refined mapping of daf's active site residues that provide for
TRANSCRIPT
STRUCTURE-BASED MAPPING OF DAF’s ACTIVE SITE RESIDUES
THAT DECAY ACCELERATE THE C3 CONVERTASES Lisa Kuttner-Kondo†, Dennis E. Hourcade§, Vernon E. Anderson‡, Nasima Muqim†, Lynne
Mitchell§, Dinesh C. Soares*, Paul N. Barlow*, and M. Edward Medof†1 †Institute of Pathology and ‡Department of Biochemistry, Case Western Reserve University,
Cleveland, OH 44106, §Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, and *Institute of Structural and Molecular Biology and School of Chemistry,
University of Edinburgh, Edinburgh EH9 3JJ, Scotland. Running Title: Refined mapping of DAF’s active site
1Address correspondence to: M. Edward Medof, M.D., Ph.D., Institute of Pathology, Case Western Reserve University School of Medicine, 2085 Adelbert Road, Room 301, Cleveland, OH 44106. Phone 1-216-368-5434; Fax 1-216-368-0495; e-mail [email protected] Supported by NIH grants R01 AI23598 (M. E. M.) and R01 AI05143 (D. E. H.)
Focused complement activation on foreign targets depends on regulatory proteins that decay the bimolecular C3 convertases. While this process is central to complement control, how the convertases engage and disassemble is not established. The 2nd and 3rd complement control protein (CCP) modules of the cell surface regulator, decay accelerating factor (DAF, CD55), comprise the simplest structure mediating this activity. Positioning the functional effects of 31 substitution mutants of DAF CCPs 2-4 on partial structures was previously reported. In light of the high-resolution crystal structure of DAF’s 4-CCP functional region, we now re-examine the effects of these and 40 additional mutations. Moreover, we map six mAb epitopes, and overlap their effects with those of the amino acid substitutions. The data indicate that DAF’s interaction with the convertases is mediated predominantly by two patches ~13 Å apart, one centered around R69 and R96 on CCP2 and the other around F148 and L171 on CCP3. These patches on the same face of the adjacent modules bracket an intermodular linker of critical length (16 Å). While the key DAF residues in these patches are present or conservative substitutions in all other C3 convertase regulators that mediate decay acceleration and/or provide factor I-cofactor activity, the linker region is highly conserved only in the former. Intra-CCP regions also differ. Linker region comparisons suggest that the active CCPs of the decay accelerators are extended while those of the cofactors are tilted. Intra-CCP comparisons suggest that
the two classes of regulators bind different regions on their respective ligands.
The C3 convertases of the classical and alternative pathways are the central enzymes of the complement cascade (1). These bimolecular complexes, C4b2a and C3bBb, produce anaphylatoxin C3a, locally amplify C3b deposition, and serve as sites for the assembly of the C5 convertases, C4b2a3b and C3bBb3b. These trimeric convertases, in turn, generate anaphylatoxin C5a and initiate the terminal pathway leading to formation of lytic C5b-9 membrane attack complexes (MAC). The relative rates of assembly and disassembly of the C3 convertases lie at the heart of complement regulation and their physiologic modulation ensures that complement acts in a proportionate and targeted fashion.
To focus complement activation on foreign targets, prevent nearby activation in the fluid phase, and simultaneously protect self tissues from complement-mediated injury, the C3 convertases are controlled by both serum- and cell-associated regulatory proteins (2,3). Because the convertases assemble on C4b and C3b fragments that condense indiscriminately with free hydroxyl and amino groups on foreign targets and these same acceptor groups are present on all biological membranes, their formation on self cells at the single enzyme level cannot be avoided. To prevent further propagation of the cascade, which would induce self-cell injury, self cells possess decay accelerating factor (DAF or CD55). By virtue of its glycosylphosphatidylinositol (GPI)-anchor which allows it to move rapidly in the plane of
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http://www.jbc.org/cgi/doi/10.1074/jbc.M611650200The latest version is at JBC Papers in Press. Published on March 29, 2007 as Manuscript M611650200
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
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the membrane, this regulator immediately decays the single C3 convertase enzymes wherever they form on self-cell surfaces (4).
DAF is ~70 kDa in size (4) . Its functional region consisting of four ~60 amino acid long repeating units, termed complement control protein modules (CCPs) (a.k.a. short consensus repeats (SCRs) or sushi domains), is suspended above the cell surface on a heavily O-glycosylated, S- and T-rich stalk(5-7). Studies with antibody-sensitized sheep erythrocytes (EshA) bearing defined complement components have shown that DAF’s decay accelerating activity (DAA) against the alternative pathway (AP) convertase, C3bBb, lies in its CCPs 2, 3, and 4, while its activity against the classical pathway (CP) enzyme, C4b2a, resides entirely within its CCPs 2-3 (8).
Although an NMR-derived solution structure of CCPs 2-3 (9) and a crystal structure of CCPs 3-4 (10) were reported earlier, an X-ray crystal structure of the full DAF CCPs 1-4 regulatory region has become available more recently (11). In the present study, we have employed this full-length structure to map the effects of the 31 previously reported principally-alanine substitution mutants and to guide the design of 40 additional mutants. The new mutations include four insertions/substitutions of residues in the linkers between CCPs 2-3, and CCPs 3-4 (12). In conjunction with this analysis, we localized the epitopes of six mAbs and considered, in light of the 3D structural information, the effects of their binding on DAF’s function. Since other regulators that mediate decay acceleration of the C3 convertases, i.e. C3b receptor (CR1 or CD35), factor H, and C4 binding protein (C4bp), are more complicated in that 1) their decay accelerating regions are larger (three or four CCP modules) and 2) they also mediate a second regulatory function, i.e. cofactor activity for factor I cleavage of C3b (factor H), C4b (C4bp) or both (CR1), the information derived from the simpler DAF CCP 2-3 structure, through sequence and structure comparisons between other decay accelerators and factor I cofactors, sheds light on differences between the two classes of regulators.
Experimental Procedures
Preparation of the cDNA encoding the native DAF CCPs 1-4 and substitution mutants - The DAF cDNA derivative HuDAF-6H, the native
DAF parental control, encoding CCP1 through CCP4 with a C-terminal hexahistidine (6H) tag was prepared as described in (12) using human DAF 13:2 cDNA in pBT-KS+ (13). For generation of the substitution mutants, the QuikChange (II) site-directed mutagenesis kit (Stratagene) was used in conjunction with HuDAF-6H. Sense primers are listed in Table I. All mutations were confirmed by sequencing. Additionally, CCP1 through CCP4 was sequenced in full for all of the following mutants that adversely affected function: R69C, R69W, R69WL70P, R96L, P97G, G98S, R100C, K126C, K126E, K127GS128 (glycine inserted between K127 and S128), T151A, F169C, D181A, R187GE188 (glycine inserted between R187 and E188), E205A, and D207A. Recombinant DAF protein expression by transient transfection - Samples of 7.5 to 15 µg of cDNA, preincubated for 20-30 min at 20 °C with 54 µl of lipofectamine (Invitrogen, Carlsbad, CA) in 1.8 ml of OptiMEM I (Gibco), were mixed with 7.2 ml of OptiMEM I and added to COS1 cells grown to near confluence in Falcon 3084 flasks (BD Biosciences, San Jose, CA). After 5 h at 37 °C, 30 ml of DMEM (10% fetal bovine serum (FBS), with or without 1% glutaMAX I (Gibco) and 1% penicillin/streptomycin) was added, and the cells were incubated overnight. The following day, the cells were washed twice with phosphate buffered saline (PBS) (Dulbecco’s), and 30 ml OptiMEM I was added. The OptiMEM I supernatant containing the recombinant protein was harvested 2 to 3 days later.
Supernatants were concentrated and buffer-exchanged into PBS using a Millipore (Bedford, MA) ultrafree-15 centrifugal filter device, biomax-5kDa molecular weight cut-off. A two-site immunoradiometric assay (IRMA) (14) and densitometry (UN-SCAN-IT gel Gel Digitizing Software, v. 5.1 for Windows, Silk Scientific, Orem, UT and Dell AIO Printer/Scanner’s A940 and A920) were used to quantitate DAF protein concentrations. The two-site immunoradiometric assay used CCP1-specific mAb IA10 at 5 µg/ml as capture mAb and 125I-labeled CCP4-specific mAb 2H6 for detection. As a second method for quantitating mutants, all of which contained a C-terminal 6H tag, an HRP-conjugated anti-6H (C-terminal) mAb (mouse, clone 3D5, IgG2b; Invitrogen) was used in conjunction with densitometry. Monoclonal antibodies - To map the epitopes of the mAbs to DAF, supernatants of the native
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DAF (wild-type, or N61Q substitution) CCP 1-4 control, substitution mutants and vector-only control were run on 10% SDS-PAGE gels and proteins transferred to PVDF membranes. These were done in duplicate to allow blotting with IA10, a mAb that binds CCP1 of DAF, as antibody control, and with the mAb under investigation. The mAbs tested included 8A7 (14), 2H6 (14), BRIC216 (Biosource International, Camarillo, CA), 1C6 (Wako Chemicals USA, Richmond, VA), 2D2-2 (15), 2D2-3 (15), and 1H4 (16). Table II lists substitution mutants tested with each antibody. Lanes were compared qualitatively to determine the binding site. Hemolytic assay buffers - Isotonic veronal-buffered saline (DGVB2+) contained 72.7 mM NaCl, 1.56 mM barbital (Fisher Scientific, Fair Lawn, NJ), 0.91 mM sodium barbital (Fisher Scientific), 1 mM MgCl2, 0.15 mM CaCl2, and 2.5% (w/v) dextrose (pH 7.3-7.4) to which 0.1% gelatin was added. Isoionic veronal buffer (GVB2+) consisted of 145 mM NaCl, 3.12 mM barbital, 1.82 mM sodium barbital, 1 mM MgCl2, and 0.15 mM CaCl2 (pH 7.3-7.4) to which 0.1% gelatin was added. Metal-chelating veronal buffer (GVB-E) substituted 10 mM EDTA for MgCl2 and CaCl2 of GVB2+. Decay accelerating activity (DAA) assays - Alternative pathway C3 convertase DAA was determined by enzyme-linked immunosorbent assay (ELISA) (17). Microtiter wells were coated overnight at 4 °C with C3b (1 µg/ml; Advanced Research Technologies, San Diego, CA) and blocked for 1 h at 37 °C with PBS supplemented with 1% (w/v) bovine serum albumin (BSA) and 0.1% (v/v) Tween-20, and incubated at 37 °C for 2 h with factor B (400 ng/ml), factor D (25 ng/ml; Advanced Research Technologies), 2 mM NiCl2, 25 mM NaCl in 10 mM phosphate buffer pH 7.4 supplemented with 4% (w/v) BSA and 0.1% (v/v) Tween-20. After extensive washing of the wells, plate-bound C3bBb (Ni2+) complexes were incubated for 15 min at 37 °C with increasing concentrations of mutant or control DAF proteins in 25 mM NaCl-supplemented phosphate buffer, or with buffer alone. Remaining plate-bound C3bBb complexes were detected with polyclonal goat anti-factor B antibody followed by peroxidase-conjugated rabbit anti-goat antibody (17). In each experiment, dose-response curves for mutant and control DAF protein were generated by regression analysis, and mutant activity was
calculated from the curves as percent activity of the wild-type protein.
Classical pathway C3 convertase DAA was determined using a hemolytic C4b2a decay assay (4). In this assay, hemolysin-sensitized sheep erythrocytes (EshA; 1 X 107 in 100 µL of DGVB2+) were incubated with ~30 site-forming units (SFU) of human C1 (Advanced Research Technologies) (15 min, 30 °C). The cells were pelleted, resuspended in 100 µl of DGVB2+ and incubated for 20 min at 30 °C with ~15 SFU of human C4 (Quidel, San Diego, CA). The cells were again pelleted and resuspended in 100 µl of DGVB2+ and then treated for 5 min at 30 °C with human C2 (Advanced Research Technologies) predetermined to yield ~1 C4b2a site/cell after subsequent decay for 15 min at 30 °C. The parental DAF, mutated DAF, or DGVB2+ control was added to the cells during this decay step after which guinea pig serum (C3-9) (Colorado Serum, Denver, CO) in GVB-E was added for 1 hr at 37 °C to develop lysis. After pelleting of unlysed cells, the O.D.412 value of the supernatant was measured and residual C4b2a sites were calculated. Calculations - In each assay, a dose-response curve of percent activity vs. concentration was established for mutants and controls, and the concentration of DAF protein required for 50% activity was determined. The concentrations obtained for mutants were compared with the concentrations of their corresponding controls, and percent activity was calculated, with the native DAF control assigned a score of 100%. For classical pathway C3 convertase assays, between two and three independent experiments were performed. For alternative pathway C3 convertase assays, between two and five assays were performed. Sequence alignments - Sequences of DAF CCPs 2-4 (P08174), CR1 CCPs 1-3 and 15-17 (P17927), factor H CCPs 1-3 (CAA68704), C4bpα CCPs 1-3 (AAA36507), MCP CCPs 1-4 (P15529), Kaposica CCPs 1-4 (AAC57082), VCP CCPs 1-4 (P68638), and SPICE CCPs 1-4 (NP_042056) were initially aligned using Clustal W (1.8) (18). Subsequently, CCP4 of factor H and CCP4 of C4bpα were added, and the sequence alignment was subjected to further manual editing on the basis of conservation of residues. PSI-BLAST and position specific substitution matrix - To test whether the residues in the linker region are conserved in regulators that have decay acceleration activity, a PSI-BLAST
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(19) alignment using Cys113 to Ile142 as the seed sequence was performed using blastpgp. The PSI-BLAST alignment of the reference sequence NCBI protein database yielded an alignment of DAF homologues from a variety of species. The position-specific substitution matrix from this alignment was used in a PSI-BLAST search of the nonredundant database. The convergence of this PSI-BLAST search yielded all of the known proteins that have DAA in the classical pathway. A parallel PSI-BLAST with the homologous sequence from MCP yielded an alignment limited to MCP analogs. The cut-off score (in bits) for DAF and MCP (Tables V and VI) is 50. The DAF sequences in Table V are based on iteration 6, while the MCP sequences in Table VI are based on iteration 4. Examination of the position-specific substitution matrix identifies those residues that are strongly conserved (log odds value > 6) in the DAF and MCP alignments, and is the basis for Figure 4 panels C and D.
RESULTS Mapping of DAF’s active site by mutational analyses - The positions of our 31 previously reported and 40 new DAF mutants (a total of 71 substitutions in 60 of 190 amino acids in DAF’s functionally-relevant CCPs 2-4) on the crystal structure of the four CCP modules are shown in two views (differing by 180°) in Fig. 1, panels A-D. The activity of each mutant relative to that of unmodified recombinant DAF CCP 1-4 with respect to accelerating decay of the CP and AP pathway convertases is summarized in Table III. For mapping DAF’s interface with the C3 convertases, solvent-exposed side chains were distinguished from buried residues (L70, G98, F123, and F154). The functional effects of substitutions of these exposed residues are represented schematically by the following color code.
To grade function, we designated substitutions resulting in retention of > 33% of native function as having no significant effect (green), 17-33% as having a limited effect (yellow), 5-16% as having a considerable effect (orange) and ≤ 4% as having an essentially obliterative effect (red). Fig. 1, panels A and B show the effects of the substitutions on DAF’s CP and AP activity on one face of the CCP 2-4 modules and panels C and D show effects of the substitutions on DAF’s CP and AP activity on the opposite face of the two modules.
As seen in Fig. 1, panels A and B, solvent-exposed R69 and R96 in CCP2 and L171 in CCP3 are critical for DAF’s function in both pathways. Consistent with F148 being buried in the full length CCP 1-4 crystal structure, its alanine substitution totally abolished DAF’s function. Lying on the same face of the crystal structure, these two sets of residues, each of which is almost contiguous in the two adjacent CCPs are separated by 13 Å from each other. In contrast, on the opposite face, none of the tested residues were found to be important for DAF function except for portions of R100, K126, and K127, which wrap around the CCP 2-3 interface. Consequently on the face shown in Figs 1A and 1B, the two closely spaced clusters of 1) externally oriented, 2) functionally critical, and 3) separated but juxtaposed residues on the two adjacent CCPs provide full function in the classical pathway and contain all of the residues where function is not fully lost in the alternative pathway.
Alternative mutations of the above residues identified as being critical were subsequently made in which we a) substituted residues occupying equivalent positions in other regulators with decay acceleration activity, or b) altered either charge or hydrophobicity. The results of these substitutions are summarized in Table III. With regard to the functional patch on CCP2 (R69 and R96), while the substitution R69A abolished DAF’s CP and AP activity, R69W (as in CR1 CCP1) had little effect. In contrast, R69C, like R69A, abolished DAF’s CP and AP activity. Similarly, while alanine substitution of R96 (conserved in both CR1 CCP1 and factor H CCP1) totally abolished DAF’s CP and AP activity, the substitution R96L (present in C4bpα CCP1) retained partial activity, more in the AP than in the CP. In contrast to these findings involving the functionally important R69, R96 patch on CCP2, the substitutions K76A and E94A, 16 Å from R96, and the substitutions R100C, E102Q, K108A, K116A and E122A on the opposite face, had little effect. With respect to residues in a patch on CCP3 previously reported to affect DAF and AP activity i.e. F169, L171, and F169C, similarly to F169A, D181A abolished DAF’s AP activity. Concerning the buried residue F148, while the substitution F148A completely abolished DAF’s CP and AP activity, F148Y had no effect, implicating the phenyl ring as the important element in maintaining structure.
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To help resolve the differences in interpretations based on previous partial structures with respect to the flexibility of the CCPs 2-3 linker, we made new substitutions or insertions in this site. Elongation of the CCP 2-3 linker by insertion of a G between K127 and S128 totally abolished DAF’s CP and AP activity. The new substitutions K126C and K126E nearly abolished DAF’s AP activity, similar to the substitution K126A, while they had lesser effects on DAF’s CP activity. At the CCPs 3-4 junction (R187, E188, I189, Y190), the addition of a G between R187 and E188 greatly reduced DAF’s AP activity while not affecting DAF’s CP activity, arguing that only CCPs 2-3 are important in DAF’s CP function, and that for CP function, fixation of the CCP 2-3 spatial relationship by CCP4 precisely in its native orientation is not essential. Mapping of DAF’s active site by epitope analysis and functional effects of mAbs - The epitopes of six anti-DAF mAbs on the crystal structure (as mapped by the inability of each to bind the mutant proteins) are shown in Fig. 2, panels A-F. The amino acids comprising the epitopes of those mAbs that impair DAF’s function are combined in Fig 2, panel G. As seen in Fig. 2, panel A, the epitope of 1H4 (which completely inhibits both DAF’s CP and AP activity) (16) includes structurally relevant F148, F169, L171, and D181, that of 1C6 includes T151 (Fig. 2, panel B), that of 2D2-3 includes K126, T151, and F169 (Fig. 2, panel C), and that of 8A7 includes R69, F148, and L171 (Fig. 2, panel D). The substitution F169C was poorly recognized by BRIC216 (Fig. 2, panel E), which has been reported to have inhibitory effects on DAF’s CP activity (16), as well as by 1C6, which is able to completely block DAF’s function (16) (Fig. 2, panel B). Thus, as found by alanine substitution analysis on CCP3, the epitope mapping reinforces the proposition that L171 is a contact site for DAF’s CP activity and the three residues F169, L171, and D181 are contact sites for DAF’s AP activity. Correlation of DAF’s active site residues with corresponding residues in other C3 convertase regulators that mediate decay acceleration - The sequences of CCP modules in other C3 convertase regulators that decay accelerate the bimolecular enzymes are aligned with that of DAF CCPs 2-4 in Fig. 3, panels A and B. Focusing on the functionally critical residues identified above (seen in the orientation where
the major negative effects on function grouped together), R96-100 in CCP2 in conjunction with K125-127 in the CCP 2-3 linker and buried F148 in CCP3 are conserved in CCPs 1-2 of CR1 (DAA), factor H (DAA + CF), Kaposica, and C4bp (DAA + CF). While R96-100 and F148 are also highly conserved in C3 convertase regulators which lack substantial DAA, but control C3 convertases principally by providing cofactor activity for factor I cleavage, i.e. membrane cofactor protein (MCP), CR1 CCPs 15-17, vaccinia virus complement control protein (VCP), and the smallpox inhibitor of complement enzymes (SPICE), K125-127 are less well conserved. Relevant to this difference between the two groups of regulators, the crystal structure of DAF CCP 1-4 shows an elongated arrangement of CCP 2-3 (<25-30 tilt angle – in DAF~2,3 crystal structure), whereas the crystal and NMR structures of CCPs 1-2 of MCP, CCPs 15-16 of CR1, and CCPs 1-2 of VCP show tilt angles of 68.3°, 31.2°, and 62.8° for the respective opposing CCP modules, Fig. 4, panels A and B (20).
Armed with the above information concerning the region containing the residues in DAF’s active site that are critical for decay acceleration, we performed a PSI-BLAST alignment using Cys113 to Ile142 encompassing the CCP2-3 linker and its preceding and succeeding β strands as the seed sequence in the NCBI protein database. As shown in Table IV, this analysis yielded DAF homologs from a variety of species as well as ~10 other proteins. Among known proteins having DAA are CR1 (site 1; CCPs 1-3) (21), Kaposica (22), mouse Crry (23), and the recently reported but only partially characterized rat CSMD1 (24). The only missing decay accelerator is factor H (+ factor H related-proteins). Interestingly, this analysis identified several additional sequences of uncharacterized function. A parallel PSI-BLAST for homologous sequences with MCP, which lacks DAA but has cofactor function, did not identify any DAF orthologs other than CR1-related sequences (in particular CR1 site 2), CR1 having been shown to possess cofactor activity but also to retain DAA, and yielded an alignment primarily limited to MCP homologs (Table V).
Examination of the position specific substitution matrix identified those residues that are strongly conserved (log odds value > 6) in the DAF alignment and those strongly conserved in the MCP alignment. Those conserved in the
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DAF alignment are colored blue on the DAF CCPs 2-3 backbone structure, Fig. 4, panel C, and those conserved in the MCP alignment are colored yellow on the MCP CCPs 1-2 backbone structure, Fig. 4, panel D. The two representations show that conservation associated with decay acceleration is highest around the linker region while that associated with cofactor function extends into the opposing modular arms.
DISCUSSION
Our characterization in this study of surface
patches on DAF that mediate its DAA of the C3 convertases utilized positioning of the effects of substitution mutants that diminish its CP and AP activities, in conjunction with localization of the epitopes of mAbs reported to diminish its function (16) on the crystal structure of CCPs 1-4 (11). Armed with this information on DAF, we then analyzed the extent of conservation of residues in DAF, and in other C3 convertase regulators determined by mutagenesis to be important, either for cofactor activity, or for DAA (12,21,25-31). Whereas we found an absence of strict conservation of residues which distinguish the two functions, by focusing alignment on the sequence flanking the CCP 2-3 junction where DAF’s function resides, we identified differences between those regulators that 1) mediate DAA and 2) mediate cofactor activity.
The mutational analyses, together with the epitope mapping, revealed two functionally important patches on DAF’s tandem CCP2 and CCP3 surfaces that spatially are positioned 13 Å apart on the same face of the molecule, but on two adjacent modules as shown in Figure 1. These patches are centered around R69 and R96 of CCP2, and F148 and L171 of CCP3. The alternative pathway functional surface on CCP3 appears larger than the classical pathway surface, as mutation of the nearby residues F169 and D181 diminishes AP function more than CP function. The functional importance of these patches is corroborated by the mAb footprint data as all six inhibitory mAbs include residues from the hydrophobic patch on CCP3 and the footprint of one of these mAbs, 8A7 which spans CCPs 2-3, also includes R69.
Although mutations which have a minimal effect on function are sometimes viewed as less informative, they are important in that they rule out uninvolved residues and thereby focus
attention on the relevant region(s) of contact. The mutations near the CCP1-2 junction of CCP2 (K76A, Y79A, Y84A, K108A, and K116A), on the opposite face of CCP3 from the 148/171 patch (N131G), and near the CCP3-4 junction in CCP3 (E185S, R136S), caused no deficits in DAF function in either CP or AP. Newly generated mutations in CCP4, including E205A, D207A, H208A, Y211A, K221A, H229A and Y232A (Table III, Fig 1 and Fig 2F), likewise failed to affect DAF’s activity in either pathway. Consequently, in not finding any new functionally critical regions, we more precisely honed in on DAF’s active site.
While two amino acid residues not located in either of the above patches were previously identified (12) as being important for AP function, one is localized in the linker between CCPs 2 and 3, and the other is in CCP4. Specifically, mutation of the two positively charged linker residues K125 and K126 decreased AP DAA to 32% and 9% and mutations of R206, R212 and N220 decreased AP DAA to 25, 25, 20%. The epitope mapping with mAb 26 supports the previously hypothesized contact region in CCP4. Interpretation of the modest functional deficits introduced by mutation however are necessarily ambivalent.
The new substitutions (P97G, G98S, D181A) near the two patches on the tandem CCP2 and CCP3 surfaces previously identified as constituting the active sites of DAF (11,12) are suggestive of pathway differences in that P97G and G98S, both in the linker region close to the interface between CCPs 2 and 3, decreased DAF’s CP function to 16 and 4% while having a lesser effect on its AP function (72 and 43%). Since the P97 lies within the loop comprised of the D and E strands of CCP2 (DE loop) that is close to CCP3 and is substantially exposed, it is a potential hydrophobic contact point. G98 is a buried residue in the CCP 2-3 linker region so that no CP/AP comparison can be made. On the other hand, the finding that mutating D181 which lies downstream of the AP-only critical F169 on CCP3, negatively affected DAF’s AP function only, slightly extends the region on CCP3 associated with DAF’s AP function since F169 is contiguous with the F148/L171 patch.
Bringing the collective mutagenesis and epitope mapping data together with the crystal structure of DAF provides a number of new insights. Different substitutions at key residues
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(R69, R96, K126, and F169), by virtue of having similar deleterious effects as the alanine substitution, verify that these residues are important in function. An example is that the exposed K126 substitutions K126C and K126E have the same effect as K126A, argues that K126 is functionally important with respect to charge.
The importance of the rigidity and relative orientation of successive CCPs was evaluated by insertion of a glycine residue into the linker sequence. Glycine residues enhance the flexibility of the linker region by virtue of the conformational freedom introduced into the backbone. Additionally, insertion of the residue will necessarily extend the sequence-distance between the modules and, in the absence of strong intermodular contacts, serve to randomize the relative orientations. Insertion of a glycine into the CCP2 - CCP3 linker region between K127 and S128, rendered DAF unable to decay accelerate either C3 convertase. Conversely, insertion of a glycine in the CCP 3-4 linker between R187 and E188 left DAF’s ability to accelerate decay of C4b2a unaffected, implying that the relative orientation of CCP3 and CCP4 is unimportant for CP function. However, consistent with the mutational analysis, this orientation would affect the alignment of R212 and R206 in CCP4 relative to CCP3, and the decrease in activity for the AP was therefore consistent.
The functional orientation of the two identified patches on CCP2 and CCP3 must be determined to define DAF’s interface with the C3 convertases. As shown in Fig 1, these two patches lie on opposite sides of a concavity defined by the tilt between modules. Any significant change in either the skew or twist angles would rotate these two patches away from each other and interpose the bulk of one of the CCPs. This observation supports the proposition that the orientation determined crystallographically approximates the functional orientation.
The orientation of the two CCPs are determined in large measure by two considerations: first favorable (or unfavorable) intermodular contacts can constrain the relative orientation, and second the linker sequence may adopt preferred conformations based on interactions within the linker and with each of the CCPs. There are few intermodular contacts detected either in the crystal structure or by analysis of NOEs in the NMR spectra of DAF
CCP2-CCP3 (9). The absence of intermodular contacts suggests that key determinants of the orientation of CCP2 and CCP3 would be present in the linker region sequence and nearby loops.
To investigate whether there is a difference in conserved sequence in the linker region for complement control proteins that have DAA and CF activity, PSI-BLAST analyses were performed starting with human DAF which possesses only DAA function and MCP which possesses only CF function. The results permitted a position-specific substitution matrix to be generated that was capable of identifying proteins containing tandem CCPs that have DAA. Other attempts at aligning CCP units have focused on alignments of sequences within modules. The success of the PSI-BLAST alignment, based on the 29 residues which comprise the linker and the two adjacent strands and beta turns, that define the strands in DAF vs that in MCP, suggests that the linker region has a significant role in supporting the DAA of these proteins. Stronger conservation of the linker region and of the immediate residues in CCP3 adjacent to the linker support the proposal that these residues help constrain the relative orientations of CCP2 and CCP3 needed for its DAA activity. The same CCP domains of C4bp, factor H, and CR1 can exhibit both DAA and CF activity. The proposition that distinct functional orientations are required for CF activity and DAA requires that the linker region between these CCPs be flexible enough to permit the orientations required for both DAA and CF activity to be sampled.
The PSI BLAST analyses, in conjunction with the mutagenesis insertion data, argue that in regulators that possess DAA an open arrangement (i.e. small tilt angle) between the two CCP modules exists. The crystal structure, and mapping of the epitope of mAb 8A7, both imply this orientation is accessed in solution. This open arrangement would allow one CCP to interact with C3b or C4b, and the other CCP to interact with Bb or C2a. In contrast, the greater tilt angle (less open structure) between the two CCPs in the regulators with CF activity may provide for two-site binding to C3b or C4b. An implication of this sequence and structure comparison is that the intermodular angle and the flanking residues in the two opposing modules comprising the active sites of the two sets of regulators are structural features which distinguish the two functions. These findings are consistent with the hypothesis that DAF’s
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function, i.e. DAA, requires rigidity of the adjacent modules, while CF function, i.e.
C3b/C4b binding, requires cooperative binding of both modules to the C3b/C4b target.
ACKNOWLEDGEMENTS
We would like to thank Joann Mould of Drexel University for the DAF mAbs 2D2-2 and 2D2-3
and Douglas Lublin of Washington University for the DAF mAb 1H4.
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FIGURE LEGENDS Fig. 1. Positions of mutations are shown on DAF’s X-ray crystal structure (PDB ID: 10K3). Red ≤ 4% DAA of WT DAF, orange ≤ 16%, yellow ≤ 33%, and green > 33%. CP activity is shown in A (front) and C (back) while AP activity is shown in B (front) and D (back). Fig. 2. (A-F) Epitopes on DAF recognized by anti-DAF mAbs 1H4, 1C6, 2D2-3, 8A7, BRIC216 and 2H6 mapped on DAF’s X-ray crystal structure. Highlighted are those where western blots with mAbs were negative. (G) Overlap of epitopes mapped by mAb with loss of DAA function. Fig. 3. Amino acid alignment of DAA and cofactor CCP proteins with (A) classical pathway and (B) alternative pathway functionally important residues labelled (21,22,25-35). The designation DAF-2 refers to CCP2 of DAF, CR1-1 refers to CCP1 of CR1, and so forth. Numbering corresponds to DAF. The color scheme for DAA and for CF activity is – (red), + (orange), ++ (yellow), +++ to ++++ (green) scores in the publications cited. Darker green indicates ≥ 200% increase in function. Coloring in CR1-15, 16, 17 refers to “site 2” amino acid substitutions (including those in CR1-8, 9, 10) that affect CF. The data for SPICE are derived from CF assays. The CF data for Kaposica and C4bp were done at 10-100-fold higher concentrations than used for DAA. (18,21,22,25,27-29,31-33,36-43). Fig 4. Structural representations of the tilt angles of sequential CCP domains from proteins that (A) have prominent DAA: (red) DAF 23, (blue) C4BP-α 12 structures adapted from files 1OK3 and 2A55 model 12 from the Protein Data Bank, respectively (B) have prominent cofactor activity (red) VCP 12, (green) CR1 site 2, (blue) MCP 12 structures adapted from files 1RID, 1GKN model 1 and 1CKL from the Protein Data Bank, respectively. The highly conserved residues identified by PSI-BLAST analysis of the linker and flanking sequences are highlighted for the decay accelerating factors in (C) and for the membrane cofactor protein analogs in (D). The backbone of the residues conserved in both protein sequences, which prominently include the cysteines that form the disulfides, are colored white, while the residues more prominently conserved in the decay accelerating proteins as typified by DAF 23 are colored blue (panel C) and the residues more prominently conserved in the membrane cofactor proteins are colored yellow on the backbone of human MCP (panel D). The chain tracings were generated in RasTop and the superposition with the Swiss-PDBviewer.
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MW: CCP Alignment CP Fig. 3A ALIGNMENT OF DECAY ACCELERATING ACTIVITY AND COFACTOR CCP PROTEINS (CP) DAA: 70 80 90 100 110 120 DAF-2 RSCEVPTRLNSASLKQPYITQN--YFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWS-TAVEFCKKK CR1-1 -QCNAPEWLPFARPTN-LTDEF--EFPIGTYLNYECRPGYSGRP-FSII--CLKNSVWT-GAKDRCRRK C4bpα-1 -NCGPPPTLSFAAPMDITLTET--RFKTGTTLKYTCLPGYVRSHSTQT-LTCNSDGEWV-YN-TFCIYK Kaposica-1 -KCSQKTLIGYRLKM--SRDG---DIAVGETVELRCRSGYTTYAR-NITATCLQGGTWS-EPTATCNKK CF: CR1-15 GHCQAPDHFLFAKLKT-QTNAS--DFPIGTSLKYECRPEYYGRP-FSIT--CLDNLVWS-SPKDVCKRK MCP-1 --CEEPPTFEAMELIG--KPKP—-YYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRE C4bpα-1 -NCGPPPTLSFAAPMDITLTET--RFKTGTTLKYTCLPGYVRSHSTQT-LTCNSDGEWV-YN-TFCIYK Kaposica-1 -KCSQKTLIGYRLKM--SRDG---DIAVGETVELRCRSGYTTYAR-NITATCLQGGTWS-EPTATCNKK SPICE-1 -CCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTG-TGWTLFN--QCIKR VCP-1 -CCTIPSRPINMKFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTG-TGWTLFN--QCIKR DAA: 130 140 150 160 170 180 DAF-3 SCPNPGEIRNGQIDVPGG---ILFGATISFSCNTGYKLFG-STSSFCLISGS-SVQWSDPLPECREI CR1-2 SCRNPPDPVNGMVHVIKG---IQFGSQIKYSCTKGYRLIG-SSSATCIISGD-TVIWDNETPICDRI C4bpα-2 RCRHPGELRNGQVEIKTD---LSFGSQIEFSCSEGFFLIG-STTSRCEVQ-DRGVGWSHPLPQCEIV Kaposica-2 SCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKE CF: CR1-16 SCKTPPDPVNGMVHVITD---IQVGSRINYSCTTGHRLIG-HSSAECILSGN-TAHWSTKPPICQRI MCP-2 TCPYIRDPLNGQAVPANGT--YEFGYQMHFICNEGYYLIG-EEILYCELKGS-VAIWSGKPPICEKV C4bpα-2 RCRHPGELRNGQVEIKTD---LSFGSQIEFSCSEGFFLIG-STTSRCEVQDR-GVGWSHPLPQCEIV Kaposica-2 SCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKE SPICE-2 RCPSPRDIDNGHLDIG-G---VDFGSSITYSCNSGYYLIG-EYKSYCKLGSTGSMVWNPKAPICESV VCP-2 RCPSPRDIDNGQLDIG-G---VDFGSSITYSCNSGYHLIG-ESKSYCELGSTGSMVWNPEAPICESV
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DAA: 190 200 210 220 230 240 250 DAF-4 YCPAPPQIDNG-IIQGERDHYGYRQSVTYACNKG------FTMIGEHSIYCTVNNDE-GEWSGPPPECRG CR1-3 PCGLPPTITNGDFISTNRENFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII C4bpα-3 KCKPPPDIRNG-RHSGEENFYAYGFSVTYSCDPR------FSLLGHASISCTVENETIGVWRPSPPTCEKI Kaposica-3 KCHR-PKIKNGDFKP-DKDYYEYNDAVHFECNEG------YTLVGPHSIACAVNNT----WTSNMPTCELA CF: CR1-17 PCGLPPTIANGDFISTNRENFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII MCP-3 LCTPPPKIKNGKHTFSEVEVFEYLDAVTYSCDPAP-GPDPFSLIGESTIYCGDNSV----WSRAAPECKVV C4bpα-3 KCKPPPDIRNG-RHSGEENFYAYGFSVTYSCDPR------FSLLGHASISCTVENETIGVWRPSPPTCEKI Kaposica-3 KCHR-PKIKNGDFKP-DKDYYEYNDAVHFECNEG------YTLVGPHSIACAVNNT----WTSNMPTCELA SPICE-3 KCQLPPSISNG-RHNGYNDFYTDGSVVTYSCNSG------YSLIGNSGVLCSGGE-----WSNP-PTCQIV VCP-3 KCQSPPSISNG-RHNGYEDFYTDGSVVTYSCNSG------YSLIGNSGVLCSGGE-----WSDP-PTCQIV DAA: C4bpα-4 TCRKPDVSHGEMVSGFGPIYNYKDTIVFKCQKGFVLRGSSVIHCDADSKWNPSPPACEP Kaposica-4 GCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTE-WDPPLPKCVL CF: MCP-4 KCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLK C4bpα-4 TCRKPDVSHGEMVSGFGPIYNYKDTIVFKCQKGFVLRGSSVIHCDADSKWNPSPPACEP Kaposica-4 GCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTE-WDPPLPKCVL SPICE-4 KCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSPGNTWQPELPKCVR VCP-4 KCPHPTISNGYLSSGFKRSYSYNDNVDFKCKYGYKLSGSSSSTCSPGNTWKPELPKCVR
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MW: CCP Alignment AP Fig. 3B ALIGNMENT OF DECAY ACCELERATING ACTIVITY AND COFACTOR CCP PROTEINS (AP) DAA: 70 80 90 100 110 120 DAF-2 RSCEVPTRLNSASLKQPYITQN--YFPVGTVVEYECRPGYRREPSLSPKLTCLQNLKWS-TAVE-FCKKK CR1-1 -QCNAPEWLPFARPTN-LTDEF--EFPIGTYLNYECRPGYSGRP-FSII--CLKNSVWT-GAKD-RCRRK FactorH-1 EDCNELPPRRNTEILTGSWSDQT--YPEGTQAIYKCRPGYRSLG--NVIMVCRK-GEWVALNPLRKCQKR Kaposica-1 -KCSQKTLIGYRLKM--SRDG---DIAVGETVELRCRSGYTTYAR-NITATCLQGGTWS-EPTA-TCNKK CF: CR1-15 GHCQAPDHFLFAKLKT-QTNAS--DFPIGTSLKYECRPEYYGRP-FSIT--CLDNLVWS-SPKDV-CKRK MCP-1 --CEEPPTFEAMELIG--KPKP—-YYEIGERVDYKCKKGYFYIPPLATHTICDRNHTWLPVSDDA-CYRE Kaposica-1 -KCSQKTLIGYRLKM--SRDG---DIAVGETVELRCRSGYTTYAR-NITATCLQGGTWS-EPTA-TCNKK SPICE-1 -CCTIPSRPINMTFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTG-TGWTLFN--Q-CIKR VCP-1 -CCTIPSRPINMKFKNSVETDANANYNIGDTIEYLCLPGYRKQKMGPIYAKCTG-TGWTLFN--Q-CIKR DAA: 130 140 150 160 170 180 DAF-3 SCPNPGEIRNGQIDVPGG---ILFGATISFSCNTGYKLFG-STSSFCLISGS-SVQWSDPLPECREI CR1-2 SCRNPPDPVNGMVHVIKG---IQFGSQIKYSCTKGYRLIG-SSSATCIISGD-TVIWDNETPICDRI FactorH-2 PCGHPGDTPFGTFTLTGG-NVFEYGVKAVYTCNEGYQLLGEINYREC----D-TDGWTNDIPICEVV Kaposica-2 SCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKE CF: CR1-16 SCKTPPDPVNGMVHVITD---IQVGSRINYSCTTGHRLIG-HSSAECILSGN-TAHWSTKPPICQRI MCP-2 TCPYIRDPLNGQAVPANGT--YEFGYQMHFICNEGYYLIG-EEILYCELKGS-VAIWSGKPPICEKV Kaposica-2 SCPNPGEIQNGKVIFHGGQDALKYGANISYVCNEGYFLVGREYVRYCMIGASGQMAWSSSPPFCEKE SPICE-2 RCPSPRDIDNGHLDIG-G---VDFGSSITYSCNSGYYLIG-EYKSYCKLGSTGSMVWNPKAPICESV VCP-2 RCPSPRDIDNGQLDIG-G---VDFGSSITYSCNSGYHLIG-ESKSYCELGSTGSMVWNPEAPICESV
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DAA: 190 200 210 220 230 240 250 DAF-4 YCPAPPQIDNG-IIQGERD---HYGYRQSVTYACNKG------FTMIGEHSIYCTVNNDE-GEWSGPPPECRG CR1-3 PCGLPPTITNGDFISTNRE---NFHYGSVVTYRCNPGSGGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII FactorH-3 KCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSG------YKIEGDEEMHCSDD----GFWSKEKPKCVEI Kaposica-3 KCHR-PKIKNGDFKP-DKD---YYEYNDAVHFECNEG------YTLVGPHSIACAVNNT----WTSNMPTCELA CF: CR1-17 PCGLPPTIANGDFISTNRE---NFHYGSVVTYRCNLGSRGRKVFELVGEPSIYCTSNDDQVGIWSGPAPQCII MCP-3 LCTPPPKIKNGKHTFSEVE---VFEYLDAVTYSCDPAP-GPDPFSLIGESTIYCGDNSV----WSRAAPECKVV Kaposica-3 KCHR-PKIKNGDFKP-DKD---YYEYNDAVHFECNEG------YTLVGPHSIACAVNNT----WTSNMPTCELA SPICE-3 KCQLPPSISNG-RHNGYND---FYTDGSVVTYSCNSG------YSLIGNSGVLCSGGE-----WSNP-PTCQIV VCP-3 KCQSPPSISNG-RHNGYED---FYTDGSVVTYSCNSG------YSLIGNSGVLCSGGE-----WSDP-PTCQIV DAA: FactorH-4 SCKSPDVINGSPISQKII-YKENERFQYKCNMGYEYSERGDAVCTESG-WRP-EPSCEE Kaposica-4 GCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTE-WDPPLPKCVL CF: MCP-4 KCRFPVVENGKQISGFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLK Kaposica-4 GCKFPSVTHGYPIQGFSLTYKHKQSVTFACNDGFVLRGSPTITCNVTE-WDPPLPKCVL SPICE-4 KCPHPTILNGYLSSGFKRSYSYNDNVDFTCKYGYKLSGSSSSTCSPGNTWQPELPKCVR VCP-4 KCPHPTISNGYLSSGFKRSYSYNDNVDFKCKYGYKLSGSSSSTCSPGNTWKPELPKCVR
Figure 3
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Table I. Sense primers (5’ to 3’) used in site-directed mutagenesis Mutation Primer____________________________ R69C GCTGCGAGGTGCCAACATGCCTAAATTCTGCATCCCTC R69W GCTGCGAGGTGCCAACATGGCTAAATTCTGCATCCCTC S74A GGCTAAATTCTGCAGCCCTCAAACAGCC K76A GGCTAAATTCTGCATCCCTCGCACAGCCTTATATCACTCAG Y79A CTGCATCCCTCAAACAGCCTGCTATCACTCAGAATTATTTTCC Y84A GCCTTATATCACTCAGAATGCTTTTCCAGTCGGTACTGTTG E94A GGTACTGTTGTGGAATATGCGTGCCGTCCAGGTTACAG R96L GTTGTGGAATATGAGTGCCTTCCAGGTTACAGAAGAGAACC P97G GTGGAATATGAGTGCCGTGGAGGTTACAGAAGAGAACC G98S GAATATGAGTGCCGTCCAAGTTACAGAAGAGAACC R100C GAGTGCCGTCCAGGTTACTGCAGAGAACCTTCTCTATC E102Q CGTCCAGGTTACAGAAGACAACCTTCTCTATCACC K108A GAACCTTCTCTATCACCAGCACTAACTTGCCTTCAG K116A CTTGCCTTCAGAATTTAGCATGGTCCACAGCAGTCG E122A GGTCCACAGCAGTCGCATTTTGTAAAAAGAAATC K126C CAGCAGTCGAATTTTGTAAATGCAAATCATGCCCTAATCCGG K126E GCAGTCGAATTTTGTAAAGAGAAATCATGCCCTAATCCG N131A* GTAAAAAGAAATCATGCCCTGCTCCGGGAGAAATACGAAATG R136S GCCCTAATCCGGGAGAAATATCAAATGGTCAGATTGATGTACC F148Y CCAGGTGGCATATTATATGGTGCAACCATCTCC T151A GGCATATTATTTGGTGCAGCCATCTCCTTCTCATGTAAC S155C GGTGCAACCATCTCCTTCTGCTGTAACACAGGGTAC F169C GGCTCGACTTCTAGTTGTTGTCTTATTTCAGGCAGCTC S173A CTTCTAGTTTTTGTCTTATTGCAGGCAGCTCTGTCCAGTGG S175A GTTTTTGTCTTATTTCAGGCGCCTCTGTCCAGTGGAGTGAC S175C GTTTTTGTCTTATTTCAGGCTGCTCTGTCCAGTGGAGTGAC Q178A CAGGCAGCTCTGTCGCGTGGAGTGACCCGTTG S180A GGCAGCTCTGTCCAGTGGGCTGACCCGTTGCCAGAGTGC D181A GCTCTGTCCAGTGGAGTGCCCCGTTGCCAGAGTGC E185S GGAGTGACCCGTTGCCATCGTGCAGAGAAATTTATTG E205A CAATGGAATAATTCAAGGGGCACGTGACCATTATGG D207A CAAGGGGAACGTGCCCATTATGGATATAGACAG H208A CAAGGGGAACGTGACGCTTATGGATATAGACAGTCTG Y211A GGAACGTGACCATTATGGAGCTAGACAGTCTGTAACGTATG K221A CTGTAACGTATGCATGTAATGCAGGATTCACCATGATTGGAG H229A GGATTCACCATGATTGGAGAGGCCTCTATTTATTGTACTGTG Y232A GATTGGAGAGCACTCTATTGCTTGTACTGTGAATAATGATG Insertion Primer________________________________ G between K127 and S128 CGAATTTTGTAAAAAGAAAGGCTCATGCCCTAATCCGGGAG G between R187 and E188 CCGTTGCCAGAGTGCAGAGGCGAAATTTATTGTCCAGCAC *N131G was obtained.
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Table II. Epitope Mapping of mAbs Using Substitution Mutants mAb Substitution Mutants Tested by Western Blot 8A7 R69A; L70A; N71K; S72F; R96A; R100A; K126A; K126C; N131G; L147A;
F148A; T151A; F169A; F169C; L171A; D181A BRIC216 R69W/L70P; L147A; F148A; T151A; F154A; S155A; S165A; F169A; F169C;
L171A; S180A 2D2-3 R69A; N71K; E94A; R96A; P97G; R100A; K125A; K126A; F148A; T151A;
F169A; L171A; D181A 1H4 F148A; T151A; S155A; S165A; F169A; L171A; D181A 1C6 R69W/L70P; R96A; P97G; R100A; K126A; K127A; N131G; L147A; F148A;
T151A; F154A; S155A; Y160A; K161A; F163A; S165A; F169A; F169C; L171A; Q178S; S180A; D181A
2H6 Y160A; F163A; R187A; R187GE; E205A; R206A; D207A; H208A; Y211A; R212A; N220A; K221A; H229A; Y232A; D238A; E239A
2D2-2 R69A; R96A; P97G; R100A; F148A; T151A; F169A; L171A
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Table III. C3 convertase DAA for DAF mutants. Classical pathway hemolytic assays and alternative pathway ELISA assays.
MUTANT ALTERNATIVE PATHWAY CLASSICAL PATHWAY LINKER BETWEEN CCPS 1 AND 2
Q61N 58±8 89±4 CCP2
R69A 10±8 7.9±1.7 R69C-6H 4.2±1.4 1.8±0.2 R69W-6H 83±17 33±3
R69W,L70P-6H 15±8 1.6±0.3 L70A 25±12 98±19 N71K 29±9 83±26 S72F 64±53 105±44
S74A-6H 92±39 84±3 K76A-6H 106±37 44±18 Y79A-6H 95±34 71±4 Y84A-6H 75±15 103±43 E94A-6H 139±36 170±70
R96A 0.2±0.3 1.4±0.2 R96L-6H 27±27 1.6±1.4 P97G-6H 72±35 16±4 G98S-6H 43±9 4.3±0.2 R100A 12±8 25±4
R100C-6H 26±13 35±10 E102Q-6H 95±45 154±2 K108A-6H 68±14 52±6 K116A-6H 105±34 80±21 E122A-6H 74±26 70±4
F123A 82±65 60±11 F123A,R101I 16±3 25±7
LINKER BETWEEN CCP2 AND CCP3 K125A 32±10 62±2 K126A 9±5 122±51
K126C-6H 8.3±2.0 53±13 K126E-6H 0.7±0.2 101±14
K127A 14 11±2 K127-G-S128-6H 0±0 3.8±1.8
S128C-6H 33±18 42±0.5 CCP3
N131G-6H 136±84 67±16 R136S-6H 47±18 110±36
L147A 37±22 72±22 F148A 4±1 3.7±2.3
F148Y-6H 52±15 64±14
MUTANT ALTERNATIVE PATHWAY CLASSICAL PATHWAY CCP3
T151A-6H 42±5 100±0 F154A 43±22 80±9 S155A 128±59 115±10
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S155C-6H 79±12 105±40 Y160A 20±10 126±38 K161A 59±30 124±11 F163A 88±13 183±10 S165A 59±28 56±8 F169A 0.7±0.2 64±18
F169C-6H 4.9±3.3 53±2 L171A 4±2 16±2
S173A-6H 64±14 69±23 S175A-6H 90±28 95±7 S175C-6H 74±36 57±21
S176A 47±23 58±4 V177A 62±14 43±12
Q178A-6H 77±13 135±70 S180A-6H 68±29 62±12 D181A-6H 8.4±3.0 126±66 E185S-6H 107±59 119±46
LINKER BETWEEN CCPS 3 AND 4 R187A 53±23 73±14
R187-G-E188-6H 5.8±1.5 54±13 CCP4
E205A-6H 78±41 83±9 R206A-6H 25±13 128±11 D207A-6H 119±47 73±33 H208A-6H 46±32 95±21 Y211A-6H 74±44 74±11 R212A-6H 25±9 114±13
N220A 32±8 67±12 K221A-6H 44±33 85±3
E228A 69±9 57±8 H229A-6H 169±142 123±36 Y232A-6H 67±39 87±29 D238A-6H 50±14 71±6 E239A-6H 61±25 56±17
OTHER RhDAF-6H 28±12 92±32
Note: Substitution mutants were made in a human DAF CCP1-4 module with N61Q (no C-terminal tag) [“HuDAF(N61Q)”] or N61 + C-terminal 6XHis Tag [HuDAF-6H].
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Table IV. Protein GI Reference
I. DAF Homologs
Sus scrofa 10185705 Homo sapiens 225451 Pongo pygmaeus 459390 Cercopithecus aethiops 28300292 Pan troglodytes 8132012 Erythrocebus patas 8132020 Macaca mulatta 8132016 Gorilla gorilla 8132008 Bos taurus [DAF-1] 71044405 Macaca fascicularis 84579265 Papio hamadryas 8132014 Daf 2 (Mus musc.) 6681131 Daf 1 (Mus musc.) 15030123 Cavia porcellus 1805487 Rattus norvegius 38511538
II. Other Known C3 Convertase Regulators
CR1 (Papio hamadryas) 662829 CR1 (Homo sapiens) 809019 CR1 (Pan troglodytes) 55589354 ORF4 (HHV8) 87196822 CRRP (Cavia porcellus) 191251 MCR1 [Crry/p65] (Mus musc.) 595982 Complement receptor [Cr2] (Mus musc.) 192691 Complement regulatory membrane protein [CREMP] (also called CREM) (Gallus gallus)
45382791
Complement receptor related protein isoform 3 [Crry] (Rattus norv.) 53759105
III. Other Proteins of Unknown Function
CSMD1 (Rattus norv.) 62662966 CSMD1 (Mus musc.) 14787176 CSMD1 (Homo sap.) 38604975 mKIAA1890 (Mus musc.) 60360458 CSMD1 (Pan trog.) 55630158 Novel protein containing Sushi domain (Xenopus tropicalis) 89273853 Complement binding protein (Cercopithecine herpesvirus 17) 18653812 CSMD3 isoform 1 (Gallus gallus) 50731858 CSMD3 isoform 1 (Mus musc.) 82956973 CSMD3 isoform 1 (Homo sap.) 34330131 mKIAA1894 (Mus musc.) 28972866 KIAA1894 (Homo sap.) 34327986 Complement binding protein (Macaca mulatta rhadinovirus 25-95) 7329993 Unnamed protein product (Tetraodon nigroviridis) 47229403 Table V.
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Protein GI Reference
I. MCP Homologs
Pongo pygmaeus 18028954 Gorilla gorilla 18028952 Homo sapiens 27502415 Papio hamadryas 2330892 Pan troglodytes 55589360 Cercopithecus aethiops 1777315 Macaca mulatta 2330906 Macaca fascicularis 2330904 Saguinus oedipus 3059215 Rattus norvegius 3978555 Mus musculus 3341865 Saguinus mystax 2997607 Saimiri sciureus 2997609 Callithrix jacchus 24954161 Cavia porcellus 1679584 Sus scrofa 47522706 Bos Taurus 33086939 Predicted: similar to MCP isoform 12 precursor (Canis familiaris) 73960707
II. Other Known C3 Convertase Regulators
Complement receptor (Mus musc.) 533222 Predicted: similar to complement component (3b/4b) isoform F precursor isoform 6 (Homo sap.)
88952721
Complement receptor 1 (Homo sap.) 306680 M144R (Myxoma virus) 9633780 Complement receptor (Papio cynocephalus) [homologue of the human CR1-like genetic element]
1301611
Complement receptor [Crry] (Mus musc.) 387133 Complement receptor 1 (Pan troglodytes) 557727 Complement C3b/C4b receptor-like protein precursor (Homo sap.) 7441738 Complement component receptor type 1 (Papio hamadryas) 662829
III. Other Proteins of Unknown Function
Predicted: similar to hypothetical protein (Rattus norvegius) (Chrom 20) 34852697 Possible homologue to human CR1-like genomic element (Pan trog.) 557729 Gp144R (Rabbit fibroma virus) 6578669
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Mitchell, Dinesh C. Soares, Paul N. Barlow and M. Edward MedofLisa Kuttner-Kondo, Dennis E. Hourcade, Vernon E. Anderson, Nasima Muqim, Lynne
convertasesStructure-based mapping of DAF's active site residues that decay accelerate the C3
published online March 29, 2007J. Biol. Chem.
10.1074/jbc.M611650200Access the most updated version of this article at doi:
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