Characterization of synthetic C3a analog peptides on human eosinophils in comparison to the native complement component C3a.
The C3a anaphylatoxin is a potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like Ca2+ transients and the activation of the respiratory burst. These findings support an important role for C3a in diseases typically associated with a peripheral blood or tissue eosinophilia. Synthetic human C3a analogue peptides with variations at the C-terminal effector domain have been evaluated with respect to their binding affinity and signaling potency on human eosinophils. Flow cytometrical analysis and RT-PCR revealed that the C3a receptor is constitutively expressed on human eosinophils. Peptides bearing an N-terminal 9-fluorenylmethoxycarbonyl and the 6-aminohexanoyl motif were the most powerful peptides tested. Amino acid replacements in the conserved C-terminal pentapeptide decreased binding affinity and functional potency substantially. In addition, synthetic C3a analogue peptides induced C3aR internalization, led to transient changes of intracellular Ca2+ concentration, and did release reactive oxygen species in human eosinophils indicating the in vivo relevance of C3a-related sequences. The tripeptide LAR was found to be essential for C3a receptor binding on human eosinophils. Moreover, the putative binding motif of C3a anaphylatoxin is also crucial for the induction of biologic effects in the human system such as changes of intracellular Ca2+ concentration and the release of reactive oxygen species. This study demonstrates that the carboxyl terminus is important for the interaction with the C3aR and the biologic potency of C3a anaphylatoxin in the human system and plays a key role in the activation process of human eosinophils.
Petering
H
H
Department of Dermatology, Institute of Medical Microbiology, Hannover Medical University, Hannover, Germany.
Köhl
J
J
Weyergraf
A
A
Dulkys
Y
Y
Kimmig
D
D
Smolarski
R
R
Kapp
A
A
Elsner
J
J
D002118:Calcium / Q000378:metabolism; D015926:Complement C3a / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism; D004804:Eosinophils / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007424:Intracellular Fluid / Q000276:immunology / Q000378:metabolism; D008565:Membrane Proteins; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000276:immunology / Q000378:metabolism; D011951:Receptors, Complement / Q000096:biosynthesis / Q000378:metabolism; D016897:Respiratory Burst / Q000276:immunology; D017386:Sequence Homology, Amino Acid
D008565:Membrane Proteins; D009842:Oligopeptides; D011951:Receptors, Complement; C085567:complement C3a receptor; D015926:Complement C3a; D002118:Calcium