Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.)

Fructans, β2-1 and/or β2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively β2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed—as did the native enzyme from wheat—a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PC

CreateTarget and Analyze This! new software assisting imaging mass spectrometry on Bruker Reflex IV and Ultraflex II instruments

Two new software programs are presented which enable matrix-assisted laser desorption/ionisation imaging mass spectrometry (MALDI-IMS) on Bruker Reflex and Ultraflex instruments. The first program, CreateTarget, creates a high density raster with the dimensions of the tissue section, and converts these parameters into a virtual target plate file that can be imported in the normal mass spectrometer control software. Following automated spectrum acquisition, the second program, Analyze This!, converts the array of spectra into an Analyze 7.5 image format that can be read by image analysis software. These two tools are sufficient to allow IMS, and offer a valid alternative to commercially available software. CreateTarget and Analyze This! are available free of charge. Copyright (c) 2006 John Wiley & Sons, Ltd.status: publishe

Direct profiling and identification of peptide expression differences in the pancreas of control and ob/ob mice by imaging mass spectrometry

Imaging mass spectrometry (IMS) technology utilizes MALDI MS to map molecules of interest in thin tissue sections. In this study, we have evaluated the potential of MALDI IMS to study peptide expression patterns in the mouse pancreas under normal and pathological conditions, and to in situ identify peptides of interest using MS/MS. Different regions of the pancreas of both control and ob/ob mice were imaged, resulting in peptide-specific profiles. The distribution of ions of m/z 3120 and 3439 displayed a striking resemblance with Langerhans islet's histology and, following MS/MS fragmentation and database searching were identified as C-peptide of insulin and glicentin-related polypeptide, respectively. In addition, a significant increase of the 3120 peak intensity in the obese mice was observed. This study underscores the potential of MALDI IMS to study the contribution of peptides to pancreas pathology.status: publishe

In vitro and in silico modelling of protein hydrolysis by pepsin: A case study with lactoferrin

International audienc

In silico modeling of protein hydrolysis by endoproteases: a case study on pepsin digestion of bovine lactoferrin

International audienceThis paper presents a novel model of protein hydrolysis and release of peptides by endoproteases. It requires the amino-acid sequence of the protein substrate to run, and makes use of simple Monte-Carlo in silico simulations to qualitatively and quantitatively predict the peptides that are likely to be produced during the course of the proteolytic reaction. In the present study, the model is applied to the case of pepsin, the gastric protease. Unlike pancreatic proteases, pepsin has a low substrate specificity and therefore displays a stochastic behavior that is particularly challenging to model and predict. Two versions of the model are studied and compared with peptidomic data obtained during pepsin hydrolysis of bovine lactoferrin. The first version of the model takes into account cleavage probabilities according to the amino acids in position P1-P1' only, whereas the second version also accounts for the influence of neighbor amino acids (P4, P3, P2, P2', P3', P4') and peptide terminal ends. The second version of the model was able to reproduce many real-world features of the reported behavior of pepsin, such as the peptide size distribution, or the quantity of free amino-acids. More remarkably, 50% of the experimentally monitored peptides (44/87) lay within the 120 most abundant simulated peptides. The presented methodology has the advantage of being applicable not only to different proteins, but to different enzymes as well, as long as cleavage frequency data are available

Light-induced Fos expression in phosphate-activated glutaminase- and neurofilament protein-immunoreactive neurons in cat primary visual cortex

Previous double-stainings in the cat visual cortex [E. Van der Gucht, S. Clerens, K. Cromphout, F. Vandesande, L. Arckens, Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex, Eur. J. Neurosci. 16 (2002) 1620-1626] showed that a minority of Fos-immunoreactive nuclei was located in distinct subclasses of inhibitory neurons following sensory stimulation. This report describes double-stainings between Fos and phosphate-activated glutaminase (PAG) or Fos and neurofilament protein (SM1-32) revealing that, following a short-term visual experience, Fos is also expressed in neurochemically distinct subpopulations of non-GABAergic, pyramidal neurons in supra- and infragranular layers of cat area 17. (c) 2004 Elsevier B.V. All rights reserved.status: publishe