Paul Whitley

Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor

To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed. Mutants with one or more cysteine residues altered were constructed with a histidine residue tag for purification purposes and expressed as recombinant proteins in E. coli. Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72 77) and one mutant with all 6 cysteines altered (6 Cys). The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding. Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2. However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered. A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72 77 or the 6 Cys mutant. Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 microg/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved. The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy.

Sec-independent translocation of a 100-residue periplasmic N-terminal tail in the E. coli inner membrane protein proW.

The ProW protein, located in the inner membrane of Escherichia coli, has a very unusual topology with a 100‐residue‐long N‐terminal tail protruding into the periplasmic space. We have studied the mechanism of membrane translocation of the periplasmic tail by analysing ProW‐PhoA and ProW‐Lep fusion proteins, both in wild‐type cells and in cells with an impaired sec machinery. Our results show that the translocation efficiency is not affected by treatments that compromise the SecA and SecY functions, but that translocation is completely blocked by dissipation of the proton motive force or by the introduction of extra positively charged residues into the N‐terminal tail. This suggests that the sec machinery can act properly only on domains located on the C‐terminal side of a translocation signal, and that the N‐terminal tail is driven through the membrane by a mechanism that involves the proton motive force.

Membrane protein assembly

Publisher Summary Cells are made up of multiple membrane-bound compartments, each containing a unique set of proteins. The sorting and insertion of proteins across and into the many membrane systems of eukaryotes and prokaryotes has been the subject of a vast amount of research. This chapter discusses the assembly of proteins into the cytoplasmic (inner) membrane of prokaryotes, notably Escherichia coll. It is believed that many of the basic aspects of membrane protein assembly in E. coli are relevant to the assembly of proteins into other membrane systems such as the endoplasmic reticular (ER) membrane of eukaryotes, the thylakoid membrane of chloroplasts, and the inner mitochondrial membrane. Knowledge from E. coil can often illuminate the analogous processes in these other systems. The most important characteristics of the substrates for the secretory machinery—the nascent membrane proteins— are clarified with the discovery that positively charged amino acids act as the major topogenic determinants during membrane integration. As a measure of the understanding, one can now predict the topology of an inner membrane protein from its amino acid sequence and expect to be right 9 times out of 10.

Identification and Characterisation of Two Allergens from the Dust Mite Acarus siro, Homologous with Fatty Acid–Binding Proteins

Background: Dust mites are a major cause of allergic disease worldwide. The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non–farming populations. Methods: A degenerate primer was designed to the N–terminal amino acid sequence of a 15–kD IgE–binding protein in A. siro extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in Escherichia coli with a 6–histidine tag at its C–terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera. Results and conclusion: 15 and 17–kD allergens were identified in a fraction of A. siro extract. The cDNA of the 15–kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA–encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N–glycosylation site at position 4–6 and a cytosolic fatty acid–binding protein signature at position 5–22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid–binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17–kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.

Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the pharmacia CAP system

Background: Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE–mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts Lepidoglyphus destructor and Tyrophagus putrescentiae in the Pharmacia RAST CAP System. Methods: The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. Results: The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial L. destructor and T. putrescentiae assays. Two subjects out of 416, who tested negative in the commercial L. destructor assay, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the T. putrescentiae extract were 5/418. The possibility that these subjects were sensitised to L. destructor and T. putrescentiae could not be excluded. Conclusions: The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of L. destructor and T. putrescentiae. It appears likely that the addition of just a few more recombinant L. destructor and T. putrescentiae allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to L. destructor and T. putrescentiae.

A quantitative assay to determine the amount of Signal Peptidase I in E. coli and the orientation of membrane vesicles

The number of Signal Peptidase I (SPasel) molecules per E. coli cell was determined using western blot techniques. Different strains were found to contain approximately 1000 SPasel molecules per cell during exponential growth. Based on the activity of SPasel in vitro it could be estimated that this amount is sufficient to process all translocated precursors. SPasel did not appear to be under growth phase dependent control, but was constitutively expressed. The quantitative western blot technique was also used to establish the orientation and intactness of isolated inner membrane vesicles.

The DsbA-DsbB system affects the formation of disulfide bonds in periplasmic but not in intramembraneous protein domains

The DsbA and DsbB proteins of Escherichia coli are involved in facilitating the formation of disulfide bonds in periplasmic proteins. Here, we show that the rate of formation of a disulfide bond in the periplasmic domain of the inner membrane protein leader peptidase is reduced in dsbA and dsbB strains, whereas the rate of formation of a disulfide bond engineered into the membrane embedded domain of the same protein is completely unaffected by these mutations. We conclude that the Dsb proteins do not facilitate the formation of intramembraneous disulfides.

Glycosylation Mapping Of The Interaction Between Topogenic Sequences And The Er Translocase

Many integral membrane proteins span the hydrophobic core of a membrane with one or more a-helical segments each consisting of about 20 hydrophobic amino acids. The orientation of the hydrophobic stretch in the membrane is determined by its flanking amino acids. In general there are more positively charged residues present in cytoplasmic loops than in extra-cytoplasmic ones (von Heijne, 1986). In both prokaryotic and eukaryotic cells, proteins are targeted for secretion by N-terminal signal sequences with a common basic design: a positively charged N-terminus, a central hydrophobic stretch, and a C-terminal cleavage region that serves as a recognition site for the signal peptidase enzyme (von Heijne, 1985). Signal sequences from prokaryotes and eukaryotes look very similar and are often functionally interchangeable. They are essential for the efficient and selective targeting of the nascent protein chains either to the endoplasmic reticulum (in eukaryotes) or to the cytoplasmic membrane (in prokaryotes) (Gierasch, 1989). Signal sequences also play a central role in the interaction with the translocation machinery of the cell and in the translocation of the polypeptide chains across the membrane. Proteins are co-translationally translocated across the endoplasmic reticulum (ER) membrane. In eukaryotes two kinds of topogenic sequences are important for assembly in ER membrane: Those that initiate translocation such as signal peptides (SPs) and signalanchor sequences (SAs) and those that halt translocation, stop-transfer signals (STs). Signal peptides and signal anchor sequences differ in that SA sequences tend to have longer hydrophobic cores and lack a signal peptidase cleavage site (von Heijne, 1988).