Immunol. Additionally, a with a of C3b interact with the for the of a C3 segment in a binding interaction Immunol. and Z. Immunol. have in this to and the binding of C3 molecules in which a segment of C3 with the segment of the in to a binding mutagenesis to the of binding to important and in to the of the side chains that are required for a binding Immunol. Mol. Immunol. of the approaches have implicated the NH2-terminal segment of C3 α′-chain as contributing at to the interaction of C3b with factor B, factor H, and CR1. an this segment a in C3b and the interaction with factor B, factor H, and CR1 the 727–767 peptide with factor H and CR1 for binding to C3b and in study but not the of the interaction between C3b and factor Finally, a in which the 727–767 segment the ability to interact with all Z. Immunol. Z. Mol. Immunol. had a of and peptides the 727–767 for ability to factor B and factor H. The that the to factor B the segment to the binding of factor H. mutagenesis on C3 the of the side chains within the segment and identified of residues and as important not only for the interaction of C3b with factor B but for the ability of C3b and to to CR1 and on Immunol. with the of the mutations not the interaction with factor H. the have extended previous mutagenesis study on C3 to cover the remainder of the charged residues in the with the of residues for the interaction with factor H. factor H, CR1 and MCP as for the factor cleavages of the of mutants for ability to interact with soluble of CR1 (sCR1) and MCP We have identified Glu-744 and Glu-747 within the segment as residues side chains contribute to the interaction with factor H. We have found that the charged residue contacts with CR1, although with factor H, extend over a portion of the none of the mutations had any effect on the interaction with in the in this study have identified residues within the segment at the NH2 terminus of C3b α′-chain that contribute to the binding interaction with factor H and CR1. to any in the of MCP with any of mutant to a of of with two antibodies and the mutant molecules be to be with to the of which the to which the C3 convertase cleavage from to of is important to that there between the of the and not an I cofactor binding of cofactor binding of mutant C3 molecules in this to on of in the cofactor-dependent factor I cleavage assay using the with to I cofactor for the C3(H2O) to but cleavage I of to and a with to I cofactor for the C3(H2O) to but cleavage I of to and a with to I cofactor for the C3(H2O) to but cleavage I of to and a to on of in the cofactor-dependent factor I cleavage assay using the with to I cofactor for the C3(H2O) to but cleavage I of to and a in a the factor H and CR1 of interaction within the 727–767 segment overlap, the charged residue contacts required CR1 extend over a wider portion of the segment than is the case for factor H. the negatively charged side chains of Glu-744 and Glu-747 appear to be important for the interaction with factor H, for CR1 there of charged Glu-747, and Glu-754/Asp-755 that had effects on the cleavage of C3(H2O) to of the a contacts with Glu-744 and the Lys-757/Glu-758 to be required for CR1 to as a cofactor for the third factor is of previous using factor H as the I cofactor for the third H is not a cofactor for this cleavage at at Immunol. a for contacts in to the of to the factor I to the third cleavage of C3 α-chain. The that H and I interact with be with the that residues Glu-744 and Glu-747 are for the binding of factor H to C3(H2O) is with the of the Z. Mol. Immunol. study which that C3 residues 744–754 contribute to the binding of factor H. The only mutant in which the interaction with H to be the mutant the of is the as that with that the of the and Glu-736/Glu-737 charged on to the binding of factor H to be in assay that there is the of over a of to have had an effect on the segment which now two approaches to a to H that all of the mutant for which the I cofactor assay a interaction with factor H CR1 had at none of the of the mutations any The only the mutant which to only the third factor I cleavage with CR1 as the is that is not at all in the interaction with CR1 that at only to the cofactor binding required to the third factor I The dominance of negatively charged residues as contacts on the C3 side of the interactions with CR1 and factor H, with the of interactions suggests that to charged residues on the cofactor side of the interaction form an essential of the binding interface. with to the and within CR1, that all loss-of-function mutations from the of a the of a These are in with the that the is between charged residues of CR1 and negatively charged residues C3b and mutagenesis of factor H have to be of is that there is a to the interactions of C3b with CR1 and factor H within the peptide segment the study Z. Mol. Immunol. to a binding for factor H, there is a of of which contribute a to the binding for factor H and Z. Immunol. have the of the NH2-terminal α′-chain segment of C3 to the interactions with factor H, factor B, and all of of the segment with the from and C3 and from the factor in molecules in to to the Immunol. although not that the NH2-terminal α′-chain segment on its the The in which the 727–767 segment the ability to interact with H, B, and sCR1 but not Z. Immunol. the the of loss-of-function from a a there is general agreement between the from the study and from with to the with the mutants as the only deleterious effects on the third factor cleavage with sCR1 as the all the molecules a of a of the peptide from the with a of mutants in study to the interaction with H and is in the of effect of the interaction with factor H of the residues Glu-744 and Glu-747 are in the case of the and of the two acidic residues is its in a charged residue in and on have to the of cleavage in the assay. this not is that the assay and Z. Immunol. not have of We have cleavages in the case of the and that not to the third factor I cleavage of the of at residue in the and the of Glu-754/Asp-755 a in the in the case of the the of an at residues and for the of at residues and is not that of the Glu-736/Glu-737 in the molecules effect in the of C3(H2O) to in case a of two side chains and side in is a and have and with to factor B binding have that the be the effect Immunol. is now a of from synthetic blocking and protein approaches that the acidic segment at the terminus of C3 α′-chain a for factor H, CR1, and factor B and the The protein implicated this in contributing a binding for Immunol. there is that this is not the only binding in the C3 for as as binding and with and synthetic peptides all the as contributing to binding the functional homology the I with the that CR1, factor H, and MCP are of C3b that a at binding in The of and now as a for a mutagenic that the interactions for the protein of in the in this study have identified residues within the segment at the NH2 terminus of C3b α′-chain that contribute to the binding interaction with factor H and CR1. to any in the of MCP with any of mutant to a of of with two antibodies and the mutant molecules be to be with to the of which the to which the C3 convertase cleavage from to of is important to that there between the of the and not an I cofactor binding The the factor H and CR1 of interaction within the 727–767 segment overlap, the charged residue contacts required CR1 extend over a wider portion of the segment than is the case for factor H. the negatively charged side chains of Glu-744 and Glu-747 appear to be important for the interaction with factor H, for CR1 there of charged Glu-747, and Glu-754/Asp-755 that had effects on the cleavage of C3(H2O) to of the a contacts with Glu-744 and the Lys-757/Glu-758 to be required for CR1 to as a cofactor for the third factor is of previous using factor H as the I cofactor for the third H is not a cofactor for this cleavage at at Immunol. a for contacts in to the of to the factor I to the third cleavage of C3 α-chain. The that H and I interact with be with the The that residues Glu-744 and Glu-747 are for the binding of factor H to C3(H2O) is with the of the Z. Mol. Immunol. study which that C3 residues 744–754 contribute to the binding of factor H. The only mutant in which the interaction with H to be the mutant the of is the as that with that the of the and Glu-736/Glu-737 charged on to the binding of factor H to be in assay that there is the of over a of to have had an effect on the segment which now two approaches to a to H binding. We that all of the mutant for which the I cofactor assay a interaction with factor H CR1 had at none of the of the mutations any The only the mutant which to only the third factor I cleavage with CR1 as the is that is not at all in the interaction with CR1 that at only to the cofactor binding required to the third factor I The dominance of negatively charged residues as contacts on the C3 side of the interactions with CR1 and factor H, with the of interactions suggests that to charged residues on the cofactor side of the interaction form an essential of the binding interface. with to the and within CR1, that all loss-of-function mutations from the of a the of a These are in with the that the is between charged residues of CR1 and negatively charged residues C3b and mutagenesis of factor H have to be of is that there is a to the interactions of C3b with CR1 and factor H within the peptide segment the study Z. Mol. Immunol. to a binding for factor H, there is a of of which contribute a to the binding for factor H and CR1. Z. Immunol. have the of the NH2-terminal α′-chain segment of C3 to the interactions with factor H, factor B, and all of of the segment with the from and C3 and from the factor in molecules in to to the Immunol. although not that the NH2-terminal α′-chain segment on its the The in which the 727–767 segment the ability to interact with H, B, and sCR1 but not Z. Immunol. the the of loss-of-function from a a there is general agreement between the from the study and from with to the with the mutants as the only deleterious effects on the third factor cleavage with sCR1 as the all the molecules a of a of the peptide from the with a of mutants in study to the interaction with H and is in the of effect of the interaction with factor H of the residues Glu-744 and Glu-747 are in the case of the and of the two acidic residues is its in a charged residue in and on have to the of cleavage in the assay. this not is that the assay and Z. Immunol. not have of We have cleavages in the case of the and that not to the third factor I cleavage of the of at residue in the and the of Glu-754/Asp-755 a in the in the case of the the of an at residues and for the of at residues and is not that of the Glu-736/Glu-737 in the molecules effect in the of C3(H2O) to in case a of two side chains and side in is a and have and with to factor B binding have that the be the effect Immunol. is now a of from synthetic blocking and protein approaches that the acidic segment at the terminus of C3 α′-chain a for factor H, CR1, and factor B and the The protein implicated this in contributing a binding for Immunol. there is that this is not the only binding in the C3 for as as binding and with and synthetic peptides all the as contributing to binding the functional homology the I with the that CR1, factor H, and MCP are of C3b that a at binding in The of and now as a for a mutagenic that the interactions for the protein of We are to of and of for with and