Johnny Beaugrand

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

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.

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (Kd) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

His22 of TLXI plays a critical role in the inhibition of glycoside hydrolase family 11 xylanases

Recently, a novel wheat thaumatin-like protein, TLXI, which inhibits microbial glycoside hydrolase family (GH) 11 xylanases has been identified. It is the first xylanase inhibitor that exerts its inhibition in a non-competitive way. In the present study we gained insight into the interaction between TLXI and xylanases via combined molecular modeling and mutagenic approaches. More specifically, site-specific mutation of His22, situated on a loop which distinguishes TLXI from other, non-inhibiting, thaumatin-like proteins, and subsequent expression of the mutant in Pichia pastoris resulted in a protein lacking inhibition capacity. The mutant protein was unable to form a complex with GH11 xylanases. Based on these findings, the interaction of TLXI with GH11 xylanases is discussed.

Algerian pearl millet ( Pennisetum glaucum L.) contains XIP but not TAXI and TLXI type xylanase inhibitors

An XIP (xylanase inhibiting protein) type xylanase inhibitor was purified from Algerian pearl millet ( Pennisetum glaucum L.) grains and characterized for the first time. Cation exchange and affinity chromatography with immobilized Trichoderma longibrachiatum glycoside hydrolase (GH) family 11 xylanase resulted in electrophoretically pure protein with a molecular mass of 27-29 kDa and a pI value of 6.7. The experimentally determined N-terminal amino acid sequence of the purified XIP protein is 87.5%, identical to that of sorghum ( Sorghum bicolor L.) XIP and 79.2% identical to that of wheat ( Triticum aestivum L.) XIP-I. The biochemical properties of pearl millet XIP are comparable to those described earlier for sorghum XIP, except for the higher specific activity toward a T. longibrachiatum GH family 11 xylanase. On the basis of immunoblot neither TAXI nor TLXI type xylanase inhibitors were detected in pearl millet grains.

Quantification of Wheat TAXI and XIP Type Xylanase Inhibitors: A Comparison of Analytical Techniques

ABSTRACT To date, three different techniques are available for the quantification of TAXI and XIP type proteinaceous xylanase inhibitors in cereals. A first approach is based on the determination of the residual activities of xylanases (also referred to as endo-1,4-β-d-xylanases, EC 3.2.1.8), which are specifically inhibited by these inhibitors, after incubation with sample containing the inhibitors. The other two techniques are immunoblotting and ELISA which are based on recognition of TAXI and XIP proteins by specific antibodies. TAXI, as well as XIP, are easily extracted by aqueous buffers. Hence, the large difference in their concentrations (2–10 fold higher for XIP than for TAXI in whole meal) is not caused by differences in extractability. The repeatabilities of the three techniques are comparable. The intra-assay and inter-assay coefficients of variation were 6–7 and 10–14%, respectively, which is in the range of values described for methods to quantify other compounds in plant and animal tissues. ...