Xueni Chen

LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice

Respiratory distress syndrome (RDS), which is the leading cause of death in premature infants, is caused by surfactant deficiency. The most critical and abundant phospholipid in pulmonary surfactant is saturated phosphatidylcholine (SatPC), which is synthesized in alveolar type II cells de novo or by the deacylation-reacylation of existing phosphatidylcholine species. We recently cloned and partially characterized a mouse enzyme with characteristics of a lung lysophosphatidylcholine acyltransferase (LPCAT1) that we predicted would be involved in surfactant synthesis. Here, we describe our studies investigating whether LPCAT1 is required for pulmonary surfactant homeostasis. To address this issue, we generated mice bearing a hypomorphic allele of Lpcat1 (referred to herein as Lpcat1GT/GT mice) using a genetrap strategy. Newborn Lpcat1GT/GT mice showed varying perinatal mortality from respiratory failure, with affected animals demonstrating hallmarks of respiratory distress such as atelectasis and hyaline membranes. Lpcat1 mRNA levels were reduced in newborn Lpcat1GT/GT mice and directly correlated with SatPC content, LPCAT1 activity, and survival. Surfactant isolated from dead Lpcat1GT/GT mice failed to reduce minimum surface tension to wild-type levels. Collectively, these data demonstrate that full LPCAT1 activity is required to achieve the levels of SatPC essential for the transition to air breathing.

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants

Rationale An important property of allergens is their ability to cross‐link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized.

Direct inhibition of Bruton's tyrosine kinase by IBtk, a Btk-binding protein

Brutons tyrosine kinase (Btk) is required for human and mouse B cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency in mice. Unlike Src proteins, Btk lacks a negative regulatory domain at the COOH terminus and may rely on cytoplasmic Btk-binding proteins to regulates its kinase activity by trans-inhibitor mechanisms. Consistent with this possibility, IBtk, which we identified as an inhibitor of Btk, bound to the PH domain of Btk. IBtk downregulated Btk kinase activity, Btk-mediated calcium mobilization and nuclear factor-κB–driven transcription. These results define a potential mechanism for the regulation of Btk function in B cells.

The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.

Ara h 2 and Ara h 6, co‐purified together in a 13–25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX‐38 cells sensitized with IgE from human peanut allergic sera.

Ara h 2 and Ara h 6 have similar allergenic activity and are substantially redundant.

Background: The moderately homologous (approx. 60%) proteins Ara h 2 and Ara h 6 are the most potent peanut allergens. This study was designed to define the relative individual contributions of Ara h 2 and Ara h 6 to the overall allergenic activity of a crude peanut extract (CPE). Methods: Ara h 2 and Ara h 6 were removed from CPE by gel filtration chromatography. Ara h 2.01, Ara h 2.02 and Ara h 6 were further purified (>99%). The potency of each allergen and the ability of these allergens to reconstitute the allergenic activity of CPE depleted of Ara h 2 and Ara h 6 was measured with RBL SX-38 cells sensitized with IgE from sensitized peanut allergic patients. Results: The potency of the native proteins were significantly different (p < 0.0001) although not dramatically so, with a rank order of Ara h 2.01 > Ara h 2.02 > Ara h 6. The addition of either purified Ara h 2 or Ara h 6 independently at their original concentration to CPE depleted of both Ara h 2 and Ara h 6 restored 80–100% of the original CPE allergenic activity. Addition of both Ara h 2 and Ara h 6 consistently completely restored the allergenic activity of CPE. Conclusions: These studies indicate that either Ara h 2 or Ara h 6 independently can account for most of the allergenic activity in a CPE and demonstrate important redundancy in the allergenic activity of these related molecules.

Analysis of the Effector Activity of Ara h 2 and Ara h 6 by Selective Depletion from a Crude Peanut Extract

It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively removed from a CPE, removal of each alone from a CPE had no significant effect on the effector activity. However, removal of Ara h 2 and Ara h 6 together significantly reduced the effector activity of CPE.

Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6.

Cross‐linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE‐binding epitopes of peanut allergens have been defined, little is known about conformational IgE‐binding epitopes.

Application of phage peptide display technology for the study of food allergen epitopes

Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy.

Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations.

SCOPE Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions. METHODS AND RESULTS Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity. CONCLUSION Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.