The Sand Fly Salivary Protein Lufaxin Inhibits the Early Steps of the Alternative Pathway of Complement by Direct Binding to the Proconvertase C3b-B

Abstract

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.

Amblyomma sculptum Salivary Protease Inhibitors as Potential Anti-Tick Vaccines

“…In addition to the hemolytic assays, the results showed that specific antibodies present in the serum of animals immunized with each of the recombinant salivary proteins could interfere with their anti-complement activity, probably due to opsonization by the antibodies. Similar results were observed in hemolytic assays with SALO and lufaxin, the salivary complement inhibitors of Lutzomyia longipalpis ( 33 , 34 ). The importance of the complement in host resistance against ticks has been previously shown ( 21 , 55 , 56 ) and this effect is highly desirable if the proteins are intended to be used as vaccine antigens.…”

“…The simultaneous presence of anticoagulant and anti-complement activities performed by A. sculptum recombinant proteins is not uncommon in serine protease inhibitors produced by other invertebrates. Salivary proteins from L. longipalpis and Amblyomma americanum were previously shown to be able to delay plasma clotting time and block the activation of the complement system ( 34 , 62 , 63 ). This may be related to the non-specific inhibition of different serine protease members of the cascades.…”

“…To assess the effect of the mouse antiserum on the recombinant activities, hemolytic assays were performed as described above; however, 1 µM of each protein was incubated with its respective antiserum (produced according to the immunization procedures below) at different concentrations, at a final volume of 25 µl, before being mixed with NHS and the erythrocytes ( 34 ). Assays using rAsKunitz and rAs8.9kDa were performed with the classical pathway-mediated complement activation, whereas those with rAsBasicTail were performed using alternative pathway activation methodology.…”

Anti-complement activity in salivary glands and midgut of Chagas disease vector, Panstrongylus megistus (Hemiptera, Triatominae)

“…By acting at the beginning of the classical pathway cascade, in addition to protecting the vector midgut, this inhibitor is also responsible for the down-regulation of inflammation at the bite site as it may prevent the generation of C3a and C5a subproducts, which are potent anaphylatoxins that are responsible for recruiting leukocytes to the bite site 14 . Further studies are required to discover if P. megistus possess a single salivary inhibitor that strikes both pathways or specific molecules directed to classical and alternative pathways, as it has been recently reported for the phlebotomine sand fly, Lutzomyia longipalpis 29 , 30 .…”

Biomarkers for Zoonotic Visceral Leishmaniasis in Latin America

“…LJM11 and LJM17 and two other salivary proteins from L. longipalpis saliva, LJL143 and LJL23, were highly immunogenic in dogs (Collin et al, 2009 ; Teixeira et al, 2010 ). LJL143, also called Lufaxin, has dual anticoagulant and anti-complement activities (Collin et al, 2012 ; Mendes-Sousa et al, 2017 ), while LJL23 is an Apyrase, an inhibitor of platelet aggregation (Teixeira et al, 2010 ; Abdeladhim et al, 2014 ). Interestingly, SP01B and SP01, Apyrase homologs from saliva of P. perniciosus , one of the primary vectors of L. infantum in Europe, were also recognized as markers capable of detecting vector exposure in dogs (Abdeladhim et al, 2014 ; Lestinova et al, 2017 ) Moreover, SP03B, a yellow protein from P. perniciosus saliva was also identified as a good marker of vector exposure in dogs (Kostalova et al, 2017 ).…”