Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome

Abstract

Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS. HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo. During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo. On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR. The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g. rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2). Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified using a three-step procedure. The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley a-amylase/subtilisin inhibitor as substrates. HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR). The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a K m of 13 lM compared with 44 lM for HvTrxh2. Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles.

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Barley contains two thioredoxin isoforms as well as two isoforms of an NADPH-dependent thioredoxin reductase that reduce the disulfide formed in the thioredoxin active site motif CXXC after it has reduced a target protein disulfide bond (Maeda et al 2003;Shahpiri et al 2008). We have developed a proteomics-based procedure that allows identification of target disulfides in protein mixtures (Maeda et al 2005; P. Hägglund et al, manuscript in preparation).…”

Molecular characterization of an h-type thioredoxin gene from grape

“…Concerning to proposed classification based on the residue at position 101 [47] These 77 and 88 residues are implicated in the aromatic tetrahedral cluster important for thermostability of Trxs h [48], while 101 residues are involved in the R 101 KDD 104 motif critical for cell to cell movement [42].…”

“…It seems that the N-terminal M 1 AEE 4 motif acts like an arm appended to the main body to VvTrx h2 and may be able to mediate protein cell to cell transport through plasmodesmata. Also the charged Glu residues (Glu3 and Glu4) play a crucial role in this cell to cell transport, since replacement of Glu residues with Ala appeared to potentiate a limited interaction between protein and plasmodesmata [47].…”

“…Nevertheless, it is predicted that this mutant form of Trx h is also capable to movement from cell to cell [47]. It would appear that two clusters of charged residues, E , predicted to project from the surface of protein, function in the same way to mediate in the binding and/or transport of this protein through plasmodesmata [47]. Similarity, mutation of Gly5 had also relatively little effect on the 3D structure ( Figure 6E); the VvTrx h2G5A appeared still to be capable of cell to cell transport.…”

Interactions between Barley .ALPHA.-Amylases, Substrates, Inhibitors and Regulatory Proteins

“…In the germinating seeds the disulphide reductase thioredoxin h 33) is capable of reducing disulphide bonds in a series of target proteins, including BASI as well as different α amylase inhibitors of the CM protein family shown to act on mammalian and insect α amylases. 27) In the case of BASI, it has been demonstrated that one of its two disulphide bonds, actually situated in the three dimensional structure in the vicinity of residues interacting with AMY2 in the complex is particularly prone to reduction by thioredoxin h. The second disulphide bond in BASI is near the protease binding site and seems to be only slightly affected.…”