Selection of novel ligands from a whole-molecule randomly mutated C5a library

S.A. Cain1,, D.M. Williams2,, V. Harris1,2, and P.N. Monk1,3,

Krebs Institute of Biomolecular Science, 1 Department of Molecular Biology and Biotechnology and 2 Department of Chemistry, University of Sheffield, Sheffield S10 2UH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Novel antagonists of the proinflammatory leukocyte chemoattractant C5a have been produced from a phage display library of whole-molecule random mutants. The cDNA for the inflammatory polypeptide C5adR74 was used as template in a PCR reaction doped with the mutagenic nucleoside triphosphates dPTP {dP: 6-(2-deoxy-ß-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazin-7-one} and 8-oxodGTP (8-oxodG: 8-oxo-2'-deoxyguanosine) to allow the introduction of mutations in a highly controlled manner throughout the cDNA. The resultant library of mutants was displayed on bacteriophage M13 using a jun/fos linker sequence. Functional polypeptides were isolated by several rounds of selection against the receptor for C5a expressed on the surface of CHO cells. From this selection procedure, a limited number of variants of C5adR74 were obtained. When expressed as free polypeptide, the binding affinities of the selected C5adR74 sequences were increased 5-fold relative to wild-type protein. Site-directed mutagenesis of the C-terminus of these variants resulted in the production of antagonists of C5adR74 activity.

Keywords: C5a/phage display/polymerase chain reaction/pyrimidine analogue/random mutagenesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Complement fragment C5a is a potent pro-inflammatory 74-residue polypeptide (Gallin et al., 1991) with a well-defined structure consisting of a helical N-terminus, a disulfide-linked, antiparallel three-helix core and a loop-helix C-terminal domain (Zuiderweg et al., 1989Go; Zhang et al., 1997Go). The full agonist potential of C5a is located in the C-terminal decapeptide whereas the remainder of the molecule confers high-affinity binding to the cell surface receptor (Mollison et al., 1989Go; Bubeck et al., 1994Go; Toth et al., 1994Go). The agonist decapeptide is rapidly modified in vivo by serum carboxypeptidase activity which removes the C-terminal arginine residue (Bokisch and Muller-Eberhardt, 1974). The desarginated (C5adR74) form has a different pattern of activity to intact C5a. For instance, human mast cell degranulation is 103–104-fold less sensitive to C5adR74 than to intact C5a (El-Lati et al., 1994Go; Fureder et al., 1995Go; Werfel et al., 1996Go), whereas both forms are equipotent stimulants of fibroblast chemotaxis (Senior et al., 1988Go). Despite the ligand selectivity demonstrated by different cell types, only a single receptor for C5a (C5aR) has so far been cloned, which is a member of the G protein-coupled receptor superfamily (Boulay et al., 1991Go; Gerard and Gerard, 1991Go). The mechanism by which a single receptor type can display ligand selectivity in different cellular backgrounds has begun to be elucidated. We have shown that the mutation of receptor residue E199 to K completely inhibits activation by C5adR74 but has little effect on the response to intact C5a (Monk et al., 1995Go). The activity of C5adR74 on these mutant receptors can be partly recovered by mutating ligand residue K68 to E, presumably restoring an essential interaction with the receptor residue at position 199 (Crass et al., 1999Go).

Recently, a phage display system has been used to select functional sequences from libraries of C5a molecules that had undergone random mutation between residues 69 and 73 (Hennecke et al., 1998Go). A novel C5aR antagonist has been derived in this way which can inhibit C5a-mediated inflammatory events in vivo (Hennecke et al., 1998Go; Heller et al., 1999Go). However, the display of functional C5a sequences on the phage surface and the choice of the pentapeptide randomized region relied heavily on data from previous studies of the C5a/C5aR system. We have recently extended this approach, combining phage display with a whole-molecule random mutagenesis technique that uses synthetic nucleotide triphosphates to introduce mutations throughout a protein by polymerase chain reaction (PCR) (Cain et al., 2000Go). Here we report the production of novel ligands with increased affinity for the C5a receptor in the absence of prior assumptions about the particular residues involved in ligand–receptor interactions. Additional site-directed mutagenesis of these ligands was then used to produce C5aR antagonists.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

The C5a cDNA was kindly by provided Wilfried Bautsch (Medizinische Hochschule, Hannover, Germany). CHO cells transfected with wild-type and RBL cells transfected with wild-type C5aR (Pease et al., 1994Go) were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% (v/v) fetal calf serum and 400 µg/ml G418 (Gibco BRL, UK). Mutagenic nucleotide triphosphates dP {6-(2-deoxy-ß-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2] oxazin-7-one}, 8-oxodG (8-oxo-2'-deoxyguanosine) and 8-oxodG (8-oxo-2'-deoxyguanosine) were prepared as described previously (Zaccolo et al., 1996Go) or purchased from Amersham-Pharmacia (UK).

Construction of the C5adR74 mutant library

The phagemid vector pJ/F2SS was used for expression of the C5adR74 mutant libraries. pJ/F2SS is a modified version of the phagemid vector pJunFos (Crameri and Suter, 1993Go) with the second lacZ promoter removed and the second pelB leader sequence replaced by ompA (Cain et al., 2000Go). The mutant C5adR74 library was generated by PCR using the template of C5adR74 as described previously (Zaccolo et al., 1996Go; Cain et al., 2000Go). The structure of the modified phage is shown schematically in Figure 1aGo. Briefly, construction of the mutant C5adR74 library involved two PCR reactions. The first PCR reaction, in the presence of the mutagenic nucleotide analogues, introduced mutations at a theoretical rate of 5–10 mutations per molecule. The PCR product was purified and used as template for a second `wash-out' PCR reaction, containing no mutagenic nucleotide analogues. The size of the resultant C5adR74 library expressed as phage was estimated as 1x106 transformants.



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Fig. 1. Phage-displayed and free C5a receptor ligands. (a) Schematic diagram showing C5adR74 displayed on the surface of M13 phage. The coat protein III–Jun complex is assembled on the phage surface. The Fos–Flag–C5adR complex is attached via a Jun–Fos complex, leaving the important C-terminus of C5adR74 available for receptor interaction. (b) The N-terminal sequence of recombinant C5adR74 prior to the removal of the His6 tag, indicating the presence of the GGS tripeptide.

 
Affinity enrichment of the mutant library

Selection of receptor binding clones from the mutant C5adR74 library was performed on CHO cells transfected with human C5aR as described previously (Cain et al., 2000Go). Briefly, 5x106 cells were harvested, washed and resuspended in 0.5 ml of DMEM medium containing 1% (w/v) BSA, 20 mM HEPES pH 7.2 (DWB). The phages were added in 1 ml of DWB and incubated at room temperature for 30 min on a rotary mixer. After washing five times in 10 ml of DWB, the cells were resuspended in 0.5 ml of sterile PBS and the bound phages were eluted in 0.5 ml of 0.2 M HCl for 1 min. The supernatant was neutralized with 30 µl of 2 M Tris and a small sample of the phage was diluted to calculate the phage titre, by mixing 10 µl of diluted phages with 50 µl of freshly grown TG1 (OD600 = 1.0) and plating on to LB agar-carbenicillin. The remainder of the phages were amplified for the next round of selection by addition to 5 ml of freshly grown TG1 cells (OD600 1.0) and incubated at room temperature for 15 min without shaking.

Expression and purification of C5adR74 and mutants

Expression and purification of the recombinant His6-tagged C5a, C5adR74 and the C5adR74 variants was performed as described (Paczkowski et al., 1999Go). For cleavage of the His6 tag from C5adR74 fragments, 200–300 µg of purified recombinant protein were digested with 100 U of rTEV protease in 1 mM DTT and rTEV buffer for 4 h at 30°C. The digested polypeptides were separated from the rTEV protease and cleaved His6 tag by addition of Ni2+-NTA resin. A short sequence (GGS) remains at the N-terminus of the polypeptides in this production protocol (Figure 1bGo). The presence of this N-terminal tripeptide appears to decrease the receptor affinity for GGS-C5adR74 4-fold relative to native C5adR74 but has no effect on C5a (Crass et al., 1999Go; Paczkowski et al., 1999Go).

Measurement of receptor activation of RBL cells

Cellular activation was measured as the release of ß-hexosaminidase activity from RBL-2H3 cells transfected with human C5aR as described previously (Cain et al., 2000Go). The percentage of ß-hexosaminidase release was calculated as a percentage of the maximum release (1 µM C5a) sample. Typically, this concentration of C5a caused the release of 45% of the total cell-associated ß-hexosaminidase activity. Assay of the antagonist activity of the C5adR74 variants was performed as described above except that the antagonists were added at various concentrations for 15 min at 37°C after preincubation with release buffer. The agonists were then added at a final concentration of 10 nM (C5a) or 25 nM (C5adR74) and incubated as normal. Data were analysed by GraphPad Prism software and significance was assessed by a t-test.

[125I]C5a binding assay

Binding assays using 50 pM [125I]C5a were performed on adherent C5aR-transfected RBL cells in 96-well microtitre plates as described previously (Monk et al., 1994Go).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selection from C5adR74 mutant library

A randomly mutated C5adR74 library with an amino acid mutation rate of 12.3% or nine substitutions per molecule was generated by PCR (Figure 1aGo). The mutations were distributed evenly through the sequence (data not shown). After three rounds of selection for receptor binding, a distinct pattern of mutations emerged with an average of 5.7 substitutions per molecule (Table IaGo). The selected sequences fell into two distinct variant families: Variant 1 (K14R, C27R, P45S, I48T, G73R) and Variant 2 (K4R, S16P, C27R, V56A, V57A, D69N, M70T) (Table IaGo). Three other sequences were obtained that all contained C27R; two of these were also R73 and thus similar to Variant 1.


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Table I. Sequences of clones from C5adR74 libraries selected for binding to C5a receptor-transfected CHO cells (a) C5adR74 library: Round 3
 
To isolate only the highest affinity mutant C5adR74 sequences, two further rounds of selection were performed on Library 1, in the presence of 10 and 25 nM C5a, respectively. This limited the library to copies of Variant 2, with an average of 6.6 substitutions per molecule (Table IbGo). The single exception was similar to Variant 2 except that D69 and M70 were unchanged and G73 was mutated to R, as in Variant 1.

Receptor binding and activation by C5adR74 variants

We were interested to see how the mutations present in the two variants affected the agonist function of the free protein (Figure 1bGo). Measured as the ability to compete for receptor binding with [I125]C5a, Variant 1 and Variant 2 had similar affinities for the human C5aR on transfected RBL cells, with IC50 = 135 and 138 nM, respectively (Figure 2Go). However, the variants had affinities that were ~5-fold higher than GGS-C5adR74 (IC50 = 617 nM). The selected variants were then adapted by mutating K68 to E, a change previously shown to inhibit C5aR activation by C5adR74 but not by C5a (Monk et al., 1995Go; Crass et al., 1999Go). After mutation at K68, the IC50 values of the variants had increased (Variant 1[E68] = 3.1 µM; Variant 2[E68] = 1.6 µM) but were still much lower than that of GGS-C5adR74[E68] (IC50 > 20 µM) (Figure 2Go).



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Fig. 2. The binding of C5adR74 variants to C5aR-transfected RBL cells. Binding affinity was assessed by the ability to compete for receptor binding with 50 pM [I125]C5a. The competition curves for GGS-C5a (squares), GGS-C5adR74 (triangles) and K68 forms of Variant 1 (inverted triangles) and Variant 2 (circles) (solid symbols) are shown by solid lines; competition curves for E68 mutants (open symbols) are shown by broken lines. The results are the means ± SEM of three separate experiments performed in triplicate.

 
The agonist activity of the selected Variants was measured using RBL cells transfected with human C5aR. GGS-C5adR74 itself is only a partial agonist in this system (Figure 3Go), with a maximal response of only 50% of intact GGS-C5a but with a similar EC50 (EC50 values for GGS-C5a and GGS-C5adR74 = 4.2 and 8.8 nM, respectively). In contrast, the two Variants of GGS-C5adR74 were potent, full agonists (EC50 values for Variant 1 and Variant 2 = 8.6 and 5.7 nM, respectively) (Figure 3Go) despite their apparently poor binding affinity in competition with [I125]-C5a. After mutation at K68, Variant 2[E68] and GGS-C5adR74[E68] could not activate the receptor at any concentration tested (Figure 3Go). Variant 1[E68] activated the C5aR to 20% of the response to intact GGS-C5a at 1 µM, but at 10 µM this had declined to control levels.



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Fig. 3. Activation of C5aR-transfected RBL cells by C5adR74 variants. RBL cells transfected with the C5aR were treated with GGS-C5a (squares) or the [K68] (closed symbols) and [E68] forms (open symbols) of GGS-C5adR74 (triangles), Variant 1 (inverted triangles) and Variant 2 (circles). In all cases, cell activation was assessed as the release of ß-hexosaminidase from intracellular granules into the supernatant, relative to the effect of 1 µM intact GGS-C5a (= 100%). The results shown are the means ± SEM of three separate experiments performed in triplicate.

 
Antagonist activity of [Glu68] variants

The [E68] variants bound to the C5aR but were almost inactive and so were tested for their ability to act as antagonists of GGS-C5a and GGS-C5adR74. Both were full antagonists of GGS-C5adR74 activity (Figure 4aGo). Variant 1[E68] had an IC50 = 4.7 µM and Variant 2[E68] = 0.9 µM (significantly different: p = 0.0009). Variant 2[E68] was a considerably more effective antagonist of GGS-C5adR74 receptor activation than Variant 1[E68], but both were much more effective GGS-C5adR74 antagonists (95- and 19-fold, respectively) than GGS-C5adR74[E68]. However, the [E68] variants only weakly antagonized the degranulation response to 10 nM GGS-C5a with IC50 values of 25.3 µM (Variant 1[E68]) and 14.6 µM (Variant 2[E68]), (insignificantly different: p = 0.2887), whereas GGS-C5adR74[E68] had no detectable antagonist activity (Figure 4bGo). These data imply that the [E68] variants, particularly Variant 2[E68], have a degree of selectivity for the GGS-C5adR74–receptor interaction.



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Fig. 4. The antagonism of C5adR74 activity on C5aR-transfected RBL cells by E68 variants. RBL cells were pre-incubated with E68 forms of C5adR74 (triangles) and Variant 1 (inverted triangles) and Variant 2 (circles) for 15 min prior to the addition of either 25 nM GGS-C5adR74 (a) or 10 nM GGS-C5a (b). Cell activation was assessed as the release of ß-hexosaminidase from intracellular granules into the supernatant. Results shown are the means ± SEM from four separate experiments performed in triplicate.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
C5a has been extensively characterized and has a known structure and functionality and so it represents an excellent test system for ligand modification by whole-molecule random mutagenesis and phage display. In this study, we produced a library of randomly mutated C5adR74 molecules and subjected them to selection for binding activity for the human C5aR expressed on the surface of CHO cells.

Not surprisingly, the mutants selected from the library on the basis of receptor binding showed a complete absence of mutation of the key receptor-interacting basic residues [K19, K20, R37, R40, R46, K49, K68 (Mollison et al., 1989Go; Bubeck et al., 1994Go; Zhang et al., 1997Go)] and the C residues (C21, 22, C34, C47, C54,55) known to form intramolecular disulphide bridges (Mollison et al., 1989Go; Bubeck et al., 1994Go; Zhang et al., 1997Go). C27, which is mutated in all selected sequences, has been shown not to form a disulphide bridge (Zimmermann and Vogt, 1984Go) and the ubiquitous mutation of C27R may simply be an adaptation to phage display. This was found previously for sequences selected from a randomly mutated intact C5a library (Cain et al., 2000Go). The mutations in the C-terminal domain (G73R, D67N, M70T) are more likely to influence the binding affinity of the variants when expressed as free protein, perhaps by allowing the formation of the loop-helix found at the C-terminus of C5a (Zhang et al., 1997Go) but not C5adR74 (Zuiderweg et al., 1989Go). Certainly, these residues were not mutated in sequences selected from an intact C5a library, suggesting that the mutations are specific for C5adR74 function (Cain et al., 2000Go).

In the absence of structural information on the mutant C5adR74 molecules, it is possible only to speculate on the consequences of the particular mutations. The gain or loss of the two P residues (both of which occur in loops connecting helices) may have consequences for the relative positioning of those helix pairs. The mutation of S16P may affect the position of the N-terminal helix, which links the molecule to the phage particle, optimizing the display position of the C5a for receptor interaction. The presence of P16 is associated with K4R in Variant 2. An attractive alternative explanation may be that P16 coincidentally increases the proximity of residue 4 to the C-terminus, allowing R4 partly to replace R74 in Variant 2. In Variant 1, the mutation of G73 to R might have a similar effect. The substantial differences in the mutation patterns between selected C5a and C5adR74 libraries (Cain et al., 2000, and results presented here) are confirmation that these ligands interact with the C5aR in different ways (Crass et al., 1999Go). The smaller variety of substitutions in the latter may be due to the deleterious effect of mutations on the binding mode of C5adR74. The more optimal binding mode of C5a may allow more mutations whilst retaining sufficient affinity to be selected in these conditions.

Variants 1 and 2 had similar affinities for the human C5aR on transfected RBL cells. This contrasts with the selection of the phage-conjugated polypeptides (Figure 1aGo), when Variant 2, but not Variant 1, was selected in the presence of increasing concentrations of GGS-C5a. However, the variants had affinities that were considerably higher than that of GGS-C5adR74, suggesting that the increased affinities of the phage-conjugated variants were retained in the free polypeptides. In a previous study of selection from a whole-molecule randomly mutated library of full-length C5a, the free polypeptides had a lower receptor affinity than the phage conjugates (Cain et al., 2000Go). Interestingly, native C5adR74 (i.e. without the N-terminal tripeptide extension introduced during production as a His6-tagged protein) has a 4-fold higher receptor-binding affinity than the GGS-C5adR74 form (Figure 1bGo) used in this study. The full-length C5a is not affected by the N-terminal tripeptide extension (Crass et al., 1999Go; Paczkowski et al., 1999Go). Hence the mutagenesis and selection procedure has produced variants of GGS-C5adR74 that are able to tolerate these additions at the N-terminus and therefore which resemble intact C5a in this respect. Previous work using GGS-C5a has shown that the receptor binding affinity is decreased only slightly by the mutation of K68 to E68 whereas the affinity for GGS-C5adR74 is decreased considerably (Crass et al., 1999; results presented here). However, the affinities of the variants decreased considerably when mutated at K68, so in this respect, the variants resemble GGS-C5adR74 more closely than GGS-C5a. The almost complete loss of agonist potential of the E68 variants also suggests that the variants interact with the receptor identically with GGS-C5adR74. In contrast, Variants 1 and 2 were full agonists at the C5aR (unlike GGS-C5adR74), suggesting a closer resemblance to GGS-C5a. Overall, the selected variants appear to have properties intermediate between those of full-length C5a and C5adR74. The mutations selected in the phage display procedure may act, in part, to overcome N-terminal modification. This is likely because the mutant library is displayed as an N-terminal fusion to the bulky Fos linker protein that may interfere with the receptor binding and activation domains of C5adR74.

The E68 variants were antagonists of GGS-C5adR74, but were much less effective at antagonizing receptor activation by GGS-C5a. These data imply that the E68 variants, particularly Variant 2[E68], have a degree of selectivity for the GGS-C5adR74–receptor interaction even though GGS-C5a and GGS-C5adR74 are equally potent agonists for C5aR when expressed in RBL cells. Hence these antagonists may be useful tools in the analysis of the mechanism of ligand selectivity by C5aR and in the design of antagonists that target specific cell types. A C5aR antagonist has previously been produced from phage displayed libraries of C5adR74 randomly mutated between residues 69 and 73 (Hennecke et al., 1998Go). However, prior to mutagenesis these workers introduced mutations (A27, F67) into the molecule to increase affinity and developed an antagonist based on a Fos-fused form of C5adR74, with a molecular weight of 18 kDa. In our approach, we have produced smaller (~9 kDa) molecules with higher affinities than GGS-C5adR74, without making prior assumptions about particular sequences and then introduced a single inactivating mutation (K68E).

In summary, a combination of random and site-directed mutation has allowed the production of a novel C5aR antagonist that shows a degree of specificity for C5adR74 and which is amenable to refinement by further cycles of mutagenesis and selection. Additionally, the positions mutated in the selected library give important information on structure–function relationships that converges with a plethora of previous conventional studies.


    Notes
 
3 To whom correspondence should be addressed. E-mail: p.monk{at}shef.ac.uk Back


    Acknowledgments
 
This work was supported by grants from the Arthritis Research Campaign (M0543), British Heart Foundation (PG95119) and the Wellcome Trust (to P.N.M.).


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 Materials and methods
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 Discussion
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Received August 11, 2000; revised December 5, 2000; accepted December 20, 2000.





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