Document Detail


Plant-expressed recombinant mountain cedar allergen Jun a 1 is allergenic and has limited pectate lyase activity.
MedLine Citation:
PMID:  20559000     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Zun Liu; Shikha Bhattacharyya; Bo Ning; Terumi Midoro-Horiuti; Edmund W Czerwinski; Randall M Goldblum; Andrew Mort; Christopher M Kearney
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-06-17
Journal Detail:
Title:  International archives of allergy and immunology     Volume:  153     ISSN:  1423-0097     ISO Abbreviation:  Int. Arch. Allergy Immunol.     Publication Date:  2010  
Date Detail:
Created Date:  2010-11-16     Completed Date:  2011-03-08     Revised Date:  2013-01-07    
Medline Journal Info:
Nlm Unique ID:  9211652     Medline TA:  Int Arch Allergy Immunol     Country:  Switzerland    
Other Details:
Languages:  eng     Pagination:  347-58     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 S. Karger AG, Basel.
Affiliation:
Department of Biology, Baylor University, Waco, TX 76798, USA.
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MeSH Terms
Descriptor/Qualifier:
Agrobacterium tumefaciens / genetics*
Allergens / genetics,  immunology,  isolation & purification,  metabolism*
Animals
Antigens, Plant
Basophils / immunology,  metabolism*,  pathology
Cell Degranulation
Cell Line
Genetic Vectors / genetics
Humans
Immunoglobulin E / metabolism
Juniperus / immunology
Mutagenesis, Site-Directed
Mutation / genetics
Plant Proteins / genetics,  immunology,  isolation & purification,  metabolism*
Polysaccharide-Lyases / metabolism
Protein Binding
Rats
Recombinant Proteins / genetics
Rhinitis, Allergic, Seasonal / immunology*
Tobacco
Tobacco Mosaic Virus / genetics*
Grant Support
ID/Acronym/Agency:
ES06676/ES/NIEHS NIH HHS; K08 AI055792/AI/NIAID NIH HHS; K08 AI055792/AI/NIAID NIH HHS; R01 AI052428/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/Allergens; 0/Antigens, Plant; 0/Plant Proteins; 0/Recombinant Proteins; 0/jun a 1 protein, Juniperus ashei; 37341-29-0/Immunoglobulin E; EC 4.2.2.-/Polysaccharide-Lyases; EC 4.2.2.2/pectate lyase
Comments/Corrections
Comment In:
Int Arch Allergy Immunol. 2010;153(4):431-3   [PMID:  20628258 ]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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