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. 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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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. 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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
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the host's defence during host–parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 Å (1 Å=0.1 nm) and was used to describe the structure–inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor α and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the host's adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
Background Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism. Methodology/Principal Findings We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection. Conclusion We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.
The voltage-gated potassium (Kv) 1.3 channel plays a crucial role in the immune responsiveness of T-lymphocytes and macrophages, presenting a potential target for treatment of immune- and inflammation related-diseases. FS48, a protein from the rodent flea Xenopsylla cheopis, shares the three disulfide bond feature of scorpion toxins. However, its three-dimensional structure and biological function are still unclear. In the present study, the structure of FS48 was evaluated by circular dichroism and homology modeling. We also described its in vitro ion channel activity using patch clamp recording and investigated its anti-inflammatory activity in LPS-induced Raw 264.7 macrophage cells and carrageenan-induced paw edema in mice. FS48 was found to adopt a common αββ structure and contain an atypical dyad motif. It dose-dependently exhibited the Kv1.3 channel in Raw 264.7 and HEK 293T cells, and its ability to block the channel pore was demonstrated by the kinetics of activation and competition binding with tetraethylammonium. FS48 also downregulated the secretion of proinflammatory molecules NO, IL-1β, TNF-α, and IL-6 by Raw 264.7 cells in a manner dependent on Kv1.3 channel blockage and the subsequent inactivation of the MAPK/NF-κB pathways. Finally, we observed that FS48 inhibited the paw edema formation, tissue myeloperoxidase activity, and inflammatory cell infiltrations in carrageenan-treated mice. We therefore conclude that FS48 identified from the flea saliva is a novel potassium channel inhibitor displaying anti-inflammatory activity. This discovery will promote understanding of the bloodsucking mechanism of the flea and provide a new template molecule for the design of Kv1.3 channel blockers.
The recombinant NO-binding heme protein, nitrophorin 1 (NP1) from the saliva of the blood-sucking insect, Rhodnius prolixus, has been studied by spectroelectrochemistry, EPR, NMR, and FTIR spectroscopies and X-ray crystallography. It is found that NP1 readily binds NO in solution and in the crystalline state, but the protein is not readily autoreduced by excess NO. Likewise, dithionite is not a very effective reductant of NP1. However, the protein can be photoreduced by illumination with visible light in the presence of excess NO, deazaflavin, and EDTA. Optical spectra of the FeIIINO and FeIINO complexes of NP1 are extremely similar, which makes it difficult to characterize the oxidation state of the NO complex by UV−visible spectroscopy. The reduction potential of NP1 in the absence of NO is ∼300 mV more negative than that of metmyoglobin (metMb). In the presence of NO, the reduction potential shifts ∼+430 mV for NP1−NO, but the reduction potential of metMb−NO cannot be measured for comparison. Based on estimated values of Kd for NP1III−NO, the Kd values for the FeII−NO complex are 20.8 and 80.6 fM at pH 5.5 and 7.5, respectively. The lower driving force for NP1 reduction is qualitatively consistent with the slower rate of autoreduction of NP1−NO; the negative charges surrounding the heme probably also play a role in determining the much slower rate of autoreduction. The N−O stretching frequencies of NP1III−NO and NP1II−NO were measured by FTIR spectroscopy. The values obtained are very typical of other heme−NO stretching frequencies in the two oxidation states: νNO = 1917 and 1904 cm-1 for two species of FeIIINO and 1611 cm-1 for FeIINO; the values of νNO are consistent with 6-coordinate "base-on" heme−NO centers for both oxidation states. The breadths of the IR bands are consistent with the large solvent accessibility of the bound NO of NP1 and also with the possibility of minor dissociation of the protein-provided histidine ligand on the IR time scale. The ratio of the two FeIII−NO species changes with pH and the nature of the buffer. The CO complex of the Fe(II) form of NP1 has νCO = 1960 and 1936 cm-1, again showing the presence of two species. Both NMR and X-ray crystallography show that the protohemin center of NP1 imidazole has a very high preference for a single orientation of the unsymmetrical protoheme moiety. The structure shows the Fe−N−O unit to be quite bent, which is consistent with its being the FeII−NO form of the protein, presumably formed by photoreduction in the X-ray beam. The proximal base, His-59, is clearly coordinated to the iron in the crystalline state and in solution at ambient temperatures, based on FTIR data, but EPR studies of dithionite-reduced samples show that a percentage of the protein has lost the histidine ligand from the FeIINO center in frozen solution.
Saliva of the blood feeding sand fly Lutzomyia longipalpis was previously shown to inhibit the alternative pathway (AP) of the complement system. Here, we have identified Lufaxin, a protein component in saliva, as the inhibitor of the AP. Lufaxin inhibited the deposition of C3b, Bb, Properdin, C5b, and C9b on agarose-coated plates in a dose-dependent manner. It also inhibited the activation of factor B in normal serum, but had no effect on the components of the membrane attack complex. Surface plasmon resonance (SPR) experiments demonstrated that Lufaxin stabilizes the C3b-B proconvertase complex when passed over a C3b surface in combination with factor B. Lufaxin was also shown to inhibit the activation of factor B by factor D in a reconstituted C3b-B, but did not inhibit the activation of C3 by reconstituted C3b-Bb. Proconvertase stabilization does not require the presence of divalent cations, but addition of Ni2+ increases the stability of complexes formed on SPR surfaces. Stabilization of the C3b-B complex to prevent C3 convertase formation (C3b-Bb formation) is a novel mechanism that differs from previously described strategies used by other organisms to inhibit the AP of the host complement system.
A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0−8.3 , from 0.19 × 10-6 to 1.71 × 10-6. The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 × 10-3 to 3.0 s-1. Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by ∼280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 Å resolution.
Abstract Background The salivary glands of hematophagous animals contain a complex cocktail that interferes with the host hemostasis and inflammation pathways, thus increasing feeding success. Fleas represent a relatively recent group of insects that evolved hematophagy independently of other insect orders. Results Analysis of the salivary transcriptome of the flea Xenopsylla cheopis , the vector of human plague, indicates that gene duplication events have led to a large expansion of a family of acidic phosphatases that are probably inactive, and to the expansion of the FS family of peptides that are unique to fleas. Several other unique polypeptides were also uncovered. Additionally, an apyrase-coding transcript of the CD39 family appears as the candidate for the salivary nucleotide hydrolysing activity in X.cheopis , the first time this family of proteins is found in any arthropod salivary transcriptome. Conclusion Analysis of the salivary transcriptome of the flea X. cheopis revealed the unique pathways taken in the evolution of the salivary cocktail of fleas. Gene duplication events appear as an important driving force in the creation of salivary cocktails of blood feeding arthropods, as was observed with ticks and mosquitoes. Only five other flea salivary sequences exist at this time at NCBI, all from the cat flea C. felis . This work accordingly represents the only relatively extensive sialome description of any flea species. Sialotranscriptomes of additional flea genera will reveal the extent that these novel polypeptide families are common throughout the Siphonaptera.
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