Lack of evidence from studies of soluble protein fragments that Knops blood group polymorphisms in complement receptor-type 1 are driven by malaria

Abstract

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC(a/b) may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15-25 of its ectodomain. These eleven modules encompass a region (CCPs 15-17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24-25. All four CR1 15-25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15-25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K(D) = 0.8-1.1 µM) and C4b (K(D) = 5.0-5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Figure 1. Schematic of CR1.

(A) Main cartoon shows the 30 CCPs (ovals) in the ectodomain of the most common CR1 size-variant. Long-homologous repeats (LHRs) and positions of functional sites (dark grey) are indicated; the eight-residue linker between long-homologous repeats C and D is drawn as a dotted line along with neighbouring modules (grey); CCPs 24/25 wherein reside the Sl1/2 and McC^a/b-encoded antigens are also highlighted (thicker outline) and their modeled 3D structures drawn as cartoons with K1590 and R1601 side chains shown as spheres.

Figure 2. Recombinant CR1 truncations produced for the current study.

The Coomassie-stained polyacrylamide gels show the results of electrophoresis of successive fractions (with increasing elution volumes, V _e) from size-exclusion chromatography for CR1-21, CR1 21–22, CR1 20–23 and CR1 15–25 (the K1590,R1601 variant); reducing (R) conditions in some lanes would reveal products of intra-modular proteolytic “clipping” that would be held together by disulfide bonds under non-reducing (NR) conditions.

Figure 3. CCPs 21–24 do not exhibit extensive inter-modular contacts.

(A) ¹H,¹⁵N heteronuclear single quantum coherence (HSQC) NMR spectrum of CR1 21 overlaid on that of CR1 21–22. (B) An overlay of the HSQC spectra for CR1 21–22 and 20–23 with (C) blow-ups of two regions. Cross-peaks are color-coded according to the CR1 fragment of origin, as indicated. A dearth of chemical shift changes for CCP 21 and CCP 21–22 amides following attachment of neighbouring modules suggests chemical shift-perturbing inter-modular contacts are limited.

Figure 4. CCPs 21–24 do not exhibit extensive inter-modular contacts.

(A) SAXS profile and fit of the DAMMIF ab initio model (red surface) to the CR1 20–23 data (1.9 mg.ml⁻¹); (B) Guinier plot of the CR1 20–23 SAXS data at 1.9 mg.ml⁻¹ (open circles) and 3.3 mg.ml⁻¹ (open squares), showing no significant concentration dependence of SAXS parameters (plots displaced on the vertical axis for clarity); (C) Distance distribution function, p(r) for CR1 20–23.

Figure 5. CR1 15–25 variants bind equally well to C3b and C4b.

Representative SPR-derived binding curves for the CR1 15–25 variants (as indicated in parentheses) flowed over C3b (upper four panels) or C4b (lower four panels) that were amine-coupled to CM5 chips (see also Figs. S1 and S2). In each case, a set of sensorgrams recorded for a range of CR1 15–25 concentrations (0.25 µM, 0.5 µM, 1 µM, 2.5 µM, 5 µM, 10 µM), are shown above the plot of response versus [CR1 15–25] used to calculate (see Methods) the K _D value listed in Table 2. CR1 variants that are widespread amongst Caucasoids (KR) or Africans (EG) bind equally both to C3b and C4b.

Figure 6. All four CR1 15–25 variants have similar cofactor activity.

The products of CR1/Factor I action on (A) C3b and (B) C4b. In the schematics, disulfides linkages (S-S) are shown as solid lines, and suspected non-disulfide covalent-linkage artifacts (thought to originate from dimerisation in the C4b samples) shown as dotted lines. The gels show the results of SDS-PAGE performed on biotinylated C3b (C) or C4b (D) (see Methods) following incubation (for one hour) with factor I and the CR1 15–25 variant indicated below (KR, KE etc.). A positive control is provided by soluble CR1 (sCR1); negative controls are either bovine serum albumin (BSA) or minus factor I (-FI). Notes: the 2008-Da C3f runs off the gel; the C4b γ chain stains poorly; in the initial C4b sample, degradation products at 25 kDa and 67 kDa are present as contaminants; the reaction was deliberately stopped prior to completion to allow a comparison to be made between the variants. See Figure S3 for ELISA results.

Figure 7. Affinities for C1q.

(A) Representative SPR-derived binding curves for CR1 15–25 variants flowed over a CM5 chip loaded (via amine coupling) with C1q (see also Fig. S4). Upper: sensorgrams recorded for a range of CR1 15–25 concentrations (as in Fig. 3). Lower: plots of response versus [CR1 15–25] used to calculate (see Methods) the K _D values listed in Table 2. CR1 variants that are widespread amongst Caucasoids (KR) or Africans (EG) bind equally to C1q. (B) Data obtained by ELISA showing that C1q (140 ng.mL⁻¹, average of three experiments) bound equally to each of the four variants of CR1 15–25 (adhered to the micro-titer plate). In these experiments, sCR1 was used as a positive control. Error bars represent standard errors of the mean.

Figure 8. None of CR1 15–25 variants interact with P. falciparum protein Rh4.

(A) In an SPR experiment, no CR1 15–25 variant had significant affinity for immobilized Rh4.9, unlike positive controls sCR1 and CR1 1–3 (26). Other constructs lacking CCPs 1–3 (CR1 10–11, CR1 15–17, CR1 20–23, CR1 21–22 and CR1 24–25(KR)) were likewise lacking in affinity for Rh4.9 and, like BSA, served as negative controls. (B) The PfRh4 invasion pathway was not inhibited in the presence of CR1 15–25 variants. Parasite strains 3D7 (black bars for untreated, white bars for nm-treated erythrocytes) and W2mefΔRh4 (grey bars, untreated erythrocytes) were tested in growth assays in the presence of final concentrations of 0.5 mg/ml of the four CR1 15–25 variants (KR, ER etc.). Growth (% of control) on the y-axis refers to the % parasitemia in the presence of CR1 constructs relative to the % parasitemia with the addition of PBS (arbitrarily set to be 100%).

Figure 9. CR1 15–25 variants do not differ in ability to disrupt P. falciparum rosetting.

(A) Both CR1 10–11 (identical to CR1 17–18) and single-module CR1 17 can disrupt rosetting of P. falciparum clone IT/R29, as can anti-CR1 antibody (J3B11) and CR1 15–17 used here as positive controls. The truncation mutants corresponding to CCPs 20–23 (B) and the CR1 homologue, factor H (FH, full-length or fragments as indicated below) (C) did not disrupt rosetting, and served as negative controls. (D) A comparison of the four CR1 15–25 variants suggested they were all equally as effective as full-length sCR1 and CR1 15–17 in terms of rosette disruption. KP: 50 mM potassium phosphate buffer; The mean and standard error of at least four experiments for each graph are shown. *** p<0.001 by ANOVA and Tukey's multiple comparison test.