Search

Background: The 2S albumins are a group of storage proteins that occur widely in seeds of dicotyledonous plants. The widespread distribution and stability to digestion of allergenic 2S albumins raise the question of why some members of this family present in important food sources, such as soybean, are not regarded as major allergens. Methods: The pepsinolytic stability of two 2S albumins from soybean seed was determined using simulated gastric fluid. Using a new protein microarray system, IgE binding to these soybean 2S albumins was studied with the sera from 23 European individuals allergic to soybean. In order to validate the microarray result, two of the sera were selected and further tested using the micro-ELISA and UniCAP system. Results: Both albumins exhibited high stability to digestion similar to other allergenic members of the 2S albumin, trypsin/amylase inhibitor and lipid transfer protein superfamily. None of the patients was found to have IgE specific to soybean 2S albumins by the microarray system, and this result was in agreement with the results from the micro-ELISA and UniCAP system.Conclusions: The results from microarray, micro-ELISA and UniCAP system suggested that the 2S albumins from soybean are not major allergens within the patient population analyzed. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have cloned and expressed genes encoding the allergenic brazil nut 2 S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2 S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy. [Copyright &y& Elsevier]

We have shown that the unfolded protein response (UPR) in Pichia pastoris requires splicing of a non-conventional intron in the HAC1(u) mRNA in common with other eukaryotes. P. pastoris is a favoured yeast expression host for secreted production of heterologous proteins and the regulation of the UPR in P. pastoris may hold the key to its effective folding and secretion of proteins. We have also shown that the C-terminal region of the Hac1p from P. pastoris is required for functionality. Although the C-terminal regions of Hac1p from both S. cerevisiae and P. pastoris are rich in phenylalanine residues, the P. pastoris Hac1p lacks a C-terminal serine that is known to be important in the efficient functionality of Hac1p from S. cerevisiae., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

A protein microarray system containing different dilutions of 77 related and non-related proteins was used to show that IgE from subjects allergic to Brazil nut specifically recognise the seed 2S albumin protein (Ber e 1). Further, correctly folded chimaeric 2S albumin proteins containing structural epitope replacement were constructed and directed to the secretion pathway of the methylotropic yeast Pichia pastoris. Through the use of a chimaeric protein microarray system together with sera from a panel of 18 well-characterised Brazil nut allergic subjects, a structural IgE epitope of Ber e 1 was mapped to a helix-loop-helix region. The same structural region has been previously reported as the immunodominant region in related food allergens by different techniques. In conclusion, the combination of chimaeric proteins and protein microarrays will greatly facilitate the screening of a large number of individuals for a particular structural epitope and help to further our understanding of how proteins are recognised by the adaptive immune system.

Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity. We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds. Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites. This internal processing provides useful information on the endoproteolytic activity in P. pastoris. We also show that, similar to many plant allergens, the 2S albumins from soybean are stable to heat and chemical treatments.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.