[49] Human C5a anaphylatoxin: Gene synthesis, expression, and recovery of biologically active material from Escherichia coli

Franke, Arthur E.; Andrews, Glenn C.; Stimler-Gerard, Norma P.; Gérard, Craig; Showell, Henry J.

doi:10.1016/0076-6879(88)62107-0

Cited by 29 publications

(21 citation statements)

References 18 publications

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“…DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGPA) was purchased from Calbiochem-Behring Corp., San DieFo, CA, and thiorphan was purchased from Peninsula Laboratories, Belmont, CA. Human C5a was obtained as a recombinant peptide from Escherichia coli lysates, reconstituted and purified as previously described (25). C3a was purified from complement-activated porcine serum as described (26).…”

Section: Methodsmentioning

confidence: 99%

Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung.

Stimler-Gerard¹

1987

J. Clin. Invest.

103

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The responsiveness ofisolated guinea pig lung parenchymal strips to substance P was enhanced by at least 100-fold in the presence of the endopeptidase inhibitors phosphoramidon (1 gM) or thiorphan (1 &M), but not with the converting enzyme inhibitor, captopril, or an inhibitor of serum carboxypeptidase N (both 1 t&M). Responses of guinea pig tracheal rings to substance P were also markedly potentiated by phosphoramidon. The increase in tissue responsiveness by these inhibitors was relatively specific for substance P among several other spasmogenic peptides, including formyl-methionyl-leucyl-phenylalanine and the complement peptides C3a and C5a. The enhanced responses appear to result from a decrease in the rate of substance P degradation in the presence of neutral endopeptidase inhibitors. Specific binding of substance P to its receptor on bronchial membranes was increased by three to fourfold in tIhe presence of phqsphoramidon.These data demonstrate an enhanced potential for substance P to contract lung tissues when degradation by a neutral endopeptidase-like enzyme is blocked.

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Section: Methodsmentioning

confidence: 99%

Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung.

Stimler-Gerard¹

1987

J. Clin. Invest.

103

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“…However, our recombinant C5a was found to be equally potent to authentic human serum C5a in binding affinity and in CSa-induced intracellular calcium rise (data not shown). A similar synthetic C5a gene was expressed and purified from E. coli in 2 other laboratories (Mandecki et al, 1985;Franke et al, 1988). The recombinant C5a from both of these groups contained a methionine at the N-terminus before the initial threonine residue of human C5a.…”

Section: Analysis Of the Recombinant C5a Proteinsmentioning

confidence: 99%

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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“…Expression of re combinant human C5a in E. coli has also been reported by others [152,153]. The fea sibility of cloning and expression of C5a anaphylatoxin in bacteria and its efficient puri fication from bacterial proteins make it pos sible to obtain sufficient quantities of C5a to allow detailed structure-function studies in volving site-specific mutagenesis of the C5a gene.…”

Section: Component C5mentioning

confidence: 99%

Recent Advances in the Study of the Molecular Structure of the Complement Proteins

Chakravarti

Kristensen

1986

Path Immunopathol Res

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[49] Human C5a anaphylatoxin: Gene synthesis, expression, and recovery of biologically active material from Escherichia coli

Cited by 29 publications

References 18 publications

Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung.

Neutral endopeptidase-like enzyme controls the contractile activity of substance P in guinea pig lung.

The pharmacophore of the human C5a anaphylatoxin

Recent Advances in the Study of the Molecular Structure of the Complement Proteins

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