Structural and functional diversity within the cystatin gene family of Hordeum vulgare.
Phytocystatins are inhibitors of cysteine proteinases from plants putatively involved in defence and as endogenous regulators of protein turnover. Seven genes encoding cystatins (HvCPI-1 to HvCPI-7), identified from EST collections and from an endosperm cDNA library, have been characterized. The intron-exon structure of their corresponding ORFs has been determined and the predicted three-dimensional models for the seven barley cystatins have been established, based on the known crystal structure of oryzacystatin I from rice. Only one out of the seven deduced proteins, HvCPI-7, had sequence variations affecting the three conserved motifs implicated in the enzyme-inhibitor interaction. In three cases, HvCPI-5, HvCPI-6, and HvCPI-7, amino acid differences lead to the prediction of important structural changes in their three-dimensional structures. Northern blot analysis indicated that the seven genes have different expression patterns in barley tissues. The recombinant proteins expressed in Escherichia coli showed distinct inhibitory properties in vitro, with different K(i) values, against the three cysteine proteinases tested: papain, cathepsin B, and cathepsin H. Moreover, these recombinant proteins presented differential fungicidal characteristics inhibiting the growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum in vitro. The resulting implications for the structural and functional diversity of the seven barley cystatins studied are discussed.
Abraham
Zamira
Z
Laboratorio de Bioquímica y Biología Molecular, Dpto. de Biotecnología-Centro de Biotecnología y Genómica de Plantas-UPM, ETS Ingenieros Agrónomos. Ciudad Universitaria s/n, E-28040 Madrid, Spain.
Martinez
Manuel
M
Carbonero
Pilar
P
Diaz
Isabel
I
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020171:Botrytis / Q000502:physiology; D017124:Conserved Sequence; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D005670:Fusarium / Q000502:physiology; D018628:Gene Dosage; D015723:Gene Library; D001467:Hordeum / Q000235:genetics* / Q000382:microbiology; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology; D016415:Sequence Alignment
D015891:Cystatins; D010940:Plant Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins