Synergistic antifungal activity of two chitin-binding proteins from spindle tree (Euonymus europaeus L.).

Two structurally different chitin-binding proteins were isolated from bark and leaves of the spindle tree (Euonymus europaeus L.). Both the small hevein-like chitin-binding protein (Ee-CBP) and the classical class-I chitinase (Ee-chitinase) possess antifungal properties, Ee-CBP being far more potent than Ee-chitinase. In addition, Ee-CBP and Ee-chitinase display a pronounced synergistic effect when added together in the test medium. Determination of the biological activities indicates that the synergism between Ee-CBP and Ee-chitinase relies on a different mode of action. Cloning and sequencing of the corresponding genes further revealed that Ee-CBP and Ee-chitinase are simultaneously expressed in bark and leaf tissues, and hence can act synergistically in planta. Moreover, analysis of the deduced sequences allowed the exact relationship between the structurally different Ee-CBP and Ee-chitinase to be corroborated. Both proteins are synthesized as similar chimeric precursors consisting of an N-terminal hevein domain linked to a C-terminal chitinase-like domain by a hinge region. However, whereas in the case of Ee-chitinase the C-terminal chitinase domain remains linked to the N-terminal hevein domain, the corresponding domain is cleaved from the Ee-CBP-precursor resulting in the formation of the hevein-type Ee-CBP. Since both precursors are--apart from the hinge region between the hevein and chitinase domains--very similar, the Ee-CBP/Ee-chitinase system offers a unique opportunity to study the importance of sequence and/or structural information comprised in the hinge region for the posttranslational processing of the respective precursor proteins.

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.).

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC(50)-values as low as 1 microg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.