Structural plasticity of the Salmonella FliS flagellar export chaperone

The Salmonella FliS flagellar export chaperone is a highly α-helical protein. Proteolytic experiments suggest that FliS has a compact core. However, the calorimetric melting profile of FliS does not show any melting transition in the 25-110 °C temperature range. CD measurements reveal that FliS is losing its helical structure over a broad temperature range upon heating. These observations indicate that FliS unfolds in a non-cooperative way and its native state shows features reminiscent of the molten globule state of proteins possessing substantial structural plasticity. As FliS has several binding partners within the cell, conformational adaptability seems to be an essential requirement to fulfill its multiple roles. This article is protected by copyright. All rights reserved

MASP-1 of the complement system enhances clot formation in a microvascular whole blood flow model.

The complement and coagulation systems closely interact with each other. These interactions are believed to contribute to the proinflammatory and prothrombotic environment involved in the development of thrombotic complications in many diseases. Complement MASP-1 (mannan-binding lectin-associated serine protease-1) activates coagulation factors and promotes clot formation. However, this was mainly shown in purified or plasma-based static systems. Here we describe the role of MASP-1 and complement activation in fibrin clot formation in a microvascular, whole blood flow model. This microfluidic system simulates blood flow through microvessels at physiological flow and shear rates and represents the closest model system to human physiology so far. It features parallel microchannels cultured with endothelial cells in a transparent microfluidic chip allowing real-time evaluation of clot formation by confocal microscopy. To test their effects on clot formation, we added the following activators or inhibitors (individually or in combination) to whole blood and performed perfusion experiments: rMASP-1cf (recombinant active form of MASP-1), complement activator zymosan, selective MASP-1 inhibitor SGMI-1 (based on the Schistocerca gregaria protease inhibitor scaffold), classical pathway inhibitor rSALO (recombinant salivary anti-complement from Lutzomyia longipalpis). Addition of rMASP-1cf resulted in accelerated fibrin clot formation while addition of SGMI-1 delayed it. Complement activation by zymosan led to increased clot formation and this effect was partially reversed by addition of rSALO and almost abolished in combination with SGMI-1. We show for the first time a strong influence of MASP-1, complement activation and pathway-specific inhibition on coagulation in a microvascular flow system that is closest to human physiology, further underpinning the in vivo relevance of coagulation and complement interactions

Soluble components of the flagellar export apparatus, FliI, FliJ, and FliH, do not deliver flagellin, the major filament protein, from the cytosol to the export gate.

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of approximately 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator

Key Components of the Complement Lectin Pathway Are Not Only Required for the Development of Inflammatory Arthritis but Also Regulate the Transcription of Factor D

The complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving lectin pathway (LP) activation, the mannan-binding lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70-95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene

Complement lectin pathway components MBL and MASP-1 promote haemostasis upon vessel injury in a microvascular bleeding model.

Background Complement lectin pathway components, in particular mannan-binding lectin (MBL) and MBL-associated serine proteases (MASPs) have been shown to interact with coagulation factors and contribute to clot formation. Here we investigated the role of MBL and MASP-1 in the haemostatic response following mechanical vessel injury in a human microfluidic bleeding model. Methods We studied haemostasis in a microvascular bleeding model in the presence of human endothelial cells and human whole blood under flow conditions. We monitored incorporation of proteins into the clot with fluorescently labelled antibodies and studied their effects on clot formation, platelet activation, and bleeding time with specific inhibitors. Platelet activation was also studied by flow cytometry. Results Upon vessel injury, MBL accumulated at the injury site in a well-defined wall-like structure. MBL showed partial colocalisation with fibrin, and strong colocalisation with von Willebrand factor and (activated) platelets. Flow cytometry ruled out direct binding of MBL to platelets, but confirmed a PAR4- and thrombin-dependent platelet-activating function of MASP-1. Inhibiting MBL during haemostasis reduced platelet activation, while inhibiting MASP-1 reduced platelet activation, fibrin deposition and prolonged bleeding time. Conclusion We show in a microvascular human bleeding model that MBL and MASP-1 have important roles in the haemostatic response triggered by mechanical vessel injury: MBL recognises the injury site, while MASP-1 increases fibrin formation, platelet activation and shortens bleeding time. While the complement lectin pathway may be harmful in the context of pathological thrombosis, it appears to be beneficial during the physiological coagulation response by supporting the crucial haemostatic system

Serum MASP-1 in complex with MBL activates endothelial cells.

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca2+ signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL-MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response