Scabies Mite Inactivated Serine Protease Paralogs Inhibit the Human Complement System

Expression of SMIPP protein in Pichia pastoris and purification

The nomenclature of the molecules investigated here is based on a phylogenetic tree illustrating the evolutionary relationships between the SMIPPs (17). Recombinant SMIPP-Ss I1 and D1 were produced in P. pastoris by cloning SMIPP-coding sequences retrieved from the original cDNA clones Yv6023A04 (GenBank accession no. AY333081; www.ncbi.nlm.nih.gov/GenBank) and YvT004A06 (GenBank accession no. AY333085) corresponding to the mature sequences predicted (15). To direct secretion of the expressed protein into the medium, all constructs placed the predicted N terminus flush with the Kex2 signal peptide cleavage site in the transfer vector pPICZαA (Invitrogen). A stop codon at the 3′ end of the SMIPP sequence was introduced to ensure that the recombinant protein did not include any of the C-terminal tags of the vector. The plasmid was propagated in Escherichia coli strain TOP10F′, linearized with SacI, and electroporated into P. pastoris KM71H (Invitrogen). Media for colony growth and biomass or induction cultures were made according to the manufacturer’s instructions. Cells were initially plated onto supported nitrocellulose filters placed on agar plates containing 100 μg/ml Zeocin (Invitrogen). After a 24-h incubation at 29°C, filters were moved onto YPDS medium containing 2 mg/ml Zeocin for selection of transformants with increased numbers of SMIPP gene copies. To ensure pure clonal isolates, single colonies were plated twice on two subsequent YPDS/Zeocin agar plates. Transformants were assessed to select for the highest expressing clone according to the manufacturer’s manual. Biomass cultures were grown for 24 h and induction cultures for 60–70 h with shaking at 29°C. SMIPP expression was detected on a Western blot using mouse antiserum generated against purified recombinant SMIPP molecules produced in E. coli. Correct processing of the recombinant protein at the Kex2 cleavage site was confirmed after purification by N-terminal sequencing at the Peptide Biology Laboratory (Monash University, Clayton, Victoria, Australia) (data not shown).

Mature SMIPP protein secreted from P. pastoris was purified as follows: expression culture supernatant was collected following centrifugation at 12,000 × g for 45 min at 4°C and stored at −80°C until required. The supernatant was thawed and prepared for hydrophobic interaction chromatography by the gradual addition of ammonium sulfate to a final concentration of 1.5 M and adjustment of pH to 5.0. The preparation was centrifuged for 30 min at 20,000 × g. The supernatant was filtered through a 0.45-μm filter and the filtrate was loaded onto a 5-ml HiTrap phenyl-Sepharose column (GE Healthcare), pre-equilibrated with 1.5 M ammonium sulfate and 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 10% (v/v) glycerol (pH 5.0). Unbound material was removed by washing with equilibration buffer. Protein was eluted stepwise with 50 ml each of 1.0, 0.5, and 0 M ammonium sulfate. Fractions were subjected to dialysis against 50 mM MES and 10% (v/v) glycerol (pH 5.0) and those containing SMIPP protein, as identified by electrophoresis on SDS-PAGE, were pooled, loaded onto a 5-ml HiTrap SP- Sepharose FF column (GE Healthcare) equilibrated in 50 mM MES (pH 5.0), and washed with 50 ml of the same buffer. A linear gradient of 0–0.4 M NaCl over 30 ml was applied to the column and purification was monitored by electrophoresis of fractions on 10% SDS-PAGE and Western blotting analysis. On average, yields of 0.9–1.3 mg of pure protein per 200 ml of culture were obtained.

Mutagenesis of N-glycosylation sites

When subjected to SDS-PAGE, each SMIPP produced a major band slightly larger than the same molecule produced in E. coli plus a smear of larger material (Fig. 1⇓, A and B). The SMIPP-Ss I1 and D1 have three and two predicted N-glycosylation sites, respectively. Function can be hindered by artifactual hyperglycosylation; therefore, we systematically mutated the predicted glycosylation sites from N to Q at positions 84, 147, and 191 of the mature protein of SMIPP-S I1 and at position 145 and 193 of the mature protein of SMIPP-S D1 singly and in combination by oligonucleotide-based site-directed mutagenesis. Among the created Pichia mutants (Fig. 1⇓, A and B), clones that expressed homogenous, nonglycosylated proteins were chosen for additional experiments. These clones were designated I1 and D1 for SMIPP-S I1 and SMIPP-S D1, respectively.

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FIGURE 1.

Characterization of purified SMIPP proteins. A and B, The purified proteins were separated on 10% SDS-PAGE gels under reducing conditions and visualized by staining with Coomassie blue R-250. A, SMIPP-S I1 expressed in P. pastoris: wild-type protein (lane 1), mutant protein N84Q/N191Q (lane 2), N147Q (lane 3) and N84Q/N147Q/N191Q (lane 4), and E. coli-expressed wild-type protein (lane 5). B, SMIPP-S D1 expressed in P. pastoris: wild-type protein (lane 1), mutant protein N145Q (lane 2), and N193Q (lane 3). SMIPP-S D1 protein with both N-glycosylation sites mutated was unstable (data not shown). Lane 4 shows E. coli-expressed wild-type protein. The proteins used for the complement assays reported here are indicated by arrows. C and D, The proteolytic activity of the SMIPPs was tested using degradation assays. Two concentrations of each SMIPP (100 and 200 μg/ml) were incubated with trace amounts of ¹²⁵I-C4b (C) or ¹²⁵I-C3b (D) for 1.5 h at 37°C. The reaction was terminated by addition of SDS-PAGE sample buffer, containing DTT, followed by heating at 95°C for 3 min. The proteins were separated on a 10–15% gradient SDS-PAGE gel and visualized using PhosphorImager. Factor I, along with its cofactors C4BP (for C4b degradation) and factor H (for C3b degradation), was used as a positive control, as was interpain A, a bacterial proteinase. In the negative controls, only ¹²⁵I-C4b or ¹²⁵I-C3b was added. (FI, factor I; FH, factor H).

Proteins

C1, C2, C3b, C4b, C5, C6, C7, C8, C9, factor B, factor D, and properdin were purchased from Complement Technology. Human MBL was purchased from Statens Serum Institutet (Copenhagen, Denmark). C1q (18), factor H (19), C4b-binding protein (C4BP) (20), and factor I (21) were purified from human plasma as described previously. The stalk and head regions of C1q were prepared by partial proteolytic digestion of intact C1q as described previously (22, 23). Pepsin (Worthington Biochemical) was used for preparation of the stalk region and purified collagenase (Clostridium histolyticum, code CLSPA) for preparation of the head region was purchased from the same company. C3b and C4b were labeled with ¹²⁵I using the chloramine-T method (24). The specific activity was 0.4–0.5 MBq/μg of protein. Interpain A was expressed and purified as described previously (25).

C3b/C4b degradation assay

To confirm that the two SMIPPs tested in this study were indeed inactive proteases and not able to cleave complement proteins, such as C3b or C4b, a degradation assay was performed. Two concentrations (100 and 200 μg/ml) of SMIPPs were mixed with trace amounts of ¹²⁵I-labeled C3b or C4b in TBS in a total volume of 40 μl. As positive controls, 15 μg/ml factor I, along with 200 μg/ml of a cofactor (C4BP for the degradation of C4b or factor H for the degradation of C3b), were added to the ¹²⁵I-C3b or ¹²⁵I-C4b. Recently, interpain A, a cysteine proteinase from Prevotella intermedia, has been shown to inhibit complement by degrading C3 and C4 (25). Therefore, interpain A was also tested here as a positive control at a concentration of 1 μM. The samples were incubated for 1.5 h at 37°C and the reaction was terminated by the addition of SDS-PAGE sample buffer with reducing agent (DTT). The samples were then incubated at 95°C for 3 min and separated on a 10–15% gradient SDS-PAGE gel. The separated proteins were visualized by using a PhosphorImager (Molecular Dynamics/GE Healthcare).

Hemolytic assay

To assess the activity of the classical pathway of complement, sheep erythrocytes (Swedish National Veterinary Institute) were washed three times with DGVB²⁺ buffer (2.5 mM Veronal buffer (pH 7.3), 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 1 mM MgCl₂, and 0.15 mM CaCl₂), resuspended to a concentration of 10⁹ cells/ml, and incubated, with gentle shaking for 20 min at 37°C, with an equal volume of amboceptor (Dade Behring) diluted 1/3000 in DGVB²⁺ buffer. Amboceptor is an Ab against sheep erythrocytes, which activates the classical pathway of complement. After two washes with DGVB²⁺, 8 × 10⁷ cells/ml were incubated for 1 h at 37°C with 0.125% normal human serum (NHS), diluted in DGVB²⁺ buffer, in a total volume of 150 μl. Before incubation with erythrocytes, NHS was preincubated with various concentrations of SMIPPs or BSA, as a negative control, for 15 min at room temperature (RT). NHS was prepared from blood of six healthy volunteers after informed consent and following review by the local ethical board at Lund University. The samples were centrifuged and the hemolytic activity (i.e., the amount of lysed erythrocytes) was determined by spectrophotometric measurement of the amount of released hemoglobin (405 nm). The lysis obtained in the absence of SMIPPs was defined as 100% hemolytic activity.

To assess the activity of the alternative pathway, rabbit erythrocytes (obtained from a local farm) were washed three times with Mg²⁺ EGTA buffer (2.5 mM Veronal buffer (pH 7.3) containing 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 7 mM MgCl₂, and 10 mM EGTA). Erythrocytes at a concentration of 6 × 10⁷ cells/ml were incubated for 1 h at 37°C with 1.3% NHS diluted in Mg²⁺ EGTA buffer in a total volume of 150 μl. Before incubation with erythrocytes, NHS was preincubated with various concentrations of SMIPPs or BSA, as a negative control, for 15 min at RT. The samples were centrifuged and the hemolytic activity was determined spectrophotometrically as described above. The lysis obtained in the absence of SMIPPs was defined as 100% hemolytic activity.

Complement activation assays

Unless stated otherwise, all incubation steps were done at RT, in 50 μl of solution, followed by extensive washing with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% (v/v) Tween 20. Microtiter plates (Maxisorp; Nunc) were incubated overnight at 4°C with 75 mM sodium carbonate (pH 9.6) containing the following: for the classical pathway, 2.5 μg/ml aggregated human IgG (Immuno); for the lectin pathway, 100 μg/ml mannan (Sigma-Aldrich); and for the alternative pathway, 20 μg/ml zymosan (Sigma-Aldrich). The wells were blocked with 200 μl of blocking solution (1% (w/v) BSA in PBS) for 2 h. For the classical and lectin pathways, respectively, 3% NHS was diluted in DGVB²⁺ buffer and incubated for 20 min (for detection of C4b and C3b) or 45 min (for detection of C1q and C9) at 37°C. For the alternative pathway, 5% NHS was diluted in Mg²⁺ EGTA buffer and incubated for 35 min for detection of C3b or for 1 h for detection of C9. NHS was preincubated for 15 min at RT with various concentrations of SMIPPs or BSA, as a negative control, before addition to the microtiter plate. Complement activation was assessed by detection of deposited complement proteins using rabbit polyclonal Abs against C1q (DakoCytomation), C4b (DakoCytomation), C3d (DakoCytomation), or goat polyclonal Abs against MBL (R&D Systems) and C9 (Complement Technology) diluted in blocking solution. After a 1-h incubation, HRP-conjugated secondary Abs against rabbit or goat Igs (DakoCytomation), diluted in blocking solution, were added for 30 min. Bound enzyme was detected using 1,2-phenylenediamine dihydrochloride tablets (DakoCytomation) and the absorbance was measured at 490 nm. The absorbance obtained in the absence of SMIPPs was defined as 100%.

Direct binding assay

Unless stated otherwise, all incubation steps were done in 50 μl of solution, followed by extensive washing, as described for the complement activation assays. Various complement proteins were coated onto microtiter plates as described above at a concentration of 10 μg/ml. BSA (1% (w/v)) was used as a negative control. The wells were blocked with 200 μl of blocking solution and incubated at RT for 2 h. When screening for binding of various complement proteins and examining binding of C1, C1q, C1q head, and C1q stalk, 200 μg/ml SMIPP preparations were added to the wells. A titration was also performed for the binding of C1q, MBL, and properdin using concentrations ranging between 12.5 and 200 μg/ml. The SMIPPs were diluted in 50 mM HEPES (pH 7.4), 100 mM NaCl, and 2 mM CaCl₂ and were allowed to bind overnight at 4°C. Specific murine polyclonal Abs against SMIPPs were then added to the wells, followed by a HRP-conjugated goat anti-mouse IgG Ab (DakoCytomation). All Abs were diluted in blocking buffer and incubated at RT for 1 h. Bound enzyme was detected as described above.

Assembly and decay of the alternative C3 convertase

To measure the assembly and decay of the alternative C3 convertase in NHS and in the presence of SMIPPs, a microtiter plate-based assay was performed as described previously (26). Briefly, microtiter plates were coated with 100 μl of 0.1% agarose in water for 48 h at 37°C. To study the assembly of the alternative C3 convertase, in the presence of SMIPPs, 5% NHS in Mg²⁺ EGTA buffer was preincubated with 0–200 μg/ml D1, I1, or BSA, as a negative control, for 15 min at RT in a total volume of 50 μl. Factor H, at a concentration of 100 μg/ml, was used as a positive control. After preincubation, the samples were added to the agarose- coated plates, incubated for 1 h at 37°C, and then washed three times with 250 μl of TBS containing 10 mg/ml BSA and 2 mM MgCl₂. The amount of assembled C3 convertases, i.e., the amount of deposited factor B and properdin, was detected by addition of 100 μl of either goat anti-human factor B (Complement Technology) or goat anti-human properdin (Complement Technology) diluted 1/1000 in TBS containing 10 mg/ml BSA and 2 mM MgCl₂. After a 1-h incubation at 37°C, the plates were washed as above and 100 μl of 1/1000 diluted HRP-conjugated rabbit anti-goat IgG (DakoCytomation) was added and incubated for 1 h at 37°C. The plates were then washed twice with TBS containing 10 mg/ml BSA, once with TBS containing 10 mg/ml BSA and 0.1% (v/v) Tween 20, and finally once with normal TBS. The amount of remaining factor B and properdin on the plate was detected as above.

To measure the decay of the alternative C3 convertase, in the presence of SMIPPs, the assay was mainly performed as above, but instead of preincubating serum and proteins and adding them together to the plate, 5% NHS was first added alone to the plate and incubated for 1 h at 37°C. After washing three times with TBS containing 10 mg/ml BSA and 2 mM MgCl₂, the SMIPPs and factor H were added and incubated for 1 h at 37°C. The amount of remaining factor B and properdin was then detected as above.

Localization of human C1q and IgG by immunohistochemistry

Human skin biopsies infested with scabies mites were formalin-fixed overnight, paraffin embedded, and cut into 4-μm serial sections. For localization studies, slides were prepared as described previously (4, 5) and serial sections underwent an Ag heat-retrieval procedure in a solution containing 1% (w/v) Revealit-Ag in 20 mM Tris (pH 7.5; ImmunoSolution) at 85°C for 5 min. The slides were cooled and washed with distilled water for 10 min followed by 15 min washes in PBS at pH 7.2. Serial sections were blocked with 10% donkey serum in 1% (w/v) BSA in PBS. Endogenous peroxidase activity was blocked with 3% (v/v) H₂O₂ in blocking buffer. The sections were incubated overnight with rabbit anti-human C1q polyclonal Ab (DakoCytomation) at a 1/200 dilution. The primary Ab was probed with the secondary Ab Dako-EnVision anti-rabbit-HRP conjugate (DakoCytomation) for 20 min. As a positive control, to identify the mite gut, adjacent sections were incubated with anti-human IgG-HRP polyclonal Ab (Abcam) in 1/500 dilutions. As a negative control, preimmune serum in 1/500 dilutions was incubated on sections of the same series. All slides were washed in PBS and the Vector NovaRed peroxidase substrate kit (Vector Laboratories) was used for staining according to the manufacturer’s recommendations. Counterstaining with hematoxylin was done as previously described (4, 5). Ethical approval for the use of shed skin crusts from the bedding of a patient with recurrent crusted scabies was obtained from the Human Research Ethics Committee of the Northern Territory Department of Health and Community Services and the Menzies School of Health Research.

Statistical analysis

All experiments were done at least three times in duplicates, if not stated otherwise. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test (GraphPad Software). Values of p < 0.05 were considered significant (∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001).

SMIPPs show no proteolytic activity

Despite their structural similarity to proteases, SMIPPs were predicted to be inactive due to the structure of their putative active sites (17). To confirm this hypothesis, we tested whether the SMIPPs could degrade the activated complement components C3b and C4b similarly to what we observed for bacterial proteases such as interpain A (24). The two SMIPPs D1 and I1 showed no proteolytic activity, since no degradation products of either C4b (Fig. 1⇑C) or C3b (Fig. 1⇑D) could be detected in contrast to what was observed for positive controls.

SMIPPs inhibit the hemolytic activity of human serum

To determine whether the two SMIPPs D1 and I1 had any inhibitory effect on the human complement system, two hemolytic assays were performed measuring activity of the classical and the alternative pathway, respectively. Human serum, as a source of all complement proteins, was pretreated with various concentrations of SMIPPs before incubation with erythrocytes. Both SMIPPs were able to fully inhibit both the classical pathway (Fig. 2⇓A) and alternative pathway (Fig. 2⇓B). Higher concentrations of SMIPPs (200 μg/ml) were required to inhibit the alternative pathway compared with the classical pathway (75 μg/ml). This is probably due to differences in serum concentration used in the two assays: 1.3% serum in the alternative hemolytic assay vs 0.125% serum in the classical hemolytic assay. These serum concentrations were chosen on the basis of an initial titration and represent conditions in which each assay is most sensitive to changes. The alternative pathway is known to require higher concentrations of serum to function properly.

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FIGURE 2.

SMIPPs inhibit the hemolytic activity of human serum. Sheep erythrocytes sensitized with Abs (A, classical pathway) or rabbit erythrocytes (B, alternative pathway) were incubated with 0.125% or 1.3% NHS, respectively. Serum was preincubated for 15 min at RT with various concentrations of SMIPPs or BSA as a negative control. After 1 h of incubation of NHS with erythrocytes at 37°C, the degree of lysis was estimated by the measurement of released hemoglobin (absorbance at 405 nm). The lysis obtained in the absence of SMIPPs was defined as 100% hemolytic activity. C, The hemolytic activity of the fully glycosylated counterparts of D1 and I1 was tested in both the classical and alternative pathways as described above. One concentration of each protein was tested: 75 μg/ml for the classical pathway and 200 μg/ml for the alternative pathway. An average of three independent experiments performed in duplicate is presented with bars indicating SD. C, The statistical significance of the differences between BSA and all tested SMIPPs were estimated for the two assays using a one-way ANOVA with Tukey’s multiple comparison test; ∗∗∗, p < 0.001.

To elucidate whether the nonglycosylated proteins used in this study behaved in the same manner as the fully glycosylated counterparts, we also tested fully glycosylated D1 and I1 in hemolytic assays. One concentration of each of the SMIPP proteins was tested for the classical pathway (75 μg/ml) and alternative pathway (200 μg/ml). Fully glycosylated D1 and I1 inhibited both complement pathways to a similar extent as their nonglycosylated counterparts (Fig. 2⇑C). However, inhibition of the classical pathway was somewhat less pronounced for glycosylated I1 compared with the nonglycosylated molecule (p < 0.001). We also tested different concentrations of the fully glycosylated D1 and I1 in the complement activation assays for the three pathways, measuring C3b deposition (data not shown). We found that the nonglycosylated and the fully glycosylated D1 and I1 inhibited all three pathways in a very similar manner. The small difference seen for I1 in the classical pathway hemolytic assay could, however, also be noticed in the classical pathway complement activation assay, but not in the assays measuring activity of the lectin and alternative pathways. Based on the similar results obtained for the non- and fully glycosylated proteins, we continued testing only the nonglycosylated variants in this study.

SMIPPs inhibit all three complement pathways

The complement system works in a cascade-like manner with consecutive proteolytic activation of a number of proteins. To assess at what level of the cascade the SMIPPs inhibit complement, a microtiter plate-based deposition assay was performed in which complement activation was initiated by specific ligands for each pathway. After addition of human serum, pretreated with SMIPPs, the deposited complement proteins were detected using specific Abs.

In the case of the classical pathway, complement was activated by aggregated IgG deposited on the plate. When adding serum together with either D1 or I1, deposition of all four tested complement factors, C1q, C4b, C3b, and C9, was decreased (Fig. 3⇓, left panel). The same pattern was observed for the lectin pathway; after activation with mannan, deposition of MBL, C4b, C3b, and C9 was inhibited by both D1 and I1 (Fig. 3⇓, right panel). This was also true for the alternative pathway; after initiation with immobilized zymosan, C3b and C9 deposition was decreased (Fig. 4⇓). Overall, D1 seemed to inhibit the pathways slightly less efficiently than I1, especially for the alternative pathway, where double the amount of D1 (400 μg/ml for D1 and 200 μg/ml for I1) was needed to completely inhibit deposition of C3b and C9.

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FIGURE 3.

SMIPPs inhibit the classical and the lectin pathways. Aggregated human IgG (classical pathway, left panel) and mannan (lectin pathway, right panel) were immobilized on microtiter plates and allowed to activate 2% or 3% NHS, respectively, containing various concentrations of SMIPPs or BSA as a negative control (preincubated for 15 min at RT). After 20 min (for C4b and C3b) or 45 min (for C1q, MBL, and C9) of incubation at 37°C, the plates were washed and the deposited C1q, MBL, C4b, C3b, and C9 was detected with specific polyclonal Abs. The absorbance obtained in the absence of SMIPPs was defined as 100%. An average of three independent experiments performed in duplicate is presented with bars indicating SD.

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FIGURE 4.

SMIPPs inhibit the alternative pathway. Zymosan was immobilized on microtiter plates and allowed to activate 5% NHS containing various concentrations of SMIPPs or BSA as a negative control (preincubated for 15 min at RT). After 35 min (for C3b) or 1 h (for C9) of incubation, the plates at 37°C were washed and the deposited C3b and C9 were detected with specific polyclonal Abs. The absorbance obtained in the absence of SMIPPs was defined as 100%. An average of three independent experiments performed in duplicate is presented with bars indicating SD.

SMIPPs bind directly to C1q, MBL, and properdin

Structural and functional analysis of the two SMIPPs analyzed in the current study found that they are devoid of proteolytic activity (17) and we could not detect any degradation of complement proteins such as C3 and C4 by the SMIPPs (Fig. 1⇑, C and D). To elucidate whether the complement inhibition seen in the previous assays is due to SMIPPs binding to complement proteins, a microtiter plate-based direct binding assay was used. A first screening was performed, where the plates were coated with various complement proteins, followed by incubation with one concentration (200 μg/ml) of SMIPPs and the binding was detected with specific murine polyclonal Abs against D1 and I1. In this direct binding assay, a clear interaction of both D1 (Fig. 5⇓A) and I1 (Fig. 5⇓B) with C1q, MBL, and properdin was observed under conditions of physiological ionic strength and pH. The strongest binding of D1 and I1 was observed to C1q. To examine these interactions further, various concentrations (from 0 to 200 μg/ml) of the SMIPPs were allowed to bind to the immobilized complement proteins and the results showed that the binding of C1q, MBL, and properdin was dose dependent for both D1 (Fig. 5⇓C) and I1 (Fig. 5⇓D). Also, under these experimental conditions, the binding to C1q was shown to be the strongest interaction. Cross-competition assays in which D1 or I1 was allowed to bind immobilized complement proteins in the presence of another complement protein as competitor showed that C1q, MBL, and properdin bind to different sites on SMIPPs (data not shown).

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FIGURE 5.

Direct binding of SMIPPs to various complement proteins. A, B, and E, Microtiter plates were coated with various complement proteins or BSA as a negative control and incubated with 200 μg/ml D1 or I1 overnight at 4°C. C and D, Microtiter plates were coated with C1q, MBL, properdin, or BSA and various concentrations of D1 (C) and I1 (D) were added. In all of the assays, bound protein was detected using specific polyclonal Abs against D1 and I1. An average of three independent experiments performed in duplicate is presented with bars indicating SD. The statistical significance of differences between BSA and the rest of the groups was estimated using a one-way ANOVA with Tukey’s multiple comparison test: ∗_*, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.

SMIPPs interact mainly with the collagenous stalk of C1q

C1q is part of the C1 complex along with two molecules of each of the smaller proteolytic subunits C1r and C1s. C1q is the recognition molecule responsible for the activation of the classical pathway and it is composed of six subunits. Each subunit has a globular head at the C terminus and a collagenous stalk at the N terminus. C1q binds many different ligands and most of the ligands that activate C1q and lead to activation of the classical pathway bind to the globular head domains. On the contrary, ligands binding to the collagenous stalk of C1q may inhibit activation via the classical pathway (27).

To identify the binding site for SMIPPs on C1q, head and stalk regions of C1q were prepared, using partial proteolytic digestion of intact C1q, with collagenase (for heads) and pepsin (for stalks). By coating these isolated parts of C1q on a microtiter plate, we could demonstrate that the binding of both D1 and I1 was localized to the collagenous stalk region of C1q (Fig. 5⇑E). The binding to the globular heads was not significantly different from the binding to the negative control BSA. We also demonstrated that the binding to C1q is significantly stronger than to the intact C1 complex. This is probably due to the fact that C1r and C1s also interact with the stalks and may be competing to some extent with SMIPPs.

SMIPPs inhibit assembly of alternative pathway C3 convertases due to their ability to bind properdin, but they do not accelerate decay of preassembled convertases

We observed that the SMIPPs inhibited the alternative pathway of complement, both in hemolytic (Fig. 2⇑B) and complement deposition assays (Fig. 4⇑). This could be due to binding of the positive regulator of this pathway, properdin, which occurs as shown in Fig. 5⇑. To investigate whether the binding of the SMIPPs to properdin would inhibit the assembly and/or accelerate the decay of the alternative C3 convertase, a microtiter plate-based assay was performed. Agarose, a polysaccharide that will activate the alternative pathway of complement (28), was coated onto microtiter plates, followed by addition of NHS, as a source of complement proteins and the formed C3 convertases were detected by measuring the amount of deposited factor B and properdin using specific polyclonal Abs. To assess the assembly of the alternative C3 convertase, in the presence of SMIPPs, D1 and I1 were preincubated with NHS before being added to the agarose- coated plates. Factor H was used as a positive control, since it is known to bind C3b and hinder the formation of the alternative C3 convertase. Both D1 and I1 significantly hindered the formation of the convertase (Fig. 6⇓A) at both tested concentrations, as indicated by the reduced amount of deposited factor B and properdin. To assess the ability of the SMIPPs to accelerate the decay of the alternative C3 convertase, the proteins were added after NHS incubation, i.e., after the assembly of the convertases. Factor H was also in this setup used as a positive control, due to its known ability to accelerate the decay of the alternative convertase, by directly binding C3b and displacing Bb from the convertase. This can be seen in Fig. 6⇓B, where the addition of factor H resulted in a decrease of deposited factor B. However, the SMIPPs were not able, at any tested concentration, to accelerate the decay of the alternative C3 convertase (Fig. 6⇓B). Together, these results demonstrate that the SMIPPs are able to inhibit the assembly of the alternative C3 convertase, but once the convertase is preformed, SMIPPs are not able to accelerate its decay.

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FIGURE 6.

SMIPPs inhibit assembly of alternative pathway C3 convertases due to their ability to bind properdin, but they do not accelerate decay of preassembled convertases. Microtiter plates were coated with agarose, followed by the addition of NHS, and the C3 convertases formed were detected by measuring the amount of deposited factor B and properdin with specific polyclonal Abs. The SMIPPs (or BSA as a negative control) were either added together with NHS to study the assembly of the convertase (A) or after NHS incubation to study the decay (B). Factor H was added as a positive control but only at one concentration; 100 μg/ml. The absorbance obtained in the absence of SMIPPs was defined as 100% and an average of three independent experiments performed in duplicate is presented with bars indicating SD. The statistical significance of differences between BSA and the rest of the groups was estimated using a one-way ANOVA with Tukey’s multiple comparison test: ∗∗∗, p < 0.001. FH, factor H.

SMIPPs colocalize with human complement factor C1q in the mite digestive system

The presence of complement factors in the scabies mite gut was clearly demonstrated by probing serial sections of human mite-infested skin with Abs against human C1q in comparison to staining with human IgG, which for the purpose of this experiment served as a positive control for gut localization (Fig. 7⇓). Human IgG was localized to the gut (Fig. 7⇓B) as previously documented (4, 5). This confirmed that, in the adjacent serial section (Fig. 7⇓C), labeling by anti-C1q Abs was indeed in the gut. Staining of the gut was the most easily seen; however, sections that were in the right plane also showed staining in the esophagus (Fig. 7⇓, B and C). The localization of SMIPP proteins to the digestive system of the mite has previously been shown in sections of scabies-infested human skin (4, 5) probed with polyclonal Abs against individual SMIPPs. In all cases, the sections probed with the preimmune serum only stained with the counterstain, as demonstrated in Fig. 7⇓D, clearly excluding any unwanted background staining.

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FIGURE 7.

Immunohistological colocalization of C1q and host IgG indicates that human complement is ingested into the mite gut. A schematic diagram (A) outlines the features visible in three serial histological sections of mite-infested epidermis. The epidermis is shown in blue, the burrows in white, the bodies of two mites in gray, and the mite gut in red. B, C, and D. The matching serial histological sections. Red staining indicates binding of Ab to protein. Section (B) was probed with anti-human IgG, a marker for the mite gut (4 ). Section C shows staining of the mite gut when probing with anti-C1q, while section D, probed with preimmune mouse serum as a negative control, remained unstained. b, Burrow wall; c, cuticle; es, esophagus; g, gut; m, mouth parts. Scale bar, 100 μm).