- Email: [email protected]
Modelling the complex between CD55 and Factor B using electron paramagnetic resonance
Molecular Immunology 44 (2007) 147–266
Abstracts from the XXIst International Complement Workshop
1 Modelling the complex between CD55 and Factor B using electron paramagnetic resonance
an accurate model of the complex between CD55 and Factor B vWF-A.
Rachel J.M. Abbott a , Janet E. Banham b , Christiane R. Timmel b , Gunnar Jeschke c , Susan M. Lea a
doi:10.1016/j.molimm.2006.07.285
a
Sir William Dunn School of Pathology, South Parks Road, Oxford, UK; b Chemistry Department, South Parks Road, Oxford, UK; c Max Planck Institut F¨ur Polymerforschung, Mainz, Germany The human complement regulator CD55 (decay accelerating factor, DAF) is an intrinsic membrane glycoprotein that protects self-cells from complement-mediated lysis. It inactivates the C3 convertases by dissociating them into their constituent proteins. To fully understand how CD55 regulates the convertases more information is needed about the interaction between the components involved. A structure for the complex formed by the interaction of CD55 and human Factor B, a major component of the alternative pathway convertase, would prove particularly informative. Unfortunately, obtaining a structure of the proteins in complex with one another by X-ray and NMR methods is very difficult due to the transient nature of the interaction and the size of the components involved. In an effort to overcome these difficulties, we have applied the four-pulsed EPR method of double electron electron resonance (DEER) to provide experimentally-derived restraints that have allowed us to produce a model for the complex. We have expressed and purified a range of CD55 and Factor B vWF-A domain mutants. The single free cysteine residue on each protein has been reacted with a methane thiosulfonate nitroxide spin-label (MTSSL). DEER has been performed with frozen solutions of CD55 and Factor B vWF-A to measure the distance between the spin-labels within the protein complex. The various mutants produced have enabled us to alter the position of the spin-label within the proteins and this has allowed us to obtain a range of different measurements. Using the distances measured in this way, together with the crystal structures of CD55 and the vWF-A domain of Factor B, we have produced 0161-5890/$ – see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.molimm.2006.07.005
2 Anaphylatoxin C5a potentiates allergic inflammation in human lungs Masayoshi Abe a , Noriko Satoh a , Takehiro Umemura a , Akinori Iwasaki b , Takayuki Shirakusa b , Takeshi Katsuragi a a
Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan; b Department of Surgery, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan Bronchial asthma is a complex inflammatory disorder of the airways. Complement activation produces anaphylatoxic polypeptides, and the anaphylatoxins C3a, C4a, and C5a, are considered to contribute to the pathophysiology of asthma. Among various mediators and cytokines involved in asthma inflammation, cysteinyl-leukotrienes (cysLTs) are one of the most important mediators. Consequently, we studied whether or not C3a and C5a were able to stimulate generation of cysLTs from human lung tissues. The lung tissues that appeared macroscopically normal were obtained from patients with lung cancer. The extraction of RNA from the tissues and then polymerase chain reaction showed C5aR-, C3aR-, and C5L2-mRNA expression in the lungs. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). Incubation with C5a or C3a alone did not stimulate cysLT production from lung tissues. However, the co-addition of C5a or C3a with anti-IgE antibody potentiated cysLT production in comparison with antibody stimulation alone. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1000 ng/ml tended to increase cysLT production, but not to any significant extent. A novel
Abstracts from the XXIst International Complement Workshop
1 Modelling the complex between CD55 and Factor B using electron paramagnetic resonance
an accurate model of the complex between CD55 and Factor B vWF-A.
Rachel J.M. Abbott a , Janet E. Banham b , Christiane R. Timmel b , Gunnar Jeschke c , Susan M. Lea a
doi:10.1016/j.molimm.2006.07.285
a
Sir William Dunn School of Pathology, South Parks Road, Oxford, UK; b Chemistry Department, South Parks Road, Oxford, UK; c Max Planck Institut F¨ur Polymerforschung, Mainz, Germany The human complement regulator CD55 (decay accelerating factor, DAF) is an intrinsic membrane glycoprotein that protects self-cells from complement-mediated lysis. It inactivates the C3 convertases by dissociating them into their constituent proteins. To fully understand how CD55 regulates the convertases more information is needed about the interaction between the components involved. A structure for the complex formed by the interaction of CD55 and human Factor B, a major component of the alternative pathway convertase, would prove particularly informative. Unfortunately, obtaining a structure of the proteins in complex with one another by X-ray and NMR methods is very difficult due to the transient nature of the interaction and the size of the components involved. In an effort to overcome these difficulties, we have applied the four-pulsed EPR method of double electron electron resonance (DEER) to provide experimentally-derived restraints that have allowed us to produce a model for the complex. We have expressed and purified a range of CD55 and Factor B vWF-A domain mutants. The single free cysteine residue on each protein has been reacted with a methane thiosulfonate nitroxide spin-label (MTSSL). DEER has been performed with frozen solutions of CD55 and Factor B vWF-A to measure the distance between the spin-labels within the protein complex. The various mutants produced have enabled us to alter the position of the spin-label within the proteins and this has allowed us to obtain a range of different measurements. Using the distances measured in this way, together with the crystal structures of CD55 and the vWF-A domain of Factor B, we have produced 0161-5890/$ – see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.molimm.2006.07.005
2 Anaphylatoxin C5a potentiates allergic inflammation in human lungs Masayoshi Abe a , Noriko Satoh a , Takehiro Umemura a , Akinori Iwasaki b , Takayuki Shirakusa b , Takeshi Katsuragi a a
Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan; b Department of Surgery, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan Bronchial asthma is a complex inflammatory disorder of the airways. Complement activation produces anaphylatoxic polypeptides, and the anaphylatoxins C3a, C4a, and C5a, are considered to contribute to the pathophysiology of asthma. Among various mediators and cytokines involved in asthma inflammation, cysteinyl-leukotrienes (cysLTs) are one of the most important mediators. Consequently, we studied whether or not C3a and C5a were able to stimulate generation of cysLTs from human lung tissues. The lung tissues that appeared macroscopically normal were obtained from patients with lung cancer. The extraction of RNA from the tissues and then polymerase chain reaction showed C5aR-, C3aR-, and C5L2-mRNA expression in the lungs. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). Incubation with C5a or C3a alone did not stimulate cysLT production from lung tissues. However, the co-addition of C5a or C3a with anti-IgE antibody potentiated cysLT production in comparison with antibody stimulation alone. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1000 ng/ml tended to increase cysLT production, but not to any significant extent. A novel