Biologically Active Conformer of the Effector Region of Human C5a and Modulatory Effects of N-Terminal Receptor Binding Determinants on Activity

Finch, Angela M.; Vogen, Shawn M.; Sherman, Simon; Kirnarsky, Leonid; Taylor, Stephen M.; Sanderson, Sam D.

doi:10.1021/jm960727r

Cited by 53 publications

(97 citation statements)

References 25 publications

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“…pD 2 transforms [Ϫlog EC 50 (M)] were calculated for each concentration-response curve and reported as the mean Ϯ SE. Peptide binding affinity to the C5aR was evaluated on intact human PMNs by a competition assay using [ 125 I]-C5a according to previously described methods (7,8). Statistical analysis of the values obtained from pharmacologic assays was performed using one-way ANOVA.…”

Section: Pharmacologic Assaysmentioning

confidence: 99%

Induction of an Antigen-Specific CTL Response by a Conformationally Biased Agonist of Human C5a Anaphylatoxin as a Molecular Adjuvant

Ulrich¹,

Cieplak

Paczkowski

et al. 2000

The Journal of Immunology

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A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating an Ag-specific CTL response against murine P815S target cells expressing an Ld-restricted CTL epitope of the hepatitis B surface Ag (HBsAg). Groups of BALB/c mice (H-2d) were immunized with aqueous solutions of the HBsAg CTL epitopes (IPQSLDSWWTSL and IPQSLDSWWTSLRR); the C5a agonist (YSFKPMPLaR); the C5a agonist and HBsAg CTL epitopes admixed (IPQSLDSWWTSL and IPQSLDSWWTSLRR + YSFKPMPLaR); the C5a-active, HBsAg CTL epitope-C5a agonist constructs (IPQSLDSWWTSLYSFKPMPLaR, IPQSLDSWWTSLRRYSFKPMPLaR, and IPQSLDSWWTSLRVRRYSFPMPLaR); a C5a-inactive, reverse-moiety construct (YSFKPMPLaRRRIPQSLDSWWTSL); and a C5a-attenuated, carboxyl-terminal-blocked construct (IPQSLDSWWTSLRRYSFKPMPLaRG). Ag-specific CD8+ CTL responses were observed after the secondary boost in the absence of any added adjuvant only in mice that were immunized with C5a-active contructs, IPQSLDSWWTSLRRYSFKPMPLaR and IPQSLDSWWTSLRVRRYSFKPMPLaR. These two C5a-active immunogens contained potential subtilisin-sensitive linker sequences between the HBsAg CTL epitope and the C5a agonist; i.e., a double-Arg (RR) and a furin protease sensitive sequence (RVRR). The introduction of these potentially cleavable sequences may be a method of increasing the likelihood of liberating the CTL epitope from the C5a agonist by intracellular proteases, thereby facilitating entry of the epitope into Ag-processing pathways via an exogenous route.

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Section: Pharmacologic Assaysmentioning

confidence: 99%

Induction of an Antigen-Specific CTL Response by a Conformationally Biased Agonist of Human C5a Anaphylatoxin as a Molecular Adjuvant

Ulrich¹,

Cieplak

Paczkowski

et al. 2000

The Journal of Immunology

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“…The vaccines used in this study were composed of three basic components, the first being the molecular adjuvant, YSFKPMPLaR (EP54), which is a conformationally biased, response-selective agonist of complement component C5a [65][66][67][68][69][70][71][72][73][74] . EP54 retains C5a-like immune stimulatory properties, but with significantly reduced C5a-like inflammatory properties [17][18][19][20]. The role of the molecular adjuvant EP54 in these vaccines is to target covalently attached Ags to and activate the Ag processing and presentation capacity of C5a receptor-bearing antigen presenting cells [21][22][23], particularly dendritic cells (DCs) [24].…”

Section: Introductionmentioning

confidence: 99%

Immune responses to methamphetamine by active immunization with peptide-based, molecular adjuvant-containing vaccines

Duryee

Bevins

Reichel

et al. 2009

Vaccine

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a b s t r a c tVaccines to methamphetamine (meth) were designed by covalently attaching a meth hapten (METH) to peptide constructs that contained a conformationally biased, response-selective molecular adjuvant, YSFKPMPLaR (EP54). Rats immunized with EP54-containing meth vaccines generated serum antibody titers to authentic meth, an immune outcome that altered meth self-administration. Immunization increased meth self-administration suggesting pharmacokinetic antagonism. The ability of immune sera to bind a METH-modified target protein dramatically decreased during and shortly after the meth selfadministration assay, suggesting effective sequestration of free meth. However, the binding ability of immune sera to the METH-modified target protein was recovered 34 days after meth-free clearance time.

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“…7). The backbone conformation of C5a-(65-69) was extracted from the NMR/calculation data on YSFKPMPLaR, a known agonist of C5aR (27,28). Docking was performed by a systematic search on the six-dimensional grid (three translations and three rotations) that covered a total of 2,268 starting orientations of C5a-(65-69) within the TM region of C5aR.…”

Section: Methodsmentioning

confidence: 99%

“…Then, possible orientations of the entire structure of C5a were restored by overlapping the rigid core C5a-(1-62) from PDB entry 1KJS with each of the four selected orientations of C5a-(59 -74) within C5aR. It was found that only one specific orientation of C5a satisfied the requirements of spatial proximity of positions 15,18,19,20,27, and 46 in C5a to specific residues in the N-terminal segment 8 -41 of C5aR, as was suggested by available site-directed mutagenesis data (31,32). Seventeen conformations of NT 8 -41 (out of 185) and 11 conformations of the EC1 ϩ EC2 ϩ EC3 loop package (out of 29) did not show steric clashes with this particular orientation of C5a; all these conformations together with the selected orientation of C5a and the TM region of C5aR make up the final model of the C5a-C5aR complex.…”

Section: Methodsmentioning

confidence: 99%

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

Hagemann

Miller

Klco

et al. 2008

Journal of Biological Chemistry

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Complement factor 5a (C5a) is an anaphylatoxin that acts by binding to a G protein-coupled receptor, the C5aR. The relative orientation of this ligand-receptor pair is investigated here using the novel technique of disulfide trapping by random mutagenesis (DTRM) and molecular modeling. In the DTRM technique, an unpaired cysteine is introduced in the ligand, and a library of randomly mutagenized receptors is screened to identify mutants that introduce a cysteine at a position in the receptor that allows functional interactions with the ligand. By repeating this analysis at six positions of C5a, we identify six unique sets of intermolecular interactions for the C5a-C5aR complex, which are then compared with an independently developed computational three-dimensional model of the complex. This analysis reveals that the interface of the receptor N terminus with the cysteine-containing ligand molecules is selected from a variety of possible receptor conformations that exist in dynamic equilibrium. In contrast, DTRM identifies a single position in the second extracellular loop of the receptor that interacts specifically with a cysteine probe placed in the C-terminal tail of the C5a ligand.One of the most biologically important families of receptors is the G protein-coupled receptors (GPCRs), 2 which eukaryotic cells use to sense signals as diverse as light, odorants, small molecules, and polypeptide hormones (1). The GPCRs, which number ϳ1000 in the human genome (2), have a shared topology made up of seven transmembrane domains (TMs) linked by intracellular and extracellular (EC) loops, along with an extracellular N terminus and intracellular C terminus (Fig. 1). These receptors also have a shared mechanism of action, whereby the activated receptor serves as a guanosine exchange factor on the ␣ subunit of a heterotrimeric G protein, thus transmitting the signal to the interior of the cell. Many drugs are directed against GPCRs, including adrenergic, muscarinic, dopaminergic, serotonergic, GABAergic, and histaminergic receptors. Taken together, drugs acting on GPCRs make up an estimated 50% of the current pharmacopoeia (3).The structural basis of receptor-ligand interaction is of great interest in both basic and applied pharmacology. In the case of GPCRs, a central question is how receptors with a single common topology can be activated by such a wide variety of ligands. A related question is how specificity is built into these receptors, so that each is activated only by the appropriate ligands.Many GPCRs are activated by polypeptide ligands, including chemokines such as SDF-1 (CXCL12), physiologic modulators such as angiotensin II, glycoprotein hormones such as luteinizing hormone, and chemotactic factors such as complement factor 5a (C5a). These ligands are too large to fit entirely in an interhelical cleft of a GPCR, so their bulk must serve some adaptive function other than receptor activation per se. In some cases, the ligand is recruited by its affinity for the receptor extracellular domains, and a second discret...

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Biologically Active Conformer of the Effector Region of Human C5a and Modulatory Effects of N-Terminal Receptor Binding Determinants on Activity

Cited by 53 publications

References 25 publications

Induction of an Antigen-Specific CTL Response by a Conformationally Biased Agonist of Human C5a Anaphylatoxin as a Molecular Adjuvant

Induction of an Antigen-Specific CTL Response by a Conformationally Biased Agonist of Human C5a Anaphylatoxin as a Molecular Adjuvant

Immune responses to methamphetamine by active immunization with peptide-based, molecular adjuvant-containing vaccines

Structure of the Complement Factor 5a Receptor-Ligand Complex Studied by Disulfide Trapping and Molecular Modeling

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