Primary Structure of ω‐Hordothionin, a Member of a Novel Family of Thionins from Barley Endosperm, and Its Inhibition of Protein Synthesis in Eukaryotic and Prokaryotic Cell‐Free Systems

Abstract

A new sulfur-rich basic polypeptide, so called ru-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified o-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of cu-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteolytic cleavage. cu-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by electrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues, and has strong sequence identity (66 %) with the sorghum SIcrl a-amylase inhibitor. cu-hordothionin, like y-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to y-hordothionin, w-hordothionin did not inhibit plant systems such as Triticurn aestivunz, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;i-hordothionin also inhibited the a-amylase activity from human saliva, while w-hordothionin and the other different genetic variants of thionins, ahordothionin and [j-hordothionin, failed to show any inhibitory effect.Keywords: w-hordothionin; y-thionin; barley; a-amylase inhibitor.Thionins are basic and cysteine-rich low-molecular-mass polypeptides of about 5 kDa, prevalently found in the endosperm of several Gramineae such as wheat and barley (a and /? purothionins and hordothionins), as well as in a variety of plant species, including leaves and stems (e.g. viscotoxin, pyrularia). Although orally innocuous, the thionins are toxic to injected animals [ l , 21, some yeast strains [3, 41, bacterial [3, 51, animal and plant cells [6, 71, fungi [8, 91 and insect larvae [lo]. They modify membrane permeability in cultured mammalian cells [ I l l and inhibit in vitro protein synthesis in cell-free systems derived from wheat germ, rabbit reticulocytes and Artemiu [ 121. Despite the fact that thionins are all believed to play a role in the defense system of plants against pathogens, the role and precise functional relationship between these polypeptides is still unclear.Recently, we described a new family of thionins, named 1'-thionins of about 5 kDa, found in the endosperm of wheat [13] and barley 1121. The three-dimensional structure of two members of the y-thionin family, y,-hordothionin (y,-H) and y,-puro- In this paper, we report the complete amino acid sequence of a new basic and cysteine-rich polypeptide isolated from barley endosperm, which we h...

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

“…Whether RsAFP2-induced fungal growth arrest is a result of the induced membrane permeabilization or results from its interaction with a putative intracellular target is currently not known. Several plant defensins isolated from barley have been shown to inhibit protein synthesis in eukaryotic as well as prokaryotic cell-free systems (39,40), supporting the theory of intracellular targets for plant defensins. Studies are currently initiated to address the subcellular localization of fluorescently labeled RsAFP2 in susceptible yeast cells and as such to provide crucial information regarding such putative intracellular RsAFP2 targets.…”

1H NMR structure of an antifungal γ-thionin protein SIα1: Similarity to scorpion toxins

“…Sequential (i, i ϩ 1) NOESY cross-peaks of types d␣ N , d NN , and d ␤N , correlating adjacent amino acids, were used to assign systems to specific residues in the SI␣1 sequence. Figure 2a illustrates the sequential d␣ N observed for residues 1-12, while Figure 2b shows the sequential d NN to d NN connectivities for residues [16][17][18][19][20][21][22][23][24][25]. In experiments performed in D 2 O, resonances for several slowly exchanging amide NH protons in the helix and strands were clearly observed, as indicated in Figure 3.…”

“…9a,c,d, respectively) 60,61 shows that these two families of proteins do indeed adopt the same secondary structure and overall topology, although there are two major sequence deletions in the thionins. The long C-terminal tail of the scorpion Variant 3 protein has been deleted and the long loop 18,24 RsAFP1-4 from Raphanus sativus. L., 19,52 Ah-AMP1 from Aesculus hippocatanum, Dm-AMP1 from Dahlia merkii, Ct-AMP1 from Clitorea termatea, Hs-AMP1 from Hucheria sanguinea, Cb-AMP1 from Cnicus benedictus, LC-AMP from Lathyrus ochrus, 28 ␥1/2-Zeathionins from Zea mays L., 51 PSAS from Vigna unguiculata, 53 pI230 from Pisum sativum, 54 p322 from polypeptide fragment (29-74) from potato tuber mRNA Solanum tubersum, 55 FST from Nicotina tabacum, 56 1PTX, scorpion ␣-toxin II (AaH_II) from Andoctonus austalis hector, 58 2CRD (Charybdotoxin) scorpion toxin from Leiurus quinquestriatus hebraeus, 61 2SN3 (Variant-3) scorpion toxin from Centruroides sculpturatus ewing.…”

“…A protein sequence search found similarity between the thionin family, [16][17][18][19][20][21]24,51 several plant defensins, 28,52 several polypeptides from Vigna unguiculata (pSAS) (sequence identity; 28%), 53 Pisum sativum (pI230) 54 (28%), Solanum uberum (p322) (32%), 55 Nicotina tabacum (FST) (33%) 56 and a group of scorpion venom proteins, 57-68 whose sequence identities were low (less than 30%), and depended primarily on the alignment of the eight cysteine residues, (Fig. 8).…”

Plant defensins

“…25 Certain plant defensins also display other biological activities, including proteinase, 30 α-amylase inhibitory activity, and inhibition of protein translation that may contribute to their role in defense. 5,6,20,31 …”