Salivary complement inhibitors from mosquitoes: Structure and mechanism of action
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.015230
ISSN1083-351X
AutoresEthan C. Strayer, Stephen Lu, José M. C. Ribeiro, John F. Andersen,
Tópico(s)Vector-borne infectious diseases
ResumoInhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family. Inhibition of the alternative pathway (AP) of complement by saliva from Anopheles mosquitoes facilitates feeding by blocking production of the anaphylatoxins C3a and C5a, which activate mast cells leading to plasma extravasation, pain, and itching. We have previously shown that albicin, a member of the SG7 protein family from An. Albimanus, blocks the AP by binding to and inhibiting the function of the C3 convertase, C3bBb. Here we show that SG7.AF, the albicin homolog from An. freeborni, has a similar potency to albicin but is more active in the presence of properdin, a plasma protein that acts to stabilize C3bBb. Conversely, albicin is highly active in the absence or presence of properdin. Albicin and SG7.AF stabilize the C3bBb complex in a form that accumulates on surface plasmon resonance (SPR) surfaces coated with properdin, but SG7.AF binds with lower affinity than albicin. Albicin induces oligomerization of the complex in solution, suggesting that it is oligomerization that leads to stabilization on SPR surfaces. Anophensin, the albicin ortholog from An. stephensi, is only weakly active as an inhibitor of the AP, suggesting that the SG7 family may play a different functional role in this species and other species of the subgenus Cellia, containing the major malaria vectors in Africa and Asia. Crystal structures of albicin and SG7.AF reveal a novel four-helix bundle arrangement that is stabilized by an N-terminal hydrogen bonding network. These structures provide insight into the SG7 family and related mosquito salivary proteins including the platelet-inhibitory 30 kDa family. Feeding by mosquitoes and other hematophagous arthropods elicits host responses aimed at preventing blood loss and controlling microbial infection (1Ribeiro J.M. Mans B.J. Arca B. An insight into the sialome of blood-feeding nematocera.Insect Biochem. Mol. Biol. 2010; 40: 767-784Crossref PubMed Scopus (119) Google Scholar). 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An inhibitor of complement C5 provides structural insights into activation.Proc. Natl. Acad. Sci. U S A. 2020; 117: 362-370Crossref PubMed Scopus (8) Google Scholar, 16Valenzuela J.G. Charlab R. Mather T.N. Ribeiro J.M. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis.J. Biol. Chem. 2000; 275: 18717-18723Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The complement system is a proteolytic cascade that is rapidly induced by microbial infection or tissue damage leading to opsonization of microbes, synthesis of microbicidal membrane attack complexes, and production of proinflammatory anaphylatoxins. These processes lead to the phagocytosis of invading pathogens, lysis of pathogen membranes, and induction of antimicrobial inflammatory responses. Arthropod-produced complement inhibitors attack various points in the classical (CP), lectin (LP), alternative (AP), and common pathways of the complement cascade. These pathways of complement activation are initiated differently but result in the conversion of the plasma protein C3 to its activated form, C3b, by proteolytic convertase complexes (17Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2135) Google Scholar). Newly formed C3b reacts with nucleophilic groups by means of its labile thioester moiety and becomes covalently linked at the microbial surface. Once attached, it binds with a serine protease zymogen, factor B, which is then cleaved by a second serine protease, factor D to form a complex known as the alternative C3 convertase, C3bBb, which itself cleaves C3 to form C3b. At this point, the complement response is enormously amplified as each C3bBb complex produces many C3b molecules, each with the potential of binding to a microbial surface and forming a new alternative convertase complex. Downstream steps of the common pathway of complement include the activation of C5, a key component of the membrane attack complex, by the C5 convertase, which also requires C3b and thus depends on amplification of the alternative convertase (17Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2135) Google Scholar). The plasma protein properdin is essential for normal C3bBb function as it acts as a pattern recognition molecule for binding of the convertase complex to microbial surfaces and stabilizes covalently bound C3bBb, greatly extending its active lifetime (18Kemper C. Atkinson J.P. Hourcade D.E. Properdin: emerging roles of a pattern-recognition molecule.Annu. Rev. Immunol. 2010; 28: 131-155Crossref PubMed Scopus (159) Google Scholar, 19Spitzer D. Mitchell L.M. Atkinson J.P. Hourcade D.E. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J. Immunol. 2007; 179: 2600-2608Crossref PubMed Scopus (213) Google Scholar). Salivary inhibitors from ixodid ticks are effective complement inhibitors that function by scavenging properdin, thereby preventing its binding with convertase complexes (20Hourcade D.E. Akk A.M. Mitchell L.M. Zhou H.F. Hauhart R. Pham C.T. Anti-complement activity of the Ixodes scapularis salivary protein Salp20.Mol. Immunol. 2016; 69: 62-69Crossref PubMed Scopus (23) Google Scholar, 21Tyson K.R. Elkins C. de Silva A.M. A novel mechanism of complement inhibition unmasked by a tick salivary protein that binds to properdin.J. Immunol. 2008; 180: 3964-3968Crossref PubMed Scopus (50) Google Scholar). Natural inhibitors of the complement pathway and their mechanisms of inhibition are of continuing interest. Several diseases, including age-related macular degeneration (AMD), atypical hemolytic uremic syndrome (aHUS), and paroxysmal nocturnal hemoglobinuria (PNH) occur as a result of unregulated complement activation, and drugs targeting the complement pathway are currently being studied or are in use for their treatment (22Park D.H. Connor K.M. Lambris J.D. The challenges and promise of complement therapeutics for ocular diseases.Front. Immunol. 2019; 10: 1007Crossref PubMed Scopus (31) Google Scholar, 23Woodruff T.M. Nandakumar K.S. Tedesco F. Inhibiting the C5-C5a receptor axis.Mol. Immunol. 2011; 48: 1631-1642Crossref PubMed Scopus (205) Google Scholar). Previously, we isolated albicin, a member of the salivary SG7 protein family from females of the malaria mosquito An. albimanus, which inhibits activation of the AP in human serum as measured by lysis of rabbit erythrocytes, blocks the cleavage of C3 and factor B in serum, and binds specifically to the C3bBb complex (14Mendes-Sousa A.F. Queiroz D.C. Vale V.F. Ribeiro J.M. Valenzuela J.G. Gontijo N.F. Andersen J.F. An inhibitor of the alternative pathway of complement in saliva of new world anopheline mosquitoes.J. Immunol. 2016; 197: 599-610Crossref PubMed Scopus (10) Google Scholar). On surface plasmon resonance (SPR) surfaces of properdin, albicin stabilizes binding of the complex but also prevents C3 cleavage by reconstituted solution phase C3bBb in the absence of properdin (14Mendes-Sousa A.F. Queiroz D.C. Vale V.F. Ribeiro J.M. Valenzuela J.G. Gontijo N.F. Andersen J.F. An inhibitor of the alternative pathway of complement in saliva of new world anopheline mosquitoes.J. Immunol. 2016; 197: 599-610Crossref PubMed Scopus (10) Google Scholar). It does not directly inhibit the enzymatic activity of factor D, nor does it block the cleavage of factor B. Salivary gland extracts of a second Anopheles species, An. freeborni, also inhibit the activation of complement in serum (14Mendes-Sousa A.F. Queiroz D.C. Vale V.F. Ribeiro J.M. Valenzuela J.G. Gontijo N.F. Andersen J.F. An inhibitor of the alternative pathway of complement in saliva of new world anopheline mosquitoes.J. Immunol. 2016; 197: 599-610Crossref PubMed Scopus (10) Google Scholar). In this study, we describe the structures of albicin and its orthologs SG7.AF (from An. freeborni) and anophensin (from An. stephensi), characterize the binding and mechanism of AP inhibition by these inhibitors, and determine a role for properdin in modulating the inhibitory activity of SG7.AF. We also demonstrate that while the SG7 protein family is distributed throughout the species of Anopheles, not all variants possess strong anti-AP activity. Complement activation can be quantified by observing the lysis of rabbit erythrocytes when incubated with human serum. These cells consistently activate the AP in human serum and are considered as surrogates for foreign cells encountered during infection in vivo. Albicin was previously shown to prevent activation of the AP in the lysis assay using human serum. The protein was equally effective in normal human (NHS) and properdin-depleted (PDS) serum indicating that properdin is not involved in its inhibitory mechanism (Fig. 1, A–B). We performed the same assays with recombinant SG7.AF from An. freeborni and found it to block erythrocyte lysis effectively in NHS but less potently in PDS (Fig. 1, A and C). The IC50 value for SG7.AF was similar to that of albicin in NHS but increased by sixfold in PDS suggesting a role for properdin in its mechanism of action (Fig. 1, B–C). Identically to albicin, SG7.AF showed no ability to inhibit the CP (Fig. S1A). The fact that SG7.AF remained inhibitory, albeit less so, in the absence of properdin indicates that it does not function simply as a scavenger of properdin, but rather is made more potent in its anti-C3bBb activity by properdin. Mechanistic differences between properdin-independent and -dependent SG7 inhibitors were probed by comparison of SG7.AF with albicin in a variety of additional assays. In supernatants of erythrocyte lysis assay preparations, the cleavage of C3, as measured by the appearance of its cleavage product C3a on western blots, was inhibited in the presence of SG7.AF as was the cleavage of factor B, indicating that SG7.AF interferes with the production of the alternative C3 convertase and prevents amplification of complement activation in a manner similar to albicin (Fig. 2). Like albicin, SG7.AF prevents C3b, factor B, and properdin deposition from serum onto agarose-coated plates, which are considered to mimic the foreign surfaces activating complement in vivo by supporting covalent linkage of C3b through hydroxyl groups on agarose (16Valenzuela J.G. Charlab R. Mather T.N. Ribeiro J.M. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis.J. Biol. Chem. 2000; 275: 18717-18723Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). This verifies that SG7.AF blocks AP activation at the point of the C3bBb complex or before (Fig. 3, A–C). No significant binding of individual protein components of the AP to plate bound SG7.AF was observed when measured by ELISA, suggesting that the inhibitor binds to the assembled complex rather than a single protein (Fig. S1B). Additionally, SG7.AF did not induce dissociation of immobilized C3bBb complexes preassembled on agarose plates, while addition of factor H causes release of factor B and properdin (Fig. 3, D–F). This demonstrates that, like albicin, SG7.AF does not disrupt the integrity of the complex in the manner of factor H/factor I or other endogenous complement regulators. SG7.AF did inhibit the enzymatic activity of reconstituted C3bBb complexes in solution as measured by the appearance of C3a after incubation of C3 with preassembled C3bBb, but was less potent than albicin, which completely blocks the appearance of C3a on western blots (Fig. 4A). Like albicin, SG7.AF did not block the activation of factor B by factor D (conversion of C3bB to C3bBb) demonstrating that it targets the C3bBb complex directly, rather than inhibiting the serine protease responsible for its activation (Fig. 4B). Together, these data suggest that SG7.AF acts similarly to albicin in that it inhibits the catalytic activity of the C3bBb complex by directly interacting with it. However, it is more potent in the presence of properdin than in its absence.Figure 3Deposition and displacement of C3b, factor Bb, and properdin in the presence of SG7.AF: Agarose-coated plates were incubated with NHS (20%) and different concentrations of SG7.AF at 37 °C for 30 min and probed with (A) anti-C3 (1:5000), (B) anti-factor B (1:200), or (C) anti-properdin (1:200). Each experiment was run twice and replicated three times in each run. The data were normalized to the zero concentration value and the points represent means ± standard deviation. For displacement assays, plates were incubated with NHS (20%) for 30 min at 37 °C followed by incubation with SG7.AF (800 nM) or factor H (10 μg) for 30 min at 37 °C and probed with (D) anti-C3 (1:5000), (E) anti-factor B (1:200), or (F) anti-properdin (1:200). Wells treated with serum not containing SG7.AF were used as positive control, and wells probed in the absence of both serum and SG7.AF were used as negative control. Each experiment was run twice and replicated three times in each run. The data were normalized to the buffer value, and the bars represent the mean ± standard deviation. The lack of effect of factor H on C3b dissociation is consistent with covalent attachment to the agarose surface.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Effect of SG7.AF and albicin on the activity of reconstituted C3bBb and the activation of C3bB by factor D. A, C3bBb was formed in vitro by incubation of C3b (200 nM), factor B (100 nM), and factor D (50 nM) at room temperature for 2 min followed by addition of EDTA (5 mM). The resulting C3bBb complex was incubated with C3 (0, 100, or 200 nM) in the presence of SG7.AF or albicin (2 μM) for 20 min at room temperature, separated on a 10% NuPAGE gel, and transferred to a nitrocellulose membrane. C3bBb activity was evaluated by the formation of C3a using anti-C3a (1:10,000). A C3 degradation product that appears after heating SDS-PAGE samples is labeled C3∗. B, C3b (200 nM), factor B (100 nM), and factor D (50 nM) were incubated in the presence or absence of SG7.AF or albicin (2 μM) for 0, 20, and 40 min at 37 °C. Proteins were separated on a 10% NuPAGE gel and transferred to a nitrocellulose membrane. Cleavage of factor B (B) into factor Ba (Ba) and Bb was evaluated using anti-factor B (1:10,000).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition to its role in stabilizing the binding of C3b with factor B and Bb, properdin binds to cell surfaces and acts as a matrix for assembly of the C3bBb complex (19Spitzer D. Mitchell L.M. Atkinson J.P. Hourcade D.E. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J. Immunol. 2007; 179: 2600-2608Crossref PubMed Scopus (213) Google Scholar). Properdin-coated SPR surfaces mimic this condition and support the assembly of C3bB and C3bBb in a similar manner (24Hourcade D.E. The role of properdin in the assembly of the alternative pathway C3 convertases of complement.J. Biol. Chem. 2006; 281: 2128-2132Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). We have shown previously that albicin enhances rather than blocks the accumulation of C3bBb on immobilized properdin suggesting that the inhibitory mechanism involves a strengthening of the interaction of the complex with the surface. When injected along with C3b, factor B, and factor D, SG7.AF and albicin both cause an enhanced accumulation of C3bBb on the surface, but SG7.AF was required at higher concentrations, indicating a lower-affinity binding interaction for this inhibitor (Fig. 5A). SG7.AF alone, C3bB, C3bB-SG7.AF, and C3b-SG7.AF showed little or no interaction with the surface (Fig. S2). The large increase in accumulation of C3bBb on the surface observed in the presence of inhibitors suggests a substantial increase in the overall affinity of the inhibited C3bBb complex for the properdin surface relative to C3bBb alone. Dissociation of the complex from the surface exhibited biphasic kinetics with the overall dissociation of the SG7.AF-bound complex being substantially more rapid than the albicin-bound complex (Fig. 5E–F). The dissociation data at inhibitor concentrations of 1 μM were fit to a double exponential decay function. The fast phase for release of both the SG7.AF- and albicin-bound complexes exhibited a rate constant of 0.02 s−1, but with proportional amplitudes of 0.8 for SG7.AF-bound and 0.2 for albicin-bound complexes indicating that the albicin-bound complex was present mainly as a stable, slow-dissociating form while the SG7.AF complex was present mainly as a rapidly dissociating form. The slow phase rate constant for both SG7.AF and albicin complexes was 0.003 s−1. When albicin was injected after deposition of C3bBb-SG7.AF on the chip surface, the dissociation rate for the complex was reduced to a value similar to that of the bound C3bBb-albicin complex indicating that SG7.AF and albicin are rapidly exchanged in properdin-bound C3bBb and that inhibitor binding regulates the decay rate (Fig. 5F). If 1:1:1 stoichiometry is assumed for C3b, factor Bb, and SG7.AF in the bound complex, C3b would account for 70% of the complex mass. The species dissociating in the fast phase (80% of the complex mass) must therefore contain C3b and is most likely the entire monomeric C3bBb complex being released from the properdin surface after dissociation of the inhibitor. The increased accumulation of the albicin–C3bBb complex on properdin SPR surfaces suggested that the inhibitor may induce oligomerization in the manner of the staphylococcal complement inhibitor SCIN, whose binding results in dimerization of C3bBb (25Rooijakkers S.H. Wu J. Ruyken M. van Domselaar R. Planken K.L. Tzekou A. Ricklin D. Lambris J.D. Janssen B.J. van Strijp J.A. Gros P. Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.Nat. Immunol. 2009; 10: 721-727Crossref PubMed Scopus (153) Google Scholar). We assessed the oligomeric state of the albicin-inhibited complex observed in SPR experiments using gel filtration chromatography after coincubation of C3b, factor B, and factor D in the presence and absence of albicin and nickel ion, which is known to enhance the binding of C3b with factor B (25Rooijakkers S.H. Wu J. Ruyken M. van Domselaar R. Planken K.L. Tzekou A. Ricklin D. Lambris J.D. Janssen B.J. van Strijp J.A. Gros P. Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.Nat. Immunol. 2009; 10: 721-727Crossref PubMed Scopus (153) Google Scholar). Albicin markedly reduced the retention volume for complex elution, indicating an increased molecular mass for the inhibited complex beyond that attributable to addition of the inhibitor alone (Fig. 6A). Based on chromatographic data from a series of standards, the complex has a molecular weight 360 kDa, while the calculated mass of the dimeric complex is 499 kDa, suggesting a C3bBb–albicin dimer is present that partially dissociates during chromatography (Fig. 6A, Fig. S3). Nevertheless, the monomeric C3bBb complex (250 kDa) formed in the absence of albicin appeared near its predicted elution volume and showed distinct separation from the inhibited complex (Fig. 6A). Examination of chromatographic fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also showed albicin to be present along with C3b and factor Bb in the fractions corresponding to the UV absorbance peak of the inhibited complex (Fig. 6B, Fig. S3). The structure of albicin consists of a bundle of four α helices stabilized by two disulfide bonds (Fig. 7A, Table 1). Helix α1 extends from the N-terminus to Thr 14 and is followed by a section of random coil that extends to Lys 22. Helix α2 extends from Ser 23 to Gly 47, and the loop linking α2 and α3 extends from Tyr 48 to Ser 55 followed by α3 extending from Ser 23 to Gly 47 (Fig. 7A). The loop connecting helices α3 and α4 extends from Ser 78 to Ser 88, with α4 extending from Val 89 to the C-terminus. The two disulfide bonds link α3 and α4 with Cys 58 forming a disulfide with Cys 113 and Cys 81 linking with Cys 91. The N-terminal amino group of albicin participates in numerous intramolecular electrostatic interactions that appear to be important in stabilizing the overall structure, including three hydrogen bonds with residues forming the turn linking α2 and α3 (Fig. 8). The carbonyl groups of Val 45, Gly 46, and Tyr 48 form hydrogen bonds with the N-terminal amino group as does ND1 of the imidazole group of His 4 in α1. These interactions may be important for the positioning of the helical elements and stabilization of the helical bundle. Structural searches using DALI (26Holm L. DALI and the persistence of protein shape.Protein Sci. 2020; 29: 128-140Crossref PubMed Scopus (135) Google Scholar) reveal similar structural arrangements in other proteins, especially as portions of larger molecules. The level of amino acid identity in these structures is very low (≤10 % amino acid identity), suggesting that the simple antiparallel four-helix bundle may have evolved independently in the SG7 group.Table 1Data collection, phasing and refinement statistics for albicin, SG7.AF, and anophensin (Anoph)CrystalAlbicinSG7.AFAnoph-SeAnophResolution (Å)52–1.5541–1.450–2.782–2.31Beamline22-ID22-ID22-ID22-IDWavelength (Å)0.91841.00000.97911.0000Completeness (total/high-resolution shell)96.3/53.099.2/88.699.8/99.0100/100Average redundancy (total/high-resolution shell)11.1/3.612.9/4.53.5/3.77.9/6.9Rmerge (total/high-resolution shell, %)7.8/44.95.1/22.26.9/22.75.9/57.7CC1/2 (total/high-resolution shell)99.8/84.299.9/95.299.9/99.099.7/95.1I/sigI (total/high-resolution shell)19.1/2.831.4/5.314.0/9.819.4/4.1Observed reflections757,079425,15494,980142,373Unique reflections67,10932,89511,47118,055Space groupP21212P41212P21212P21212Unit cell dimensions (Å) A56.6885.9867.6467.28 B137.6885.9882.1482.36 C61.1446.0871.3571.73α, β, γ (°)90909090No. of Se/Br sites816FOM (Phenix autosol)0.39Contrast (ShelxE)0.44RefinementTotal non-H protein atoms283210132824Total non-H solvent atoms429212RMS deviations Bond lengths (Å)0.0060.0050.007 Bond angles (°)0.8060.8380.86Mean B factors (Å2) Protein19.017.461.8 Solvent26.531.9 Bromide34.4MolProbity analysis Ramachandran plot (favored/allowed, %)97.7/99.498.3/10095.0/100 Clashscore0.884.34.1 Rotamer outliers (%)0.00.871.4Coordinate error ML (Å, Phenix)0.160.140.34Rcryst/Rfree0.18/0.200.18/0.190.23/0.26 Open table in a new tab Figure 8N-terminal hydrogen bonding network of albicin and SG7.AF. Ribbon diagram of albicin with N-terminal hydrogen bonding network shown in stick representation. Side chains are shown in green with nitrogen shown in blue and oxygen in red. Hydrogen bonds are shown as red dashed lines. Helical
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