Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution.
Skp derived from Escherichia coli attracts leukocytes as a pure chemotactic ligand of the C5a receptor. We identified the submolecular region of Skp that binds and activates the C5a receptor to be -Gln103-Asp104-Arg105- using synthetic peptide fragments and site-directed mutants of Skp. As the C5a amino acid residue equivalent to Gln103 of Skp is Leu72, we prepared a Gln103Leu-Skp mutant as a recombinant protein. With this mutation, Skp gained secretagogue functions including induction of the respiratory burst and granule release reactions and leukotriene generation, in addition to the chemoattraction displayed by C5a. However, when we substituted Leu72 with Gln in C5a, the L72Q-C5a mutant largely lost its secretagogue function. These functional conversions were reproduced using synthetic peptides mimicking the receptor-binding/-activating regions of the recombinant proteins. Receptor-binding assays using the mimicking peptides demonstrated only a small difference between the Leu72-C5a and Gln72-C5a peptides. Consistently, L72Q-C5a apparently antagonized C5a secretagogue function. These results indicate that the difference between a chemotactic response and a combined chemotactic/secretory response can be attributed not to the nature of the receptor but to guidance by the ligand, at least in the case of C5a receptor-mediated leukocyte responses.
Jia
Nan
N
Department of Molecular Pathology, Faculty of Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-0811, Japan.
Semba
Umeko
U
Nishiura
Hiroshi
H
Kuniyasu
Akihiko
A
Nsiama
Tienabe K
TK
Nishino
Norikazu
N
Yamamoto
Tetsuro
T
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002630:Chemotactic Factors / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D003593:Cytoplasm; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006168:Guinea Pigs; D006632:Histamine / Q000378:metabolism; D008297:Male; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D044087:Receptor, Anaphylatoxin C5a / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016897:Respiratory Burst; D017386:Sequence Homology, Amino Acid
D002630:Chemotactic Factors; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D044087:Receptor, Anaphylatoxin C5a; D011994:Recombinant Proteins; C072721:Skp protein, E coli; D006632:Histamine; D002118:Calcium