| 1. |
- Pang, Siew Siew, et al.
(författare)
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The structural basis for complement inhibition by gigastasin, a protease inhibitor from the giant Amazon leech
- 2017
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 199:11, s. 3883-3891
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Tidskriftsartikel (refereegranskat)abstract
- Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
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| 2. |
- Duncan, Renee C., et al.
(författare)
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Identification of a Catalytic Exosite for Complement Component C4 on the Serine Protease Domain of C1s
- 2012
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 189:5, s. 2365-2373
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Tidskriftsartikel (refereegranskat)abstract
- The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dys-regulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation. The Journal of Immunology, 2012, 189: 2365-2373.
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| 3. |
- Duncan, Renee C., et al.
(författare)
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Multiple domains of MASP-2, an initiating complement protease, are required for interaction with its substrate C4
- 2012
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 49:4, s. 593-600
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is fundamental to both innate and adaptive immunity and can be initiated via the classical, lectin or alternative pathways. Cleavage of C4 by MASP-2, the initiating protease of the lectin pathway, is a crucial event in the activation of this pathway, preceding the eventual formation of the C3 convertase (C4bC2a) complex on the pathogen surface. Interactions required for the cleavage of C4 by MASP-2 are likely to be facilitated by the initial binding of C4 to an exosite on the protease. We have shown that both proteolytically active and catalytically inactive CCP1-CCP2-serine protease (CCP1-CCP2-SP) forms bind C4 with similar affinity. Interestingly, proteins containing the CCP1-CCP2 domains or the SP domain alone bound C4 with much lower affinity than the CCP1-CCP2-SP protein, suggesting that the CCP domains cooperate positively with the active site to mediate efficient binding and cleavage of C4. In addition, mutation of residue K342 to alanine in the CCP1 domain abolished binding to both C4 and C4b in its CCP1-CCP2 form, suggesting a key electrostatic role for this amino acid. The presented data indicates that all of the domains are required in order to mediate high affinity interaction with C4. (C) 2011 Elsevier Ltd. All rights reserved.
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| 4. |
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| 5. |
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| 6. |
- Hsieh, Yves S. Y., et al.
(författare)
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Total synthesis of homogeneous variants of hirudin P6 : a post-translationally modified anti-thrombotic leech-derived protein
- 2014
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Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 53:15, s. 3947-51
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Tidskriftsartikel (refereegranskat)abstract
- Hirudin P6 is a leech-derived anti-thrombotic protein which possesses two post-translational modifications, O-glycosylation and tyrosine sulfation. In this study we report the ligation-based synthesis of a library of hirudin P6 proteins possessing homogeneous glycosylation and sulfation modifications. The nature of the modifications incorporated was shown to have a drastic effect on inhibition against both the fibrinogenolytic and amidolytic activities of thrombin and thus highlights a potential means for attenuating the biological activity of the protein.
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| 7. |
- Mika, Angela, et al.
(författare)
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Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:7
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Tidskriftsartikel (refereegranskat)abstract
- Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage.
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| 8. |
- Reynolds, Simone L, et al.
(författare)
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Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.
- 2014
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Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2735. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.
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