Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent allergy in Japan. Recently, the Japanese cedar pollen allergen Cry j 3 was cloned as a homologue of Jun a 3, which is a major allergen from mountain cedar (Juniperus ashei) pollen. However, native Cry j 3 has not been isolated and there are no reports on its allergenic activity. The aims of this study were to isolate native Cry j 3 and assess its immunoglobulin E (IgE)-binding capacity in patients with Japanese cedar pollinosis. METHODS: Native Cry j 3 was purified from Japanese cedar pollen by multidimensional chromatography. We assessed the IgE-binding capacity using sera from patients allergic to Japanese cedar pollen by immunoblot analysis and ELISA. Moreover, we assayed the capacity of Cry j 3 to induce histamine release from the patients' leukocytes. We cloned cDNA corresponding to purified Cry j 3 from a cDNA library of Japanese cedar pollen. RESULTS: We isolated native Cry j 3 as a 27-kDa protein. The IgE-binding frequency of Cry j 3 from the sera of patients allergic to Japanese cedar pollen was estimated as 27% (27/100) by ELISA. Cry j 3 induced the release of histamine from leukocytes. We cloned the cDNA and named it Cry j 3.8. Cry j 3.8 cDNA encoded 225 amino acids and had significant homology with thaumatin-like proteins. CONCLUSIONS: Cry j 3 is a causative allergen in Japanese cedar pollinosis and may play crucial roles in the cross-reactivity with oral allergy syndrome.

Identification of mutations in the genes for the pollen allergens of eastern red cedar (Juniperus virginiana).

BACKGROUND: Cedar pollens are important causes of seasonal allergic disease in diverse geographical areas. However, pollens from different families and species vary in their propensity to induce allergic responses. OBJECTIVE: To compare the structure of potential allergens from eastern red cedar (Juniperus virginiana) pollen with those of the highly allergenic cedar (mountain cedar, J. ashei) pollens. MATERIALS AND METHODS: The cDNAs for potential pollen allergens, Jun v 1 and Jun v 3, were amplified by reverse transcriptase-polymerase chain reaction, cloned and sequenced. Expression of the native proteins in pollen was characterized by SDS-PAGE and immunoblotting. RESULTS: The cDNA sequence for one potential major allergen, Jun v 1, was highly homologous to those of the other cedar pollens. The second potential allergen, Jun v 3, was also highly homologous to its counterpart in mountain cedar, but a stop codon in the mRNA would result in a protein of only 91 amino acids, which would lack potential N-glycosylation sites and the IgE binding epitopes of the 199 amino acid homologue from mountain cedar pollen, Jun a 3. IgE from the sera of patients with hypersensitivity to cedar pollen bound to eastern red cedar proteins of four different sizes. N-terminal amino acid sequence analysis indicated that two of these proteins (43 and 30 kDa) were either isoforms or processed Jun v 1. No Jun v 3 protein was detected. The N-terminal sequence of an additional 145-kDa allergen, termed Jun v 4, was not homologous to any previously described allergens. CONCLUSION: These findings suggest that mutations in the genes or post-translational modifications of two potentially allergenic proteins might help to explain why the pollen of eastern red cedar is reported to be less allergenic than those of other members of Cupressaceae and Taxodiaceae families.

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen.

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.

Isolation and characterization of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND: Cedar pollens are important causes of seasonal allergic disease in diverse geographic areas. OBJECTIVE: A major allergen from mountain cedar (Juniperus ashei) pollen, termed Jun a 1, was isolated and characterized. METHODS: Water-soluble pollen glycoproteins were extracted, salt precipitated, and purified with use of concanavalin A affinity chromatography or HPLC. The purified fractions were characterized by SDS-PAGE, immunoblotting, and N-terminal amino acid sequence analysis. Binding of allergen-specific IgE from the sera of cedar-hypersensitive patients was detected by ELISA and antigen-specific responses of peripheral blood T cells by tritiated thymidine incorporation. RESULTS: The major extractable cedar pollen glycoprotein had a molecular weight and N-terminal amino acid sequence that was similar to that of the major allergen Cha o 1, from Japanese cypress (Chamaecyparis obtusa), and Cry j 1, from Japanese cedar (Cryptomeria japonica). IgE from cedar-hypersensitive patients' sera bound to the isolated glycoprotein. CONCLUSION: The predominance of Jun a 1 in the soluble proteins of mountain cedar pollen and its high degree of homology with Cha o 1 and Cry j 1 make it likely to be the major allergen of this pollen. Amino acid sequence conservation also makes Jun a 1 a potential target for cross-reactivity between these pollen allergens. The observed reactivity of IgE from the sera of Japanese cedar-sensitive patients with Jun a 1 is consistent with this proposition.

Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND: Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1. OBJECTIVE: A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed. METHODS: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera. RESULTS: Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis. CONCLUSION: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

Rapid high-temperature treatment of human milk.

Increasing interest in feeding human milk to low-birth-weight infants raises concern about microbial contamination of milk that is pooled or stored. We examined the effect of rapid high-temperature treatment on bacteria and viruses and on the nutritional and immunologic quality of pooled human milk. Growth of endogenous bacteria and infectivity of added cytomegalovirus were undetectable after heating at 72 degrees C for 15 and 5 seconds, respectively. Folic acid and vitamins B1, B2, B6, and C were not affected, whereas bile salt-stimulated lipase was inactivated by these conditions. The concentration of lactoferrin and secretory IgA, and SIgA antibody activity were not changed by heating at 72 degrees C. Lysozyme concentration and enzymatic activity were increased significantly by heat treatment, suggesting that this component may be largely sequestered in milk. Our findings suggest that rapid high-temperature treatment can reduce microbial contamination without destroying the unique nutritional and immunologic qualities of human milk.

Secretion of immunoglobulin A by human milk leukocytes initiated by surface membrane stimuli.

Some of the requirements for release of immunoglobulin A (IgA) from human milk leukocytes during phagocytosis were investigated. The role of particle adherence in IgA release was studied by using an in vitro model of frustrated phagocytosis in which human milk leukocytes were incubated with latex particles too large to ingest. Release of IgA was significantly increased from control values within 30 min in these leukocytes. A similar increment in IgA release also occurred when human milk leukocytes were incubated with other surface membrane stimuli, e.g., NFMP and phorbol. No increase in IgA release was found, however, in cells incubated with zymosan-activated serum. In addition, the release of IgA was blocked by inhibitors of actin filaments (cytochalasin B) and microtubules (colchicine). Thus, IgA is released from human milk leukocytes by secretory mechanisms that are initiated by certain membrane stimuli, some of which are shared by peripheral blood neutrophils and monocytes. Because human milk leukocytes appear to be refractory to C5a or other activated complement components and are blocked by cytochalasin B, it appears that these unusual cells may be uniquely adapted to play a role in the immunologic protection of the neonate.

Relationship between phagocytosis and immunoglobulin A release from human colostral macrophages.

Macrophages and neutrophils that contain mainly secretory immunoglobulin A (IgA) comprise the majority of cells in human colostrum. These cell populations were separated and analyzed for their ability to release total IgA and secretory IgA when stimulated to phagocytose. Colostral macrophages phagocytosed opsonized bacteria and nonopsonized latex particles; at the same time, IgA was released. Neutrophils poorly phagocytosed opsonized bacteria but actively phagocytosed latex particles. In contrast to the macrophages, the neutrophils did not release IgA, even after active phagocytosis of latex. Consequently, colostral macrophages are the main source of IgA released from colostral leukocytes when these cells are exposed to organisms or particles that are phagocytosed. A function for colostral neutrophils which sequester IgA is proposed.

Immunoglobulin A and secretory immunoglobulin A antibodies to purified type 1 Klebsiella pneumoniae pili in human colostrum.

Immunoglobulin A antibodies with binding specificity for purified Klebsiella pneumoniae type 1 pili were detected by an enzyme-linked immunosorbent assay in 20 of 21 human samples (95%). The concentrations of secretory immunoglobulin A antibody in colostrum directed against the pili were calculated by comparison of experimental enzyme-linked immunosorbent assay values with values obtained from known secretory immunoglobulin A concentrations. The presence of antibodies to K. pneumoniae type 1 pili was confirmed by double diffusion-gel studies with selected specimens of colostrum. This study shows that in the majority of human colostral samples examined, secretory immunoglobulin A antibodies with specificity for K. pneumoniae type 1 pili can be commonly found in variable, but frequently high, concentrations.

Immunologic factors in human milk during the first year of lactation.

The effects of the duration of lactation upon lactoferrin, lysozyme, total IgA, SIgA, SIgA antibodies to Escherichia coli somatic antigens and leukocytes in human milk were investigated. Longitudinal and cross-sectional studies were performed with milk collected from women 20 to 35 years of age during te first year of lactation. Collection and storage conditions and immunologic analyses were controlled to minimize confounding variables. The concentrations of lactoferrin, total IgA, and leukocytes and the uptake of 3H-thymidine by phytohemagglutinin-stimulated lymphocytes fell during the first several weeks of lactation; afterward, the levels of lactoferrin and IgA stabilized. Approximately 90% of total IgA in human milk during the year was SIgA. Secretory IgA antibody titers to E. coli increased in some individuals studied longitudinally suggesting that the enteromammary gland pathway of SIgA antibody production was active after several weeks of lactation. Moreover, the concentrations of lysozyme, after falling to a nadir of 20 to 30 micrograms/ml at 2 to 4 weeks, rose to 200 to 300 micrograms/ml by six months and remained elevated. The immunologic system in human milk undergoes remarkable changes which may represent adaptations for the recipient infant.

Enhanced immunoglobulin, A release from human colostral cells during phagocytosis.

Human colostral leukocytes were investigated for their ability to release immunoglobulin A during phagocytosis of latex particles, heat-killed Candida albicans, or live Escherichia coli. Leukocytes readily phagocytosed latex or serum-opsonized candida or the E. coli. Colostral fluid was also opsonic for yeast and bacteria. Immunoglobulin release, which consisted mainly of secretory immunoglobulin A, began during the first 15 min of incubation with latex, opsonized yeast, or opsonized bacteria. Release was significantly increased from control levels by 30 or 60 min. The release of immunoglobulin A could be inhibited by incubating leukocytes at 4 degrees C. We conclude that phagocytosis and immunoglobulin A release by human colostral leukocytes are related. The data support the hypothesis that colostral leukocytes may play an active role in protecting infants from pathogenic microorganisms.

Antibody production by human colostral cells. I. Immunoglobulin class, specificity, and quantity.

The production of antibody by human colostral cells was assayed by the hemolysis in-gel technique. When sheep erythrocytes coated with O antigens from frequently encountered Escherichia coli bacteria were used as detector cells and anti-IgA serum was added for development, numerous plaque-forming cells (PFC) were demonstrated in all samples tested. In contrast, plaques were rarely seen in the presence of anti-IgG developing serum. The direct (IgM) plaques occasionally noted with both antigen-coated and uncoated sheep erythrocytes were mainly due to the production of heterophil antibodies, since they were not formed when human erythrocytes were used as O-antigen carriers. A strikingly high number of the colostral lymphocytes formed antibodies to the E. coli antigens, up to 8%. This suggests that these cells represent a rather selective population--possibly cells from the gastrointestinal tract exposed to enteric bacteria. The large number of plaques observed, the predominance of the cells forming IgA antibodies, and the marked changes in PFC number in relationship to parturition pose a number of questions relevant to the antibody-producing colostrum cells and their relationship to the secretory immune system.