Characterisation of C5a receptor agonists from phage display libraries

被引:11
作者
Cain, SA [1 ]
Higginbottom, A [1 ]
Monk, PN [1 ]
机构
[1] Univ Sheffield, Sch Med, Acad Neurol Unit, Sheffield S10 2RX, S Yorkshire, England
关键词
complement receptor; phage display; random mutagenesis; pyrimidine analogue; PCR; 8-oxodeoxyguanosine;
D O I
10.1016/S0006-2952(03)00473-8
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
C5a des-Arg(74) has a 10- to 100-fold lower receptor binding affinity than intact C5a and is only a partial agonist. We have used phage display selection from randomly mutated C5a des-Arg74 libraries to isolate variant proteins that can activate C5a receptors with similar potency to C5a. Here we explore the interactions of three variants (V1-3) with C5aR mutated at residues involved in the differential response. The mutant Asp(282)Arg-C5aR is preferentially activated by C5a des-Arg74, probably due to repulsion between Arg(74) of C5a and the substituent Arg(282). In accordance with this hypothesis, V2 (with a polar C-terminus which has no Arg residue) but not V1 (with a C-terminal Arg residue at position 73) could activate Asp(282)Arg-C5aR. V3, with a very hydrophobic C-terminus, was the most potent agonist at Asp(282)Arg-C5aR. Arg(175) is a potential counterion for the C-terminal carboxylate of C5a. C5aR mutated to either Ala or Asp at this position lost nearly all responsiveness to both C5a and C5a des-Arg(74), suggesting that mutation of Arg(175) caused a non-specific loss of receptor conformation and a loss of signalling capacity. However, V3 could still activate Arg(175)Asp/Ala-C5aR with the same potency as wild-type C5aR. demonstrating that the mutant receptors retained high signalling capability and showed a specific loss of responsiveness. Thus C5a des-Arg(74) variants produced by phage display are potentially useful tools for the dissection of ligand-receptor interactions. (C) 2003 Elsevier Inc. All rights reserved.
引用
收藏
页码:1833 / 1840
页数:8
相关论文
共 27 条
[1]   A small molecule C5a receptor antagonist protects kidneys from ischemia/reperfusion injury in rats [J].
Arumugam, TV ;
Shiels, IA ;
Strachan, AJ ;
Abbenante, G ;
Fairlie, DP ;
Taylor, SM .
KIDNEY INTERNATIONAL, 2003, 63 (01) :134-142
[2]   ANAPHYLATOXIN INACTIVATOR OF HUMAN PLASMA - ITS ISOLATION AND CHARACTERIZATION AS A CARBOXYPEPTIDASE [J].
BOKISCH, VA ;
MULLEREB.HJ .
JOURNAL OF CLINICAL INVESTIGATION, 1970, 49 (12) :2427-&
[3]   SITE-SPECIFIC MUTAGENESIS OF RESIDUES IN THE HUMAN C5A ANAPHYLATOXIN WHICH ARE INVOLVED IN POSSIBLE INTERACTION WITH THE C5A RECEPTOR [J].
BUBECK, P ;
GROTZINGER, J ;
WINKLER, M ;
KOHL, J ;
WOLLMER, A ;
KLOS, A ;
BAUTSCH, W .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1994, 219 (03) :897-904
[4]   Molecular profile of a human monoclonal antibody Fab fragment specific for Epstein-Barr virus gp350/220 antigen [J].
Bugli, F ;
Bastidas, R ;
Burton, DR ;
Williamson, RA ;
Clementi, M ;
Burioni, R .
HUMAN IMMUNOLOGY, 2001, 62 (04) :362-367
[5]   Analysis of receptor/ligand interactions using whole-molecule randomly-mutated ligand libraries [J].
Cain, SA ;
Ratcliffe, CF ;
Williams, DM ;
Harris, V ;
Monk, PN .
JOURNAL OF IMMUNOLOGICAL METHODS, 2000, 245 (1-2) :139-145
[6]   Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor [J].
Cain, SA ;
Woodruff, TM ;
Taylor, SM ;
Fairlie, DP ;
Sanderson, SD ;
Monk, PN .
BIOCHEMICAL PHARMACOLOGY, 2001, 61 (12) :1571-1579
[7]   Selection of novel ligands from a whole-molecule randomly mutated C5a library [J].
Cain, SA ;
Williams, DM ;
Harris, V ;
Monk, PN .
PROTEIN ENGINEERING, 2001, 14 (03) :189-193
[8]   Mapping the ligand-binding site on the C5a receptor:: Arginine74 of C5a contacts aspartate282 of the C5a receptor [J].
Cain, SA ;
Coughlan, T ;
Monk, PN .
BIOCHEMISTRY, 2001, 40 (46) :14047-14052
[9]   Receptor activation by human C5a des Arg74 but not intact C5a is dependent on an interaction between Glu199 of the receptor and Lys68 of the ligand [J].
Crass, T ;
Bautsch, W ;
Cain, SA ;
Pease, JE ;
Monk, PN .
BIOCHEMISTRY, 1999, 38 (30) :9712-9717
[10]   Requirements for C5a receptor-mediated IL-4 and IL-13 production and leukotriene C4 generation in human basophils [J].
Eglite, S ;
Plüss, K ;
Dahinden, CA .
JOURNAL OF IMMUNOLOGY, 2000, 165 (04) :2183-2189