TLXI, a novel type of xylanase inhibitor from wheat (Triticum aestivum) belonging to the thaumatin family

Abstract

Wheat (Triticum aestivum) contains a previously unknown type of xylanase (EC 3.2.1.8) inhibitor, which is described in the present paper for the first time. Based on its >60% similarity to TLPs (thaumatin-like proteins) and the fact that it contains the Prosite PS00316 thaumatin family signature, it is referred to as TLXI (thaumatin-like xylanase inhibitor). TLXI is a basic (pI> or =9.3 in isoelectric focusing) protein with a molecular mass of approx. 18-kDa (determined by SDS/PAGE) and it occurs in wheat with varying extents of glycosylation. The TLXI gene sequence encodes a 26-amino-acid signal sequence followed by a 151-amino-acid mature protein with a calculated molecular mass of 15.6-kDa and pI of 8.38. The mature TLXI protein was expressed successfully in Pichia pastoris, resulting in a 21-kDa (determined by SDS/PAGE) recombinant protein (rTLXI). Polyclonal antibodies raised against TLXI purified from wheat react with epitopes of rTLXI as well as with those of thaumatin, demonstrating high structural similarity between these three proteins. TLXI has a unique inhibition specificity. It is a non-competitive inhibitor of a number of glycoside hydrolase family 11 xylanases, but it is inactive towards glycoside hydrolase family 10 xylanases. Progress curves show that TLXI is a slow tight-binding inhibitor, with a K(i) of approx. 60-nM. Except for zeamatin, an alpha-amylase/trypsin inhibitor from maize (Zea mays), no other enzyme inhibitor is currently known among the TLPs. TLXI thus represents a novel type of inhibitor within this group of proteins.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

“…XIP-and TLXI-type XIs are basic, monomeric proteins with a molecular mass of ~30 and ~18 kDa, respectively [16,17]. Multiple putative TAXI-[18, 21, 22] and XIP-type [19,23] as well as one TLXI-type gene(s) [17] have been identified in wheat and some have been confirmed. For the three types of XIs, the existence of various forms as well as differences in their spatio-temporal location, due to distinct regulatory control mechanisms, have been suggested.…”

“…To allow detection of eventually truncated N-and C-termini, the custom database was subsequently N-and C-terminally 'ragged' using DBToolkit prepared by spotting 1 µl of a mixture of sample and matrix (7 mg/ml CHCA in 50% ACN containing 0.1% TFA) on a stainless steel (192-well) MALDI target plate and allowed to air-dry at RT. Prior to MALDI-MS analysis, the instrument was externally calibrated with a mixture of Angiotensin I, Glu-fibrino-peptide B, ACTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and ACTH (18-39). For MS/MS experiments, the instrument was externally calibrated with fragments of Glu-fibrino-peptide.…”

“…The levels of TAXI-and XIP-type inhibitors were measured using a specific GH family 11 B. subtilis xylanase and a GH family 10 P. purpurogenum xylanase, respectively. 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 Affinity-purified and desalted XIs were lyophilized in small glass vials to create a moisture-free atmosphere for deglycosylation with trifluoromethanesulfonic acid (TFMS) [17,45]. Briefly, dry sample aliquots (500 µg) were incubated (180 min) on ice with a pre-cooled 10.0% anisole in TFMS solution and neutralized by gradually adding droplets of a 60% pyridine solution, thereby keeping the samples at -15°C in a MeOH/dry ice bath.…”

“…They occur simultaneously as a ~40 kDa single polypeptide and as a processed form existing of two disulfide-linked polypeptides of ~30 and ~10 kDa [20]. XIP-and TLXI-type XIs are basic, monomeric proteins with a molecular mass of ~30 and ~18 kDa, respectively [16,17]. Multiple putative TAXI-[18, 21, 22] and XIP-type [19,23] as well as one TLXI-type gene(s) [17] have been identified in wheat and some have been confirmed.…”

Characterization of Thaumatin-like Gene Family and Identification of Pectobacterium carotovorum subsp. carotovorum Inducible Genes in Brassica oleracea

“…Purified TLPs from several plant species have in vitro anti-fungal activity. Several antifungal modes of action have been described such as membrane permeabilization (Vigers et al 1991), β-glucan binding and degradation (Sakamoto et al 2006), inhibition of enzymes such as xylanases (Fierens et al 2007), α-amylase, or trypsin (Schimoler-O'Rourke et al 2001, as well as an apoptosisinducing mechanism reported in yeast (Narasimhan et al 2005). A PR5 protein from Arabidopsis thaliana ATLP3, showed considerable antifungal activity against pathogenic fungi such as Verticillium alboatrum, V. dahliae and Fusarium oxysporum (Hu and Ready 1997).…”