Decapeptide Agonists of Human C5a: The Relationship between Conformation and Neutrophil Response

A series of decapeptide analogues corresponding to the C-terminal region of the human C5a anaphylatoxin (C5a) was synthesized with residue substitutions to restrict conformational flexibility in the C-terminal region (residues 71-74). These analogues behaved as full agonists of natural C5a in their ability to induce shape change (polarization) and the release of enzyme (β-glucuronidase) from human neutrophils (PMNs). There was a significant pharmacological correlation between the polarization and enzyme-release assays, suggesting similarities in PMN responsiveness toward these constrained peptides. Good correlations were also observed between these two PMN responses and spasmogenic activity (smooth muscle contraction of human fetal artery). A structure-function analysis for PMN polarization and enzyme release led to the identification of the following preferred backbone conformations: a twisted, helixlike conformation for residues 65-69, an extended conformation for residues 70-71, and a β-turn of type V for residues (71)72-74. The existence of a C-terminal, type V β-tum is supported by the NOE (nuclear Overhauser effect) results of two peptides from this series. These conformational features are reminiscent of those that were shown to correlate with the expression of spasmogenic and platelet aggregatory activities in an earlier investigation (Sanderson, S. D.; et al. J. Med. Chem. 1994, 37, 3171). These results suggest that PMNs and the cells responsible for smooth muscle contraction possess C5a receptors that respond to similar topochemical features presented by the agonist peptide ligand

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 (IC(50) 0.2, 0.1 μM respectively). In PMNs, the affinity of YSFKPMPLaR (IC(50) 0.1 μM) was similar to that in macrophages, whereas the affinity of YSFKD(NMeNle)PLaR for the PMN C5aR was >100 μM. 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

GRP94 Is Essential for Mesoderm Induction and Muscle Development Because It Regulates Insulin-like Growth Factor Secretion

Because only few of its client proteins are known, the physiological roles of the endoplasmic reticulum chaperone glucose-regulated protein 94 (GRP94) are poorly understood. Using targeted disruption of the murine GRP94 gene, we show that it has essential functions in embryonic development. grp94−/− embryos die on day 7 of gestation, fail to develop mesoderm, primitive streak, or proamniotic cavity. grp94−/− ES cells grow in culture and are capable of differentiation into cells representing all three germ layers. However, these cells do not differentiate into cardiac, smooth, or skeletal muscle. Differentiation cultures of mutant ES cells are deficient in secretion of insulin-like growth factor II and their defect can be complemented with exogenous insulin-like growth factors I or II. The data identify insulin-like growth factor II as one developmentally important protein whose production depends on the activity of GRP94. Keywords: chaperone/HSP90/Insulin-like growth factors/mouse development