Complement-derived mediator, called anaphylatoxins, play a major role in adherence, chemotaxis (i.e. mobilization) and sequestration of leukocytes and macrophages. These factors, C3a and C5a, have receptors on selected circulating and tissue cell types that activate cells via a trans-membrane signal mechanism. Since these humoral factors stimulate inflammatory cells, thus promoting their migration and sequestration to sites of injury, the cellular receptors for these factors are a prime target for modulating and controlling the inflammatory process. Understanding the ligand-receptor interactions at the molecular level will help to elucidate mechanisms of trans-membrane cellular activation and provide, an avenue for manipulating the subsequent physiologic response (i.e. inflammation). Synthetic analogs of factor C3a have now been successfully designed that exhibit 10-fold greater activity than does the natural factor. Model synthetic peptides for C5a will be designed based on both the C3a analog studies and the three-dimensional model structure of the C5a molecule. Synthetic C-terminal C5a peptides up to 21 residues in length have been made and are weakly active (0.01 - 1%). Synthetic C5a peptide activity will be optimized by incorporating the primary and multiple sub-site binding domains into ligand analogs. Sub-site domains will be identified by site-specific mutagenesis of the human C5a molecule. A combination of mutagenesis and synthetic peptide studies will be employed to map surface interactions between C5a and the C5a receptor. We anticipate designing complex linear or; branched C5a peptides by incorporating multiple distinct binding sites in order to optimize cooperative binding interactions and thereby enhance binding affinity. If this concept of simplified analog design proves successful, functionally active synthetic analogs may be designed for numerous lymphokines and various other bioactive ligands. Synthetic agonists of C5a will be used for receptor mapping, receptor-isolation and cell identification. Reporter groups and probes attached to the synthetic analogs will be used to analyze cellular trafficking of the ligand and mechanisms of activation. Another goal of this project will be to design site-specific antagonists of C5a based on the agonist data. Effective antagonists for C5a promise to have potential clinical value as selective antiinflammatory agents useful in acute injury, rheumatoid and autoimmune diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI031963-03
Application #
2066887
Study Section
Allergy and Immunology Study Section (ALY)
Project Start
1991-12-01
Project End
1996-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Jagels, M A; Chambers, J D; Arfors, K E et al. (1995) C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo. Blood 85:2900-9
Jagels, M A; Hugli, T E (1994) Mechanisms and mediators of neutrophilic leukocytosis. Immunopharmacology 28:1-18
Hetland, G; del Zoppo, G J; Mori, E et al. (1994) Uptake of C5a by polymorphonuclear leukocytes (PMNs) after focal cerebral ischemia. I. Effect of tirilazad mesylate intervention on C5a uptake by PMNs. Immunopharmacology 27:191-8
Cui, L; Carney, D F; Hugli, T E (1994) Primary structure and functional characterization of rat C5a: an anaphylatoxin with unusually high potency. Protein Sci 3:1169-77
Ember, J A; del Zoppo, G J; Mori, E et al. (1994) Polymorphonuclear leukocyte behavior in a nonhuman primate focal ischemia model. J Cereb Blood Flow Metab 14:1046-54
Tsai, W M; Roos, G; Hugli, T E et al. (1992) Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen. J Immunol 149:2227-33

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