NORMAL GENOMIC

• Taylor SM, Sherman SA, Kirnarsky L, Sanderson SD
• Development of response-selective agonists of human C5a anaphylatoxin: conformational, biological, and therapeutic considerations.
• Curr Med Chem. 2001; 8: 675-84
• Display abstract

• 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.

• Czermak BJ et al.
• Protective effects of C5a blockade in sepsis.
• Nat Med. 1999; 5: 788-92
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• Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. In this study, we induced sepsis in rats using cecal ligation and puncture (CLP). In rats depleted of the complement factor C3, CLP led to very short survival times (about 4 days). Of the rats that underwent CLP ('CLP rats') that were C3-intact and treated with preimmune IgG, most (92%) were dead by 7 days. Blood neutrophils from these rats contained on their surfaces the powerful complement activation product C5a. This group had high levels of bacteremia, and their blood neutrophils when stimulated in vitro had greatly reduced production of H2O2, which is known to be essential for the bactericidal function of neutrophils. In contrast, when companion CLP rats were treated with IgG antibody against C5a, survival rates were significantly improved, levels of bacteremia were considerably reduced, and the H2O2 response of blood neutrophils was preserved. Bacterial colony-forming units in spleen and liver were very high in CLP rats treated with preimmune IgG and very low in CLP rats treated with IgG antibody against C5a, similar to values obtained in rats that underwent 'sham' operations (without CLP). These data indicate that sepsis causes an excessive production of C5a, which compromises the bactericidal function of neutrophils. Thus, C5a may be a useful target for the treatment of sepsis.

• Regan L
• What determines where alpha-helices begin and end?
• Proc Natl Acad Sci U S A. 1993; 90: 10907-8

• Or YS, Clark RF, Lane B, Mollison KW, Carter GW, Luly JR
• Improvements in the minimum binding sequence of C5a: examination of His-67.
• J Med Chem. 1992; 35: 402-6

• Kawai M, Quincy DA, Lane B, Mollison KW, Luly JR, Carter GW
• Identification and synthesis of a receptor binding site of human anaphylatoxin C5a.
• J Med Chem. 1991; 34: 2068-71
• Display abstract

• C5a is a 74 amino acid polypeptide that likely plays an important role in the pathogenesis of a number of inflammatory diseases. Therefore, the discovery of a C5a antagonist is of considerable therapeutic interest. A series of peptides designed to survey various regions of the molecule was synthesized by solid-phase peptide synthesis and evaluated for receptor-binding activity with polymorphonuclear leukocyte membranes. The C-terminal octapeptide (Ac-His-Lys-Asp-Met-Gln-Leu-Gly-Arg-OH) was identified as the smallest fragment which possessed reasonable C5a receptor binding activity.

• Greer J
• Comparative structural anatomy of the complement anaphylatoxin proteins C3a, C4a and C5a.
• Enzyme. 1986; 36: 150-63
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• The anaphylatoxins are a family of proteins produced during the course of complement activation as the result of cleavage by specific serine proteases. These proteins are involved in a variety of biological functions, including inflammation. Comparative modeling techniques have been used to produce structures for C4a and C5a from the crystal structure of C3a. All three structures have conserved interior residues but very different external side chains and surface shapes and properties. Comparison of the anaphylatoxin structures and of the sequence conservation among different species suggests possible locations for their receptor binding sites and for their specificity residues which permit regulated proteolytic cleavage from precursor.

• Hoeprich PD Jr, Dahinden CA, Lachmann PJ, Davis AE 3rd, Hugli TE
• A synthetic nonapeptide corresponding to the NH2-terminal sequence of C3d-K causes leukocytosis in rabbits.
• J Biol Chem. 1985; 260: 2597-600
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• Numerous biologically active fragments have been described that are derived from the C3 molecule. Recently, a polypeptide (Mr 41,000) generated from the alpha chain of human iC3b by limited proteolysis with plasma kallikrein was shown to exhibit several biological functions. This C3-derived cleavage product, C3d-K, suppresses mitogen- and antigen-induced proliferation of human T-lymphocytes and induces leukocytosis in rabbits. We have identified and synthesized a portion of C3d-K that is associated with the leukocytosis phenomenon. A nonapeptide corresponding to the amino-terminal nine residues of C3d-K was synthesized using conventional Merrifield solid-phase peptide chemistry; the structure of this peptide is Thr-Leu-Asp-Pro-Glu-Arg-Leu-Gly-Arg (TLDPERLGR). At a final concentration of 4 X 10(-6) M, both the nonapeptide and the des-Arg octapeptide (TLDPERLG) were capable of inducing leukocytosis in rabbits. Additionally, both peptides enhance vascular permeability when injected in guinea pig skin. These activities are similar to those previously attributed to a C3 fragment identified as C3e by Ghebrehiwet and Muller-Eberhard (Ghebrehiwet, B., and Muller-Eberhard, H.J. (1979) J. Immunol. 123, 616-621). We conclude that the nonapeptide TLDPERLGR represents the active center of the C3-derived leukocytosis factors C3e and C3d-K. This active synthetic analogue of C3d-K should prove valuable in elucidating the mechanism of action for complement-dependent leukocyte mobilization in vivo.

• Johnson RJ, Chenoweth DE
• Structure and function of human C5a anaphylatoxin. Selective modification of tyrosine 23 alters biological activity but not antigenicity.
• J Biol Chem. 1985; 260: 10339-45
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• Reaction of either human C5a or its des-Arg74 derivative (des-Arg74-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyrosyl23-C5a and -des-Arg74-C5a were separated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyrosyl23-C5a and -des-Arg74-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a derivatives serving as competing ligands and a murine antihuman C5a monoclonal antibody employed as first antibody demonstrated that selective modification of tyrosine23 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyrosyl23-C5a was approximately 3-fold less active than native C5a in both bioassays and competitive ligand-receptor binding assays. Additionally, photoreactive derivatives prepared by coupling either N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate to aminotyrosyl23-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These observations suggest that the tyrosyl23 residue of C5a may participate importantly in the binding interactions of this chemotactic factor and its granulocyte receptor.

• Kim PS, Baldwin RL
• A helix stop signal in the isolated S-peptide of ribonuclease A.
• Nature. 1984; 307: 329-34
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• The isolated S-peptide (residues 1-20 of ribonuclease A) is known to show partial alpha-helix formation in aqueous solutions at low temperatures. We show here that the helix is limited to certain residues, including ones that are helical in the intact protein, and that a functional helix termination signal exists in the isolated peptide.

• Gerard C, Hugli TE
• C5a: a mediator of chemotaxis and cellular release reactions.
• Kroc Found Ser. 1981; 14: 147-60

• Hugli TE
• Chemical aspects of the serum anaphylatoxins.
• Contemp Top Mol Immunol. 1978; 7: 181-214