Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Mollison, Karl W.; Mandecki, Włodek; Zuiderweg, Erik R. P.; Fayer, L; Fey, Thomas A.; Krause, R. A.; Conway, Richard G.; Miller, L.S.; Edalji, Rohinton; Shallcross, Mary Ann

doi:10.1073/pnas.86.1.292

Cited by 114 publications

(67 citation statements)

References 32 publications

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“…Other groups have published data indicating the crucial role of Arg 74. His 67, Lys 68, and Leu 72 of the C-terminal region have also been identified as important for C5a activity (Mollison et al, , 1991. Our results corroborate and extend some of these findings by showing that Arg 74, and to a lesser degree Leu 72 and Gly 73, are important for binding affinity and for C5a-induced intracellular calcium rise.…”

Section: Discussionsupporting

confidence: 81%

“…Because both of these amino acids are buried in our model of C5a, we agree with those authors' interpretation that mutants of these 2 positions probably affect proper folding of the protein. Mollison et al (1989Mollison et al ( , 1991 Our results also imply that the connection between the critical Arg 74 and the core domain can be variable because the 64-71 peptide bridge is somewhat arbitrary in terms of amino acid composition and number. Thus, the pharmacophore of C5a probably consists of 10 mostly charged amino acids embedded in a 4-helix core structure that is unconstrained in its connection to the critical C-terminal arginine-containing tripeptide.…”

Section: Discussionmentioning

confidence: 53%

“…In addition, a synthetic octapeptide corresponding to the C-terminus of C5a was able to mimic some of the C5a-induced functional responses but with a greatly reduced potency (Kawai et al, 1992). Using a mutant analysis of the full-length protein, 2 groups have identified some of the other amino acids of C5a involved with receptor binding (Mollison et al, , 1991Carney & Hugli, 1993). Here we have used more extensive mutagenesis to better define the pharmacophore of C5a.…”

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confidence: 99%

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The pharmacophore of the human C5a anaphylatoxin

Toth¹,

Huwyler²,

Boyar³

et al. 1994

Protein Science

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We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of CSa-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and sitedirected mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 53%

mentioning

confidence: 99%

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The pharmacophore of the human C5a anaphylatoxin

Toth¹,

Huwyler²,

Boyar³

et al. 1994

Protein Science

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“…One is at the core of C5a and is centered around Arg 40 (19). The other is contained in the eight amino acids of the C terminus (20).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of C5a Anaphylatoxin by a Peptide That Is Complementary to a Region of C5a

Fujita

Farkas

Campbell

et al. 2004

The Journal of Immunology

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PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37–53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all. C5a was inactivated by concentrations of 7 nM or more of Pep-A, and this concentration of Pep-A inhibited induction of intracellular Ca2+ influx in neutrophils. Patch clamp electrophysiology experiments also showed the effectiveness of Pep-A in C5aR-expressing neuroblastoma cells. Furthermore, Pep-A administration prevented rats from C5a-mediated rapid lethal shock induced by an Ab to a membrane inhibitor of complement after LPS sensitization.

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“…In that study, removal of residues 1-17 from porcine C5ades Arg by cyanogen bromide cleavage at Met-17 resulted in a 1,000-fold loss in potency. In a detailed site-directed mutagenesis study later presented by Mollison et al (1989), several rC5a mutants that contained residue substitutions in the NTR were examined for changes in potency and receptor binding. Only minor decreases in potency and receptor binding were noted for all of the NTR mutants examined, which included substitutions at residues 1-5, 7-9, 11, and 12.…”

Section: De Carney and T E Huglimentioning

confidence: 99%

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

Carney

Hugli

1993

Protein Science

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C5a is an inflammatory mediator that evokes a variety of immune effector functions including chemotaxis, cell activation, spasmogenesis, and immune modulation. It is well established that the effector site in C5a is located in the C‐terminal region, although other regions in C5a also contribute to receptor interaction. We have examined the N‐terminal region (NTR) of human C5a by replacing selected residues in the NTR with glycine via site‐directed mutagenesis. Mutants of rC5a were expressed as fusion proteins, and rC5a was isolated after factor Xa cleavage. The potency of the mutants was evaluated by measuring both neutrophil chemotaxis and degranulation (β‐glucuronidase release). Mutants that contained the single residue substitutions Ile‐6 → Gly or Tyr‐13 → Gly were reduced in potency to 4–30% compared with wild‐type rC5a. Other single‐site glycine substitutions at positions Leu‐2, Ala‐10, Lys‐4, Lys‐5, Glu‐7, Glu‐8, and Lys‐14 showed little effect on C5a potency. The double mutant, Ile‐6 → Gly/Tyr‐13 → Gly, was reduced in potency to <0.2%, which correlated with a correspondingly low binding affinity for neutrophil C5a receptors. Circular dichroism studies revealed a 40% reduction in α‐helical content for the double mutant, suggesting that the NTR contributes stabilizing interactions that maintain local secondary or tertiary structure of C5a important for receptor interaction. We conclude that the N‐terminal region in C5a is involved in receptor binding either through direct interaction with the receptor or by stabilizing a binding site elsewhere in the intact C5a molecule.

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Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

Cited by 114 publications

References 32 publications

The pharmacophore of the human C5a anaphylatoxin

The pharmacophore of the human C5a anaphylatoxin

Inactivation of C5a Anaphylatoxin by a Peptide That Is Complementary to a Region of C5a

Site‐specific mutations in the N‐terminal region of human C5a that affect interactions of C5a with the neutrophil C5a receptor

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