Major mountain cedar allergen, Jun a 1, contains conformational as well as linear IgE epitopes.

We have previously identified four linear IgE epitopes on Jun a 1, the dominant allergen in mountain cedar pollen, and mapped these to the surfaces of a molecular model and to the crystal structure of this glycoprotein. The aim of the present study was to determine if Jun a 1 also displays conformational IgE epitopes. Jun a 1 was denatured by heating at 75 degrees C for 1h, exposure to 6M guanidine or by reductive alkylation in the presence and absence of guanidine. The effects of these manipulations on the binding to IgE from patients with mountain cedar hypersensitivity was evaluated by an ImmunoCAP inhibition assay, using Jun a 1-specific caps. Treatment-associated changes in the 3D-structure were assessed by dynamic light scattering and CD spectroscopy. IgE binding to native Jun a 1 was inhibited 92+/-9% by soluble native protein and 92+/-9% by reduced and alkylated Jun a 1. However, the capacity of Jun a 1 to inhibit the binding of IgE antibodies was significantly diminished upon denaturation by heat, guanidine alone, or reduction and alkylation in guanidine, compared to native Jun a 1. Reductive alkylation treatment under denaturing conditions also increased the Stoke's radius, suggesting that the protein was partially unfolded. Analysis of the circular dichroism (CD) spectra suggested that heating and treatment with guanidine caused a loss of alpha-helical structure. Guanidine also caused an increase in random coil structure. Thus, at least a portion of the anti-Jun a 1 IgE antibodies produced by allergic humans recognize conformational epitopes and it is likely that some of these epitopes reside in alpha-helical structures of Jun a 1.

Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5.

PURPOSE: To investigate the effect of annexin A5, a calcium-dependent phospholipid-binding protein, on corneal epithelial wound healing. METHODS: The effect of annexin A5 on migration of rabbit corneal epithelial (RCE) cells in vitro was examined in scrape-wounded cell monolayers. The effect of annexin A5 on the release of urokinase-type plasminogen activator (uPA) from cultured RCE cells was determined by zymography, fluorogenic assay of PA activity, and enzyme-linked immunosorbent assay. The proliferation of RCE cells was assessed by measurement of [3H]thymidine incorporation. The effect of annexin A5 on corneal wound closure in rabbits was investigated after removal of the corneal epithelium, either by exposure to iodine vapor or surgically. Eye drops containing annexin A5 were instilled into one eye and vehicle into the other. The area of the epithelial defect was measured at various times after wounding, and the healing rate was calculated by linear regression analysis. RESULTS: Annexin A5 significantly promoted the migration of RCE cells in a wounded monolayer. However, annexin A5 had no effect on RCE cell proliferation. Annexin A5 also increased the release of uPA both from wounded RCE cell monolayers and from nonwounded semiconfluent RCE cells. In both models of corneal wound closure, the healing rate was significantly increased by instillation of eye drops containing annexin A5 compared with that apparent in the eyes that received vehicle. CONCLUSIONS: Annexin A5 promoted corneal epithelial wound healing both in vitro and in vivo. Upregulation of uPA release from corneal epithelial cells may contribute to this effect of annexin A5.

Structural basis for epitope sharing between group 1 allergens of cedar pollen.

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site.

Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens. Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens. In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes. A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements. This allowed mappings of four IgE binding sites on Jun a 1. Two of the epitopes mapped to turns or loops on the surface of the model structure. The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL. This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs. The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.

Up-regulation of urokinase-type plasminogen activator in corneal epithelial cells induced by wounding.

PURPOSE: To investigate the possible role of urokinase-type plasminogen activator (uPA) in corneal epithelial wound healing by examining its expression both in the rabbit corneal epithelium in situ and in rabbit corneal epithelial (RCE) cells in vitro. METHODS: The rabbit cornea was subjected to mechanical wounding, and frozen sections of the tissue were subsequently prepared and subjected both to immunostaining with antibodies to uPA and to in situ zymography for the detection of PA activity. RCE cell monolayers were also subjected to scrape wounding, after which they were immunostained for uPA. The amounts of uPA protein in the culture medium and of uPA mRNA in cell lysates were also determined by enzyme-linked immunosorbent assay and by reverse transcription and real-time quantitative polymerase chain reaction analysis, respectively. RESULTS: Immunostaining and in situ zymography of the wounded cornea revealed that uPA was restricted to the leading edge of the migrating corneal epithelium. In contrast, tissue-type PA was expressed throughout the corneal epithelium. Scraping of RCE cell monolayers induced the expression of uPA in the migrating cells at the wound edge. The amount of uPA in the culture medium of RCE cells increased with the number of scrape wounds applied. Wounding also induced a time-dependent increase in the abundance of uPA mRNA in the cell monolayers. The migration of RCE cells was inhibited by antibodies to uPA. CONCLUSIONS: Mechanical wounding induces up-regulation of uPA at both the protein and mRNA levels in corneal epithelial cells. uPA may thus contribute to epithelial cell migration during corneal epithelial wound healing.

Affinity improvement of the high-affinity immunoglobulin E receptor by phage display.

The immunoglobulin E (IgE)-binding site of its high-affinity receptor is localized in the second immunoglobulin-like domain (D2) of the alpha-subunit (Fc epsilon RI alpha). In this study, the randomized pentapeptides were introduced between Glu(132) and Ile(138) of Fc epsilon RI alpha D2 and displayed on a filamentous phage. After eight rounds of panning, a phage clone having a mutation of Asp(135)Tyr(136)Met(137) in Fc epsilon RI alpha D2 was obtained. The binding affinity of the mutant phages to immobilized IgE was approximately 500 times higher than that of the wild type. The mutant phages competitively inhibited the binding of IgE to the soluble receptor at a 50% inhibition (IC(50)) value of 116 pM. The mutant Fc epsilon RI alpha D2, which had been expressed as a fusion protein with glutathione S-transferase in Escherichia coli, also showed higher IgE-binding capacity than the wild type. The mutant Fc epsilon RI alpha D2 is expected to manifest its improved IgE-binding affinity together with any fusion partner.

Immuno-Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa-like protein is present in germ line cells of Xenopus, antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N- or C-termini or with one including the DEAD box of the Drosophila vasa protein. Only the antibody against the oligopeptide including the DEAD box reacted strongly with germ plasm (GP) or with cytoplasm of germ line cells of Xenopus embryos by immunofluorescence microscopy. By immunoelectron microscopy, the antibody was demonstrated to react with the GP-specific structure, germinal granules, in cleaving embryos, and with their derivatives in the germ line cells of embryos at stages extending from gastrula to feeding tadpole. It also reacted with mitochondria not only in the GP and the germ line cells but also in somatic cells, and with myofibrils in muscle cells. By Western blotting, the antibody was shown to react with several bands of Mr 42-69 ± 103 in protein samples from Xenopus embryos. In samples from Drosophila ovaries, it reacted with a Mr 71 ± 103 band which was probably the vasa protein. This indicates the possibility that Xenopus embryos contain several DEAD family proteins. One of these is present on germinal granules, resembling the vasa protein on polar granules of Drosophila.