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- Bergseth, Grethe, et al.
(författare)
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An international serum standard for application in assays to detect human complement activation products
- 2013
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 56:3 SI, s. 232-239
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Forskningsöversikt (refereegranskat)abstract
- The importance of the complement system in clinical medicine has become evident during the last decades and complement therapeutics has now reached the clinic. Thus, there is an increased interest in and need for assays to evaluate complement activity and dysfunction. Pathologically increased complement activation can indirectly be evaluated by quantification of complement components, but in order to exactly measure such activation, assays for quantification of products formed during activation are required. Progress in this field is hampered by lack of standardization. Therefore, members of the International Complement Standardization Committee, a joint initiative of the International Complement Society and the International Union of Immunological Societies (IUIS), prepared a defined standard for application in assays for complement activation products. We here report on the production and properties of this International Complement Standard #2 (ICS#2). ICS#2 was made from a pool of sera from healthy blood donors (ICS#1) that was activated with a combination of heat-aggregated IgG and zymosan, and subsequently stabilized by adding EDTA and nafamostat mesylate. The protocol was optimized to make the standard applicable in the following activation product assays: C1rs-C1-inhibitor complexes, C4a, C4bc, C4d, Bb, C3bBbP, C3a, C3bc, C3dg, C5a and the soluble terminal C5b-9 complement complex (SC5b-9, TCC). ICS#2 was defined as containing 1000 complement activation units (CAU)/mL for all activation products measured. All activation products were stable after 10 times thawing and freezing and most of the activation products were stable during storage at 4 degrees C for up to 21 days. ICS#2 was produced large-scale and is considered a valuable tool for standardization, calibration and reference control for complement activation assays, providing the necessary prerequisite for quality assessments between complement laboratories.
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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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| 5. |
- Edin, Sofia, et al.
(författare)
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Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.
- 2010
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Ingår i: Molecular Immunology. - : Elsevier. - 0161-5890 .- 1872-9142. ; 47:11-12, s. 2057-2064
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Tidskriftsartikel (refereegranskat)abstract
- Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca(2+)) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-kappaB. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca(2+) dependent and of high affinity, with a K(d) of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca(2+) level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-kappaB. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-kappaB reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca(2+) sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-kappaB.
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| 10. |
- Gjelstrup, Louise Carstensen, et al.
(författare)
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The role of higher-order protein structure in supporting binding by heteroclitic monoclonal antibodies: The monoclonal antibody KIM185 to CD18 also binds C4-binding protein
- 2011
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 49:1-2, s. 38-47
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Tidskriftsartikel (refereegranskat)abstract
- Heteroclitic monoclonal antibodies are characterized by the ability to bind multiple epitopes with little or no similarity. Such antibodies have been reported earlier, but insight into to the molecular basis of this propensity is limited. Here we report that the KIM185 antibody to human CD18 reacts with the plasma protein C4b-binding protein (C4BP). This was revealed during affinity purification procedures where human serum was incubated with surfaces coated with monoclonal antibodies to CD18. Other monoclonal antibodies to CD18 (KIM127 and TS1/18) showed no such interaction with C4BP. We constructed a sandwich-type time-resolved immunofluorometric assay using KIM185 both as capture and developing antibody. By use of proteolytic fragments of KIM185 and recombinant deletion mutants of C4BP the interaction sites were mapped to the variable region of KIM185 and the oligomerization domain of C4BP, respectively. C4BP is a large oligomeric plasma protein that binds activated complement factor C4b and other endogenous ligands as well as microorganisms. By use of the recent crystallographic data on the structure of CD11c/CD18 and prediction of the secondary structure of the C4BP oligomerization domain, we show that epitopes bound by KIM185 in these proteins are unlikely to share any major structural similarity. However, both antigens may form oligomers that would enable avid binding by the antibody. Our report points to the astonishing ability of heteroclitic antibodies to accommodate the binding of multiple proteins with no or little structural similarity within the confined space of the variable regions. (C) 2011 Elsevier Ltd. All rights reserved.
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