TY - JOUR
T1 - Plant-expressed recombinant mountain cedar allergen jun a 1 is allergenic and has limited pectate lyase activity
AU - Liu, Zun
AU - Bhattacharyya, Shikha
AU - Ning, Bo
AU - Midoro-Horiuti, Terumi
AU - Czerwinski, Edmund W.
AU - Goldblum, Randall M.
AU - Mort, Andrew
AU - Kearney, Christopher M.
PY - 2010/11
Y1 - 2010/11
N2 - Background: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. Method: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient's sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. Results: Recombinant Jun a 1 was recovered in good quantity (100 μg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. Conclusions: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.
AB - Background: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. Method: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient's sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. Results: Recombinant Jun a 1 was recovered in good quantity (100 μg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. Conclusions: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.
KW - Agrobacterium
KW - Apoplast
KW - Immunoglobulin E
KW - Jun a 1
KW - Juniperus ashei
KW - Necrosis
KW - Nicotiana benthamiana
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U2 - 10.1159/000316345
DO - 10.1159/000316345
M3 - Article
C2 - 20559000
AN - SCOPUS:77953594353
SN - 1018-2438
VL - 153
SP - 347
EP - 358
JO - International Archives of Allergy and Immunology
JF - International Archives of Allergy and Immunology
IS - 4
ER -