Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation.

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia.

A region of tapasin that affects L(d) binding and assembly.

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes.

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor.

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has approximately 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P(103) in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants (P(103)Y, G(105)D and the double mutant, P(103)Y/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and P(103)Y/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR.

Development of response-selective agonists of human C5a anaphylatoxin: conformational, biological, and therapeutic considerations.

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.

C5a modulation of interleukin-1 beta-induced interleukin-6 production by human osteoblast-like cells.

Periodontal bone resorption is controlled by osteoblast products, including interleukin (IL)-6, which are stimulated by other cytokines and complement components in the pro-inflammatory milieu. This study demonstrated that human osteoblast-like osteosarcoma cells (MG-63) responded to human recombinant (hr) C5a by releasing significant amounts of the bone-resorbing cytokine IL-6. C5a-induced release of IL-6 was enhanced 330% when cells were exposed to IL-1beta prior to C5a challenge at optimal concentrations (1.0 microg/ml C5a, 0.1 ng/ml IL-1beta). Cells simultaneously challenged with these concentrations of C5a and IL-1beta produced a 700% increase in IL-6 release relative to cells challenged with IL-1beta alone. Incubation of IL-1beta-treated cells with anti-human C5a receptor (C5aR) Ab resulted in a 78% suppression of the C5a-induced release of IL-6, but C5aR neutralization did not affect C5a/IL-1beta co-stimulation of IL-6. In addition, neither IL-1beta nor C5a significantly altered the other's cell-surface receptor relative to binding affinity or density. These results indicate that while MG-63 cells express functional C5aRs, the synergistic effect of C5a and IL-1beta on osteoblast IL-6 production is probably controlled by post-receptor signaling events. C5a agonists and antagonist used to alter critical C5a concentrations may present a new point of therapeutic intervention for the treatment of inflammatory bone resorption such as is found in periodontitis.

Induction of an antigen-specific CTL response by a conformationally biased agonist of human C5a anaphylatoxin as a molecular adjuvant.

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.

Response-selective C5a agonists: differential effects on neutropenia and hypotension in the rat.

Some in vivo activities of two complement C5a agonist analogues have been evaluated by measuring changes in blood pressure and neutropenia in the rat and comparing the results with their receptor affinities in peritoneal macrophages and polymorphonuclear leucocytes (PMNs). In vitro C5a receptor (C5aR) binding experiments showed that YSFKPMPLaR and YSFKD(NMeNle)PlaR had similar affinities for the macrophage C5aR (IC50 0.2, 0.1 microM respectively). In PMNs, the affinity of YSFKPMPLaR (IC50 0.1 microM) was similar to that in macrophages, whereas the affinity of YSFKD(NMeNle)PLaR for the PMN C5aR was >100 microM. Given i.v., YSFKD(NMeNle)PLaR had similar activity to YSFKPMPLaR on blood pressure but did not cause neutropenia. These results demonstrate selectivity of a new C5a agonist in vitro, which is paralleled in vivo. The results suggest the possibility of developing selective agonists of C5a for in vivo use in humans.

Determination of structural elements related to the biological activities of a potent decapeptide agonist of human C5a anaphylatoxin.

The structural features related to the biologic activities of a potent, response-selective decapeptide agonist of human C5a, YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73), were identified by NMR analysis in H2O, DMSO and TFE. This investigation showed that the KPM residues in H2O and the SFKPM residues in DMSO exhibited an extended backbone conformation, whereas a twisted conformation was found in this region in TFE. In H2O, the C-terminal region (PLaR) adopted a distorted type II beta-turn or a type II/V beta-turn. In the type IIN beta-turn, Leu72 exhibited a conformation typical of a type II beta-turn, whereas D-Ala73 exhibited a conformation characteristic of a type V beta-turn. Furthermore, a gamma-turn involving residues LaR overlapped with the type II/V beta-turn. In DMSO, the C-terminal region had the analogous turn-like motif (type II/V beta-turn overlapping with gamma-turn) found in H2O. In TFE, no beta-turn motifs were formed by the PLaR residues. These turn-like motifs in the C-terminal region of the peptide in both H2O and DMSO were in agreement with the biologically important conformations predicted earlier by a structure-function analysis of a related panel of decapeptide analogs. The motifs determined by the NMR analysis of YSFKPMPLaR in H2O and DMSO may represent structural elements important for C5a agonist activity and thus can be used to design the next generation of C5a agonist, partial agonist and antagonist analogs.

Protein kinase C activation is required for cigarette smoke-enhanced C5a-mediated release of interleukin-8 in human bronchial epithelial cells.

Complement-derived anaphylatoxin C5a is a glycopolypeptide important in the regulation of inflammation. Previously, we have shown that C5a receptors (C5aR) are constitutively expressed on human bronchial epithelial cells (HBECs) grown in culture. We have also shown that the expression of C5aR is increased upon exposure of HBECs to 5% cigarette smoke extract (CSE), and that this subtoxic dose of CSE significantly enhances C5a-stimulated interleukin (IL)-8 release. To determine the intracellular signaling pathway of CSE + C5a-mediated IL-8 release, we assayed protein kinase C (PKC) activity of HBECs after exposing the cells to CSE and/or C5a. No increase in PKC activity was observed when HBECs were treated with 50 nM C5a for various times. However, PKC activity was increased by 2- to 3-fold in HBECs stimulated with 5% CSE for 1 h, as compared with cells incubated with medium only. No additional increase in PKC was observed when HBECs were treated with CSE and C5a together. When HBECs were pretreated with the PKC-specific inhibitor calphostin C (1 microM), no CSE-mediated PKC activation was observed. We then correlated PKC activation with IL-8 release in the same cells. Although HBECs required stimulation by both CSE and C5a to release maximal levels of IL-8, preincubation of CSE-stimulated HBECs with calphostin C inhibited IL-8 release by CSE + C5a. These results suggest that PKC activation by CSE alone does not result in IL-8 release, but that CSE-stimulated PKC activation is required for C5a-mediated IL-8 release from HBECs.

The influence of Lys68 in decepeptide agonists of C5a on C5a receptor binding, activation and selectivity.

The potent, conformationally biased C5a agonist peptide YSFKPMPLaR (C5a65-74, Y65, F67, P69, P71, D-Ala73) was used as a template to gain insight into the nature and importance of lysine at position 68 in the peptide-receptor interaction. A panel of YSFKPMPLaR analogs with systematic substitutions for Lys68 was evaluated for C5a receptor (C5aR) binding affinity and activation in two well-characterized assay systems: human polymorphonuclear leukocytes (PMNs) and human fetal artery. In addition, we determined the activity of these new analogs in transfected rat basophilic leukemia (RBL) cells in which the Glu at position 199 of the C5aR (wtGlu199) was replaced by a Gln (C5aR-Gln199) or a Lys (C5aR-Lys199). Our results indicated that Lys68 in YSFKPMPLaR plays an important role in binding the C5aR expressed on PMNs and RBL cells. Furthermore, the data indicated that Lys68 interacted with Glu199 of the C5aR in PMNs and RBL cells. In human fetal artery, however, Lys68 substitutions had little or no effect on activity, which suggested that the receptor conformation may be different in this tissue. Thus, the interaction between Lys68 of the decapeptide agonist and Glu199 of the C5aR may be cell type-specific and may form the molecular basis for tissue-specific responses to C5a agonists.

Arterially perfused eye model of uveitis.

OBJECTIVE: To develop an in vitro model of uveitis based on an ex situ perfused eye to evaluate the anti-inflammatory activity of new pharmacological products. PROCEDURE: Eyes were removed from more than 60 dogs and 9 horses immediately after euthanasia and perfused with nutrient medium through the lateral long ciliary artery. Perfused eyes produced aqueous humour, and perfusion pressure was adjusted to obtain an intraocular pressure in the physiological range. When the eyes were treated with histamine, a complement C5a analogue peptide and hydrogen peroxide, typical signs of uveitis were produced. These included miosis, vascular leakage, reduced intraocular pressure, reduced flow of perfusate and, in some eyes, conjunctival oedema. RESULTS: Canine eyes showed a decrease in intraocular pressure and a decrease in perfusate flow rate when challenged with 100 mumol/L hydrogen peroxide. Flunixin meglumine (5 mumol/L), ketoprofen (5 mumol/L), indomethacin (5 mumol/L) as well as a new drug pirfenidone (10 mumol/L) prevented changes in intraocular pressure induced by hydrogen peroxide, but did not significantly moderate the mediator-induced changes in perfusate flow. CONCLUSIONS: This model is suitable for evaluating potential anti-inflammatory activity of drugs without having to induce uveitis in an experimental animal. The technique is suitable for species that range in size from cats to horses.

Expression of receptors for C5a anaphylatoxin (CD88) on human bronchial epithelial cells: enhancement of C5a-mediated release of IL-8 upon exposure to cigarette smoke.

Results are presented that demonstrate a heightened responsiveness of human bronchial epithelial cells (HBECs) toward the complement-derived anaphylatoxin C5a when these cells are exposed to cigarette smoke. This C5a response is possible because we show at both the protein and mRNA levels that HBECs constitutively express receptors for C5a (C5aR, CD88). Control (untreated) HBECs responded to C5a (50 nM) by releasing the proinflammatory cytokine IL-8 at low but significant levels. However, exposure of HBECs to 5% cigarette smoke extract (CSE) for at least 15 min resulted in an increase in the ability of an anti-human C5aR Ab to bind to the cell surface. CSE-treated HBECs responded in a dose-dependent fashion to human recombinant C5a and to a conformationally biased decapeptide agonist of C5a (YSFKPMPLaR) by releasing IL-8. The levels of IL-8 released in response to C5a were significantly greater in CSE-treated HBECs than in control HBECs. Moreover, this C5a-mediated release of IL-8 from CSE-treated HBECs was significantly reduced in the presence of the anti-human C5aR Ab. These results indicate that HBECs constitutively express C5aRs and that exposure to environmental irritants such as cigarette smoke modulates the expression and responsiveness of these C5aRs toward the C5a-mediated release of IL-8.

NMR analysis of a potent decapeptide agonist of human C5a anaphylatoxin.

A NMR investigation in H2O, TFE and DMSO of a conformationally constrained, potent decapeptide agonist of human C5a, YSFKDMPLaR (C5a65-74, Y65, F67, P71, D-Ala73) showed that its N-terminal region (YSFKD) exhibited an extended backbone conformation in H2O and a more twisted conformation in both TFE/H2O (30:70, v/v; referred to as TFE) and DMSO. The C-terminal region (MPLaR) of the peptide adopted compact, turn-like structures. In H2O, the C-terminal region adopted a type II beta-turn or a distorted type V/II beta-turn involving residues PLaR. In the distorted type V/II beta-turn, Leu72 exhibited a conformation typical of a type V beta-turn, whereas D-Ala73 exhibited a conformation typical of a type II beta-turn. The distorted type V/II beta-turn overlapped with an inverse gamma-turn involving residues MPL. In DMSO, the C-terminal region had the analogous inverse gamma-turn and the V/II beta-turn found in H2O. In many of the DMSO structures, two inverse gamma-turns in the MPL and PLa positions formed a double-inverse gamma-turn. None of the turns observed in H2O were present in TFE. However, in TFE, the PLa residues formed an inverse gamma-turn. Overall, the turn-like structural motifs in the C-terminal region of the peptide in both H2O and DMSO (but not in TFE) agreed with the biologically important conformations obtained earlier by the structure-function analysis of a panel of C5a agonist peptides. These motifs may represent key structural elements important for C5a agonist activity and may be used to design the next generation of C5a agonist and antagonist analogues.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity.

A conformationally biased decapeptide agonist of human C5a (C5a55-74Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a12-20-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Anti-human kappa opioid receptor antibodies: characterization of site-directed neutralizing antibodies specific for a peptide kappa R(33-52) derived from the predicted amino terminal region of the human kappa receptor.

Site-directed polyclonal Abs specific for a synthetic peptide with sequence homology to the predicted N-terminal sequence of the human kappa opioid receptor [anti-kappa R-(33-52)] are capable of binding to normal human cells and cell lines expressing mRNA specific for the human kappa receptor. Flow cytometric analysis of 1) a neuronal cell line (NT2), 2) blood-derived CD14+ monocytes, 3) monocyte-like cell lines (U937 and THP 1), 4) blood-derived CD3+ T cells and a T cell line, and 5) human B cell lines bound anti-kappa R-(33-52) in a specific manner. Anti-kappa R-(33-52) was also found to specifically neutralize the immunosuppressive activities associated with the kappa R-selective agonist U50,488H. This antiserum was found to block U50,488H-mediated inhibition of 1) Staphylococcus aureus Cowen strain I-induced B and T lymphocyte proliferation, 2) PHA-induced T lymphocyte proliferation, and 3) S. aureus Cowen strain I-induced IgG production. However, this antiserum failed to neutralize mu R-selective agonist (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol)-mediated suppression of IgG synthesis. Finally, the kappa R-selective antagonist nor-binaltorphimine hydrochloride inhibits the binding of anti-kappa R-(33-52) to the U937 cell line. These results suggest that anti-kappa R-(33-52) specifically interacts with the human kappa R molecule. Studies conducted with anti-kappa R-(33-52) indicated that this antiserum effectively blocked U50,488H-mediated immunosuppression, but by itself did not enhance or suppress lymphocyte activation. These data suggest that anti-kappa R-(33-52) 1) does not interact with the effector binding site of the receptor, but sterically interferes with U50,488H binding to the receptor; and/or 2) the antiserum interacts with a secondary binding site that is important for ligand binding, but may not be involved in signal transduction.

Molecular adjuvant effects of a conformationally biased agonist of human C5a anaphylatoxin.

A conformationally biased decapeptide agonist of human C5a anaphylatoxin (YSFKPMPLaR) was used as a molecular adjuvant in stimulating Ab responses against peptide epitopes derived from human MUC1 glycoprotein and the human mu and kappa opioid receptors. C57BL6 mice were immunized with the MUC1 epitope (YKQGGFLGL); the C5a agonist (YSFKPMPLaR); YSFKPMPLaR and YKQGGFLGL together, but unconjugated; a C5a-active, MUC1 epitope construct (YKQGGFLGLYSFKPMPLaR); and a C5a-inactive, reversed moiety construct (YSFKPMPLaRYKQGGFLGL). High Ab titers specific for the MUC1 epitope were observed only in mice immunized with the C5a-active epitope construct. Similar results were obtained in BALB/c mice immunized with the C5a-active, MUC1 epitope construct. Abs from the sera of the C57BL6 mice were predominately of the IgG2a, IgG2b, and IgM isotypes and were reactive against human recombinant MUC1 and MUC1 expressed by the Panc-1 M1F.15 pancreatic cell line. When compared with the corresponding KLH-epitope conjugates in C57BL6 mice, the epitope-C5a agonist constructs produced titers of specific IgG Abs of isotypes distinct from those generated by the keyhole limpet hemocyanin-epitope conjugates. Rabbits immunized with a mu opioid receptor epitope-C5a agonist construct (GDLSDPCGNRTNLGGRDSLYSFKPMPLaR) or a kappa opioid receptor epitope-C5a agonist construct (FPGWAEPDSNGSEDAQLYSFKPMPLaR) generated high titer, epitope-specific Ab responses. Ab titers generated in response to the opioid epitope-C5a agonist constructs were comparable to those generated by the opioid KLH-epitope conjugates. The results of this study are discussed in terms of possible mechanisms by which the conformationally biased C5a agonist serves as a molecular adjuvant.

Conformationally biased analogs of human C5a mediate changes in vascular permeability.

A panel of conformationally constrained, decapeptide agonists corresponding to the C-terminal "effector" region of human C5a (C5a65-74 or ISHKDMQLGR) was evaluated for the ability to increase vascular permeability. One constrained analog, acyl-YSFKPMPLaR, expressed between 2 and 10% of full C5a activity in increasing vascular permeability, as measured by the extravasation of Evans blue dye in guinea pig skin. This analog was at least 10-fold more potent than its unconstrained sister analog C5a65-74465, F67++ (YSFKDMQLGR), which was used as an internal standard in these assays. Neither acyl-YSFKPMPLaR nor YSFKDMQLGR changed the transvascular equillibrium of an electrolyte, 86Rb, at the peptide injection site. However, both peptides effected a significant increase in the extravasation of two macromolecules, 125I-labeled bovine serum albumin and 131I-labeled monoclonal antibody BL-3. The extravasation of Evans blue dye mediated by 0.03 to 0.1 nmol of acyl-YSFKPMPLaR was nearly abolished by 1 to 10 nmol of the antihistamine diphenhydramine. For YSFKDMQLGR, however, the sensitivity toward diphenhydramine was observed only at low concentrations of the peptide (1 nmol). When incubated in human and mouse sera, acyl-YSFKPMPLaR was shown to be stable toward the actions of serum carboxypeptidases. However, the unconstrained analog YSFKDMQLGR was rapidly converted to the des-Arg form under the same conditions. Taken together, these results support a growing body of evidence that unique topochemical features expressed in conformationally constrained agonist analogs of C5a contribute favorably to their ability to modulate vascular permeability and to their stability in serum.

Stabilization of an isolated helical capping box in solution by hydrophobic interactions: evidence from the NMR study of bioactive peptides from the C-terminus of human C5a anaphylatoxin.

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Jounral of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70 -Gln71- Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of (3)J(NH-alphaH) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered (3)J(NH-alphaH) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stabilizing capping box (E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605; H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18, p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (Ile65-Ser66-His67-Lys68-Asp-69- Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74 (Tyr65, Phe67) in receptor-binding assays.

The effect of C5a and U46619 on the isolated, perfused human placental lobule: development of a method for the online estimation of tissue fluid accumulation.

A method for the automatic and simultaneous determination of perfusion pressure and fluid accumulation in the isolated, perfused human placental lobule is described. We demonstrated that the inflammatory mediator, C5a, a C5a agonist analogue peptide, and the thromboxane mimetic U46619 caused increased fetal perfusion pressure and increased tissue weight when administered via the fetal arterial circulation. Occlusion of the fetal venous effluent tubing caused significantly greater increases in tissue weight than the pharmacological agents. Detectable increases in tissue weight occurred within 47 +/- 3 sec (n = 21) following pressure increases caused by the pharmacological agents. In each case, the increase in tissue weight was accompanied by an increased permeability of the materno-fetal barrier, shown by the transfer of Evans blue albumin from the fetal circulation to the maternal compartment.