Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Bubeck, Peter; Grötzinger, Joachim; Winkler, M; Köhl, Jörg; Wollmer, Axel; Klos, Andreas; Bautsch, Wilfried

doi:10.1111/j.1432-1033.1994.tb18571.x

Cited by 42 publications

(39 citation statements)

References 41 publications

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“…Sitedirected mutagenesis studies in which either C5a (44,50,51), CD88 (52, 53), or both (54,55) were mutated provide evidence that the C-terminal amino acids from positions 65-74 confer binding and agonist activity. The core segment, comprising the remainder of the C5a molecule, contributes significantly to high affinity binding but not to biologic activity.…”

Section: Discussionmentioning

confidence: 99%

C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

Otto

Hawlisch

Monk

et al. 2004

Journal of Biological Chemistry

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The anaphylatoxin C5a exerts a plethora of biologic activities critical in the pathogenesis of systemic inflammatory diseases. Recently, we reported on a C5a mutant, jun/fos-A8, as a potent antagonist for the human and mouse C5a receptor (CD88). Addressing the molecular mechanism accounting for CD88 receptor antagonism by site-directed mutagenesis, we found that a positively charged amino acid at position 69 is crucial. Replacements by either hydrophobic or negatively charged amino acids switched the CD88 antagonist jun/fos-A8 to a CD88 agonist. In addition to CD88, the seven-transmembrane receptor C5L2 has recently been found to provide high affinity binding sites for C5a and its desarginated form, C5adesArg 74 . A jun/fos-A8 mutant in which the jun/ fos moieties and amino acids at positions 71-73 were deleted, A8 ⌬71-73 , blocked C5a and C5adesArg 74 binding to CD88 and C5L2. In contrast, the cyclic C5a C-terminal analog peptide AcF-[OP-D-ChaWR] inhibited binding of the two anaphylatoxins to CD88 but not to C5L2, suggesting that the C5a core segment is important for high affinity binding to C5L2. Both receptors are coexpressed on human monocytes and the human mast cell line HMC-1; however, C5L2 expression on monocytes is weaker as compared with HMC-1 cells and highly variable. In contrast, no C5L2 expression was found on human neutrophils. A8 ⌬71-73 is the first antagonist that blocks C5a and C5adesArg 74 binding to both C5a receptors, CD88 and C5L2, making it a valuable tool for studying C5L2 functions and for blocking the biological activities of C5a and C5adesArg 74 in mice and humans.

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Section: Discussionmentioning

confidence: 99%

C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

Otto

Hawlisch

Monk

et al. 2004

Journal of Biological Chemistry

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“…In the case of the human CSaR, such residues are especially attractive as they recommend themselves as suitable counterparts to bind and neutralize the carboxylate and guanidinium function of the C-terminal residue Arg74 in the C5a ligand which is generally supposed to bind within the transmembrane region of the CSaR. Removal of the C-terminal Arg in the C5a ligand reduces receptor affinity and activity almost 100-fold Bubeck et al, 1994). Using sequence comparison and molecular modelling, we have selected candidate positions for an interaction with the ligand to be analyzed for involvement in receptor fimcti on.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis of the cloned C5aR (Kroll et al, 1991) was performed by the polymerase chain reaction (PCR) as described elsewhere for mutagenesis of rhC5a (Bubeck et al, 1994). Briefly, two overlapping PCR fragments were generated which were subsequently linked by a second fusion PCR.…”

Section: Materials Carrier-free N a ' T Was Obtained From Amershammentioning

confidence: 99%

Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor

Raffetseder

Méry

et al. 1996

European Journal of Biochemistry

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(Rcccived 25 Julyi2 October 1995) ~ E3B 95 122XilThe human CSa receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human CSa. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asnll9, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the CSaR (Asp37, Asp82, Arg175, Arg206, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282--+Ala) or partial loss of ligandbinding capacity (Argl75+Ala, Arg206+Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln206]CSaR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population ( < l o % ) of high-affinity sites ( K p 2 nM), which was functionally active at 20 nM in the heterologous Xencyus oocyte expression system after coexpression of Ga-16. The number of high-affinity sites of wild-type and [Gln206]C5aR in 293 cells could be up-regulated by coexpression of Gia-2 and downregulated by GTP[ yS ]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg206 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human CSaR by affecting the intracellular G-protein coupling.Keywords: C5a receptor; receptor structure; guanine-nucleotide-binding-regulatory-protein coupling; site-directed mutagenesis.Human complement factor C5a (C5a) is a 74-amino-acid glycopeptide which is generated by proteolytic cleavage from the fifth component of complement. Binding of the CSa peptide to its receptor triggers major cell activation and degranulation events, such as chemotaxis of polymorphonuclear cells, induction of the oxidative burst, histamine and interleukin release rrom monocytes. C5a is therefore thought to be an important inflammation-promoting agent. It interacts with the C5a receptor (CSaR) found in the membrane of polymorphonuclear cells, monocytes, macrophages and the histiocytic U937 and HL-60 cell lines (for a recent review, see KBhl and Bitter-Suermann, 1993, and the literature cited therein).

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“…plasmids pBLCATS-l(lM), pBLCAT5-112M) and pBLCATS-l(3M), were obtained by sequential PCR steps using mutated oligonucleotides essentially as described recently (Bubeck et al, 1994). Primers 678 and 679 served as flanking primers in both PCR steps.…”

Section: Cells and Reagents Hela Cells Were Obtained From Amentioning

confidence: 99%

Transcriptional Activation of Psoriasis‐Associated Cytokeratin K17 by Interferon‐γ

Vogel

Denecke

Troyanovsky

et al. 1995

European Journal of Biochemistry

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The acid cytokeratin K17 is inducible by interferon-? (IFN-y), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-y in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13). cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-y but not IFN-a in transient transfection assays. Sequence analysis revealed three putative y-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyltFWA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GASl (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GASl element rendered the promoter uninducible Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFF-y treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified.by IFN-y.

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Site‐specific mutagenesis of residues in the human C5a anaphylatoxin which are involved in possible interaction with the C5a receptor

Cited by 42 publications

References 41 publications

C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor

Transcriptional Activation of Psoriasis‐Associated Cytokeratin K17 by Interferon‐γ

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