Mutational analysis of a plant defensin from radish (Raphanus sativus L) reveals two adjacent sites important for antifungal activity

被引:146
作者
DeSamblanx, GW
Goderis, IJ
Thevissen, K
Raemaekers, R
Fant, F
Borremans, F
Acland, DP
Osborn, RW
Patel, S
Broekaert, WF
机构
[1] KATHOLIEKE UNIV LEUVEN,FA JANSSENS LAB GENET,B-3001 HEVERLEE,BELGIUM
[2] STATE UNIV GHENT,DEPT ORGAN CHEM,BIOMOL NMR UNIT,B-9000 GHENT,BELGIUM
[3] ZENECA AGROCHEM,JEALOTTS HILL RES STN,BRACKNELL RG42 6ET,BERKS,ENGLAND
[4] UNIV LONDON UNIV COLL,DEPT BIOCHEM & MOL BIOL,LONDON WC1E 6BT,ENGLAND
关键词
D O I
10.1074/jbc.272.2.1171
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand, When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.
引用
收藏
页码:1171 / 1179
页数:9
相关论文
共 41 条
[1]   A NEW SIGNAL PEPTIDE USEFUL FOR SECRETION OF HETEROLOGOUS PROTEINS FROM YEAST AND ITS APPLICATION FOR SYNTHESIS OF HIRUDIN [J].
ACHSTETTER, T ;
NGUYENJUILLERET, M ;
FINDELI, A ;
MERKAMM, M ;
LEMOINE, Y .
GENE, 1992, 110 (01) :25-31
[2]   MLEV-17-BASED TWO-DIMENSIONAL HOMONUCLEAR MAGNETIZATION TRANSFER SPECTROSCOPY [J].
BAX, A ;
DAVIS, DG .
JOURNAL OF MAGNETIC RESONANCE, 1985, 65 (02) :355-360
[3]  
BENNETZEN JL, 1982, J BIOL CHEM, V257, P3026
[4]   PEPTIDES FROM FROG-SKIN [J].
BEVINS, CL ;
ZASLOFF, M .
ANNUAL REVIEW OF BIOCHEMISTRY, 1990, 59 :395-414
[5]   A NEW FAMILY OF SMALL (5 KDA) PROTEIN INHIBITORS OF INSECT ALPHA-AMYLASES FROM SEEDS OR SORGHUM (SORGHUM-BICOLOR (L) MOENCH) HAVE SEQUENCE HOMOLOGIES WITH WHEAT GAMMA-PUROTHIONINS [J].
BLOCH, C ;
RICHARDSON, M .
FEBS LETTERS, 1991, 279 (01) :101-104
[6]   THIONINS [J].
BOHLMANN, H ;
APEL, K .
ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY, 1991, 42 :227-240
[7]  
BOMAN HG, 1981, TRENDS BIOCHEM SCI, V6, P306, DOI 10.1146/annurev.mi.41.100187.000535
[8]   2-DIMENSIONAL H-1-NMR STUDY OF RECOMBINANT INSECT DEFENSIN-A IN WATER - RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE AND GLOBAL FOLDING [J].
BONMATIN, JM ;
BONNAT, JL ;
GALLET, X ;
VOVELLE, F ;
PTAK, M ;
REICHHART, JM ;
HOFFMANN, JA ;
KEPPI, E ;
LEGRAIN, M ;
ACHSTETTER, T .
JOURNAL OF BIOMOLECULAR NMR, 1992, 2 (03) :235-256
[9]   ANALYSIS OF SIDE-CHAIN ORGANIZATION ON A REFINED MODEL OF CHARYBDOTOXIN - STRUCTURAL AND FUNCTIONAL IMPLICATIONS [J].
BONTEMS, F ;
GILQUIN, B ;
ROUMESTAND, C ;
MENEZ, A ;
TOMA, F .
BIOCHEMISTRY, 1992, 31 (34) :7756-7764
[10]  
BROEKAERT WF, 1990, FEMS MICROBIOL LETT, V69, P55, DOI [10.1111/j.1574-6968.1990.tb04174.x, 10.1016/S0378-1097(98)00477-7]