Defining the CD59-C9 Binding Interaction

Abstract

CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (α chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8α was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8α and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies. CD59 is a membrane glycoprotein that regulates formation of the cytolytic membrane attack complex (MAC or C5b-9) on host cell membranes. It functions by binding to C8 (α chain) and C9 after their structural rearrangement during MAC assembly. Previous studies indicated that the CD59 binding site in C9 was located within a 25-residue disulfide-bonded loop, and in C8α was located within a 51-residue sequence that overlaps the CD59 binding region of C9. By peptide screens and the use of peptides in binding assays, functional assays, and computer modeling and docking studies, we have identified a 6-residue sequence of human C9, spanning residues 365-371, as the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. The data also indicate that both C8α and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59-peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational and modeling studies. Complement activation leads ultimately to the generation and membrane insertion of a cytolytic protein assembly termed the membrane attack complex (MAC). 2The abbreviations used are: MAC, membrane attack complex; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin. The MAC is formed by the self-assembly of complement proteins C5b, C6, C7, C8, and from 1 to 18 molecules of C9. Host cells are protected from MAC-mediated lysis by CD59, an 18-21-kDa glycophosphatidylinositol-linked membrane glycoprotein. The MAC appears to play an important role in causing tissue injury following inappropriate complement activation in various ischemic and inflammatory conditions (reviewed in Refs. 1Arumugam T.V. Shiels I.A. Woodruff T.M. Granger D.N. Taylor S.M. Shock. 2004; 21: 401-409Crossref PubMed Scopus (268) Google Scholar, 2Morgan B.P. Harris C.L. Mol. Immunol. 2003; 40: 159-170Crossref PubMed Scopus (150) Google Scholar, 3Sahu A. Lambris J.D. Immunopharmacology. 2000; 49: 133-148Crossref PubMed Scopus (143) Google Scholar, 4Quigg R.J. Trends Mol. Med. 2002; 8: 430-436Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 5Lambris J.D. Holers V.M. Therapeutic Interventions in the Complement System. Humana Press, Totowa, NJ2000Crossref Google Scholar), and soluble forms of CD59 have been shown to be therapeutic in rodent models of disease (6Fraser D.A. Harris C.L. Williams A.S. Mizuno M. Gallagher S. Smith R.A. Morgan B.P. J. Biol. Chem. 2003; 278: 48921-48927Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7He C. Imai M. Song H. Quigg R.J. Tomlinson S. J. Immunol. 2005; 174: 5750-5757Crossref PubMed Scopus (63) Google Scholar). Furthermore, CD59 is sometimes overexpressed on tumor cells, and its expression has been linked to promoting tumor growth and the protection of tumor cells from mAb therapy (8Fishelson Z. Donin N. Zell S. Schultz S. Kirschfink M. Mol. Immunol. 2003; 40: 109-123Crossref PubMed Scopus (329) Google Scholar, 9Fonsatti E. Altomonte M. Coral S. De Nardo C. Lamaj E. Sigalotti L. Natali P.G. Maio M. Clin. Ter. 2000; 151: 187-193PubMed Google Scholar, 10Gelderman K.A. Tomlinson S. Ross G.D. Gorter A. Trends Immunol. 2004; 25: 158-164Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). It is therefore of interest to understand the molecular interaction between CD59 and its complement ligands for the goal of engineering effective soluble CD59-based therapeutics for treating inflammatory conditions, or for designing CD59 inhibitory molecules to enhance tumor cell susceptibility to complement. CD59 functions by binding to the α-subunit of C8 in the C5b-8 complex and preventing subsequent binding of C9, and/or by binding to C9 in the C5b-9 complex and preventing recruitment of additional C9 molecules (11Ninomiya H. Sims P.J. J. Biol. Chem. 1992; 267: 13675-13680Abstract Full Text PDF PubMed Google Scholar, 12Rollins S.A. Zhao J.I. Ninomiya H. Sims P.J. J. Immunol. 1991; 146: 2345-2351PubMed Google Scholar, 13Meri S. Morgan B.P. Davies A. Daniels R.H. Olavesen M.G. Waldemann H. Lachmann P.J. Immunol. 1990; 72: 1-9Google Scholar). CD59 can only bind to C8α and C9 in the nascent complex after conformation rearrangements of the two proteins that occur during MAC assembly. The activity of CD59 is species selective, and previous characterization of chimeric human/rabbit C8α- and C9-identified regions within the primary sequence of these proteins that interact with human CD59 (14Lockert D.H. Kaufman K.M. Chang C.-P. Huesler T. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 19723-19728Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 15Huesler T. Lockert D.H. Kaufman K.M. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 3483-3486Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). There is considerable sequence homology between C8α and C9, and the identified CD59 binding sequence in the two proteins aligned to a region just C-terminal to the proposed membrane binding domain that encompassed residues 320-415 in human C8α, and 334-415 in human C9. Peptides derived from within these sequences mapped the primary CD59 binding site to residues 359-384 in C9, a sequence contained between a Cys359-Cys405 disulfide loop in the native protein (16Huesler T. Lockert D.H. Sims P.J. Biochemistry. 1996; 35: 3263-3269Crossref PubMed Scopus (12) Google Scholar), and to residues 364-415 in C8α. In contrast to the C9 sequence, formation of the corresponding interchain disulfide between Cys375-Cys384 in C8α is required for CD59 binding (14Lockert D.H. Kaufman K.M. Chang C.-P. Huesler T. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 19723-19728Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In addition to these studies, earlier investigations in our laboratory using chimeric human/horse C9 molecules and a peptide derived from the putative hinge region of human C9-identified residues 222-271 as involved in CD59 binding (17Tomlinson S. Whitlow M.B. Nussenzweig V. J. Immunol. 1994; 152: 1927-1934PubMed Google Scholar). To better define the region of C9 that interacts with CD59 and to identify CD59-inhibitory peptides, peptides spanning the above identified regions of C9 were prepared and screened for CD59-binding and functional activity. A peptide phage display technique was also employed. A short consensus sequence was identified and allowed modeling of peptide-CD59 docking. Peptides—Linear peptides were synthesized by GenScript (Piscataway, NJ). Cyclized peptide, cp1, corresponding to sequence 359-384 of human C9 was synthesized by AnaSpec Inc. (San Jose, CA). The cp1 peptide was cyclized by a disulfide bridge formed between the two cysteines at each end of the peptide. Production of Recombinant Soluble CD59—Recombinant soluble CD59 lacking N-linked glycosylation because of a N18Q mutation was used in all binding and functional assays. N-linked glycosylation is not required for CD59 activity and the N18Q CD59 mutant protein has been previously characterized (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar). Removal of this glycosylation site facilitated Pichia expression and protein purification. The coding sequence of human CD59/N18Q was generated from a previously constructed vector (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar) by PCR and cloned into the Pichia pastoris expression vector pPICZαA (Invitrogen). The expression plasmid was transformed into P. pastoris-competent SMD1168H via electroporation, and transformed cells were plated on YPD plates with different concentrations of selecting antibiotic Zeocin (Invitrogen). After determination of the phenotype, methanol utilization plus (Mut+), or methanol utilization slow (Muts) pre-selected clones were subjected to a small scale expression test to select a high expressing clone. The level of soluble CD59 expression was determined by standard Western blot or by Sandwich ELISA using monoclonal and polyclonal antibodies to human CD59. Soluble CD59 was expressed via fermentation in a 5 liter BioFlo 110 Fermentor (New Brunswick Scientific, Edison, NJ) according to a modified protocol based on the Pichia Fermentation Process Guidelines of Invitrogen. After 1-day growth on basal medium, a 12-h glycerol feeding batch, and a 3-4-day methanol feeding batch, fermented culture was harvested and centrifuged. The supernatant was filtered with a 0.22-μm AcroPak 200 filtration filter (Pall Europe Ltd., Portsmouth, UK), concentrated and dialyzed with 50 mm Tris-HCl, pH 8.0 with a Prep/ScaleTM-TFF 1ft2 cartridge (Millipore Corporation, Bradford, MA). Soluble CD59 in the concentrated supernatant was then purified by anti-CD59 YTH53.1 antibody affinity chromatography and subsequent Mono Q 5/5 ion exchange FPLC (Amersham Biosciences). Purified protein was checked for purity and folding by 18% SDS-PAGE and Western blotting using a panel of anti-CD59 monoclonal antibodies that recognize conformational epitopes described previously (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of CD59 was using a CD59 at 1 in was with at a of at for with was on a CD59 binding assays, CD59 binding to C9, C8, and peptide, cp1, was The proteins or peptide were with ELISA pH to a of and in a for The were then on ELISA plates at at 1 as were then with in at and the plates with CD59 of was then and following were with A at was then and plates were at for 1 with with CD59 binding was by using was at and data using corresponding In binding assays, the of various peptides to the binding of CD59 to C8, C9, or cp1 was In these assays, different concentrations of peptide were with CD59 addition to protein or peptide. FPLC and of CD59 1 was with peptide in a of were in 50 mm Tris-HCl, pH 8.0 at for were a filtration to an FPLC (Amersham for were and to was prepared in with at a of of were with of in a and 1 of the to a on the was used to data in from from the was used for of the to data were Complement cell human CD59 was used in these (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). cells or expressing at were with 5 mm in PBS, and in modified (Invitrogen). at in modified were then at for with or peptides at indicated cells were with membrane serum (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) and human serum was to a cell of 1 The cells were at for and cell determined by and cells were cells as for CD59 complement inhibitory was by with shown in standard phage peptide display and (New were used to for human CD59 binding of were according to the Peptides were synthesized based on derived consensus sequences and for CD59 binding as described derived from the CD59 was used as the for docking (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The data for CD59 a of on the in Abagyan R.A. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) of the CD59 identified as the and by the conformation of was to a with the this conformation an previously identified as the putative C9 binding site (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). used both of these for our docking The conformation was the CD59 by (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) to as the conformation The CD59 conformation to as the conformation of was by selecting a of as A peptide the identified C9 consensus sequence that to and peptide peptide shown to bind were as in R.A. D.A. J. Chem. 1994; Scopus Google Scholar) and to both the and the of CD59, by the of residues within of the A was a of residues within of the previously in C9 binding from and that are also located in the of the binding (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar, Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Davies Morgan B.P. J. Exp. Med. 1997; 185: PubMed Scopus Google Scholar). and CD59 were with the to or to in to the of the docking of Peptides to studies identified two peptides that bind of residues within the putative region (17Tomlinson S. Whitlow M.B. Nussenzweig V. J. Immunol. 1994; 152: 1927-1934PubMed Google Scholar) and of residues 359-384 (16Huesler T. Lockert D.H. Sims P.J. Biochemistry. 1996; 35: 3263-3269Crossref PubMed Scopus (12) Google Scholar). In an to better define the CD59 binding sequence of C9, peptides spanning these regions were synthesized and characterized in CD59 binding assays. To for CD59 the of peptide to the binding of soluble CD59 to C9 was peptides of residues spanning the sequence and by were of the peptides with CD59 in binding not also data from a previous that the peptide a membrane that with the functional of C.-P. Huesler T. Zhao J. T. Sims P.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). A of peptides of residues each and spanning the sequence of C9 were the concentrations in this of the peptides the binding of to C9 to a similar as the and cyclized peptide, cp1 A peptide also to a of these peptides identified a 6-residue consensus sequence of of an additional peptide the consensus sequence was synthesized and To the peptide was synthesized to the and C-terminal residues on of the consensus The peptide, as cp1 and a peptide the consensus sequence, to the binding of CD59 to C9 in a A peptide of the as with sequence on CD59 binding to C9. Soluble CD59 also to peptide cp1 on and the of the interaction was by the of this binding by the addition of cp1, or to the that on a is an for at similar of peptides spanning 359-384 of C9 protein Peptides of CD59 binding to C9 are shown in sequences of are shown of C9 peptides on CD59 binding to C9. C9 was at in a was peptides to the CD59 was with peptide on of C9 peptides on CD59 binding to peptide cp1 was at in a was peptides to the CD59 was with that the peptide to CD59 in was by CD59 and peptide, by of the complex by of CD59 and at a two were by and were shown to be of and only by of C9 Peptides on of CD59 to MAC formation by binding to both C8α in C5b-8 and to C9 in the C5b-9 To C8α and C9 bind to the or an overlapping on CD59, the of the peptides to CD59 binding to C8 was The peptides cp1 and consensus sequence from cp1 that the binding of CD59 to C8 in a The peptide, on the effective cp1 at CD59 binding to of of the peptide interaction with CD59 was by the of the peptides to CD59 activity. the interaction between CD59 and the C5b-9 complex in the membrane cells expressing high of human CD59 were with antibody and in a of human The of peptides cp1, or during the in a in complement lysis of the cells peptide on It has been previously that hydrophobic peptides can in the of CD59. of the peptides at the lysis of cells that not human CD59 phage peptide display and and were using CD59, in the of CD59 binding The peptides a peptide of two of and and peptides with a consensus sequence was sequence consensus corresponding to sequence within C8α or C9, for the short in all of the of peptides that are effective CD59 peptides identified by phage display were synthesized and in CD59 binding and functional assays. of the peptides on CD59 activity and only the binding of CD59 to C9, to a peptide and to of a 6-residue consensus sequence within C9 that to CD59 allowed computer modeling of the CD59-peptide Previous studies CD59 putatively identified the binding site at the membrane in the of a hydrophobic (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar, Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Davies Morgan B.P. J. Exp. Med. 1997; 185: PubMed Scopus Google Scholar). In a computer modeling for small binding identified a small in to the binding site by the studies (19Huang Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the an in of the CD59 identified as a and by the conformation of was to a the previously identified by computer of CD59 with the binding and the binding are shown in A and and lacking a to conformation of CD59, we to use both of these in our docking The binding that have a high determined to be a for peptide binding are shown in The has a of and has and the The region of the putative C9 binding site determined by studies is shown in The the of this binding region as residues within and at a all the residues that binding docking were to for both docking using the peptide with docking to for both and in the docking the peptides within the identified binding with into the binding region identified by with the to docking with different not the that the not therefore the docking using the identified 6-residue consensus sequence within C9 that to CD59, with the to docking to the conformation of CD59, different of the to and the was with the conformation of CD59, the with to the with the and in to and CD59 using the conformation of in conformation of in in a from this indicate a sequence of human C9, spanning residues the primary CD59 recognition domain involved in CD59-mediated regulation of MAC formation. CD59 MAC formation by binding to C8α and C9 in the complex and and the data also indicate that both C8α and C9 bind to a similar or overlapping site on CD59. Furthermore, data from CD59 peptide docking models are consistent with the C9 binding site on CD59 located at a hydrophobic pocket, putatively identified previously by CD59 mutational studies (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar, Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Davies Morgan B.P. J. Exp. Med. 1997; 185: PubMed Scopus Google Scholar). of the identified peptide sequence that interacts with CD59 was shown peptide binding to CD59, in both and of CD59 binding to C9, and of CD59 The of the peptides to the binding of CD59 to C8 indicated that C8α and C9 interact with an overlapping on CD59. A previous indicated that the primary CD59 interaction in the C8α is contained between residues and (14Lockert D.H. Kaufman K.M. Chang C.-P. Huesler T. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 19723-19728Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). region of C8α with the region of C9 previously shown to be by CD59 (14Lockert D.H. Kaufman K.M. Chang C.-P. Huesler T. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 19723-19728Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and a 25-residue peptide derived from within this region of C9 was shown to bind CD59 (16Huesler T. Lockert D.H. Sims P.J. Biochemistry. 1996; 35: 3263-3269Crossref PubMed Scopus (12) Google Scholar). the aligned C8α and C9 sequences only sequence and and C8α and C9 bind to a similar CD59 site was we that C8α and C9 bind to an overlapping site on CD59. an of the sequence with the 320-415 CD59 binding sequence of C8α a sequence of high hydrophobic In these 6-residue C8α and C9 sequences to similar within the aligned sequences of C8α and C9. our data that the CD59 binding of C8α and C9 are is of that previous studies have shown that the CD59 binding site of C8α, not of C9, is disulfide formation (14Lockert D.H. Kaufman K.M. Chang C.-P. Huesler T. Sodetz J.M. Sims P.J. J. Biol. Chem. 1995; 270: 19723-19728Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, C.-P. Huesler T. Zhao J. T. Sims P.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google that the CD59 binding on the two proteins are the of and C9 that the region the identified 6-residue CD59 binding sequence to short sequence of homology It is that this to the species of CD59. The identified hydrophobic C9 sequence that interacts with CD59 is within the protein be consistent with the of of CD59 in that CD59 not bind C9, to C9 as a structural rearrangement binding to C5b-8 in the nascent with data the hydrophobic of the binding site on CD59 for C9, is therefore that binding a hydrophobic a by molecular modeling studies. The docking studies the native conformation of the C9 its for to with CD59. the studies that of the in the C9 peptide can interact with CD59. The of the to with the conformation of CD59 this conformation is for C9 the CD59 conformation with a can that the conformation of in CD59 in is to be similar to the the the data from the modeling studies to the by peptides interact with the on the human CD59 that has been in C9 binding by both CD59 (18Yu J. Abagyan R.A. Dong S. Gilbert A. Nussenzweig V. Tomlinson S. J. Exp. Med. 1997; 185: 745-753Crossref PubMed Scopus (56) Google Scholar, Y. Smith C.A. Song H. Morgan B.P. Abagyan R. Tomlinson S. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Davies Morgan B.P. J. Exp. Med. 1997; 185: PubMed Scopus Google Scholar) and C9 peptide interaction peptides by phage display that to human CD59 and identified consensus was sequence homology with C8α or C9, of the peptides with CD59 and only peptide C9 binding to CD59 only CD59 is a to Zhao J. Sims P.J. Mol. Biol. 2004; PubMed Scopus Google Scholar), cell M. J. P. V. A. J. Immunol. 1992; PubMed Scopus Google Scholar), A. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), and E. R. M. R. S. S. E. R. L. C. A. J. Immunol. 2003; PubMed Scopus Google Scholar). is the binding between CD59 and these is that phage peptides with or of these is also that of these ligands an overlapping binding site on CD59 with C8α and C9, and that peptides with the binding of these previously shown that a peptide derived from the C9 hinge region CD59 and complement lysis of human are protected by expressed (17Tomlinson S. Whitlow M.B. Nussenzweig V. J. Immunol. 1994; 152: 1927-1934PubMed Google Scholar). was that CD59 not bind to proteins the hinge sequence, and that the activity was by a membrane of the peptide C.-P. Huesler T. Zhao J. T. Sims P.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). in the we were to CD59 binding of peptides derived from within the hinge with the data on the C9 sequence, is that the hinge region of C9 is not involved in the inhibitory of CD59. CD59 is expressed on tumor cells, at and CD59 antibody and tumor cell the the MAC is to play a role in various inflammatory conditions, and MAC formation a therapeutic the molecular interaction between CD59 and its complement ligands has for the of therapeutic at CD59 activity or MAC formation. The of a 6-residue sequence of human C9 as the primary CD59 recognition domain the of and small therapeutics in the of the interaction also of CD59 to CD59-based and from the a for of with