title: Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.
abstract: Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.
authors.affiliation: Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, Austria. ines.swoboda@akhwein.at
authors.forename: Ines
authors.initials: I
authors.lastname: Swoboda
authors.forename: Monika
authors.initials: M
authors.lastname: Grote
authors.forename: Petra
authors.initials: P
authors.lastname: Verdino
authors.forename: Walter
authors.initials: W
authors.lastname: Keller
authors.forename: Mohan B
authors.initials: MB
authors.lastname: Singh
authors.forename: Nicole
authors.initials: N
authors.lastname: De Weerd
authors.forename: Wolfgang R
authors.initials: WR
authors.lastname: Sperr
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Nadja
authors.initials: N
authors.lastname: Balic
authors.forename: Rudolf
authors.initials: R
authors.lastname: Reichelt
authors.forename: Roland
authors.initials: R
authors.lastname: Suck
authors.forename: Helmut
authors.initials: H
authors.lastname: Fiebig
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta
authors.forename: Susanne
authors.initials: S
authors.lastname: Spitzauer
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification / Q000648:ultrastructure; D000595:Amino Acid Sequence; D019138:Antibodies, Blocking / Q000096:biosynthesis / Q000378:metabolism; D019306:Artemisia / Q000201:enzymology / Q000276:immunology* / Q000648:ultrastructure; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D001667:Binding, Competitive / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D017124:Conserved Sequence; D003888:Desensitization, Immunologic / Q000379:methods; D006636:Histamine Release / Q000276:immunology; D007073:Immunoglobulin E / Q000378:metabolism; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010316:Particle Size; D010368:Pectins / Q000378:metabolism; D031784:Phleum / Q000201:enzymology / Q000276:immunology* / Q000648:ultrastructure; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D011058:Pollen / Q000201:enzymology* / Q000276:immunology* / Q000648:ultrastructure; D011096:Polygalacturonase / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D012130:Respiratory Hypersensitivity / Q000175:diagnosis / Q000201:enzymology* / Q000276:immunology*; D020539:Sequence Analysis, Protein
chemical_list: D000485:Allergens; D019138:Antibodies, Blocking; D015415:Biomarkers; D007074:Immunoglobulin G; D010940:Plant Proteins; C407403:phl p 13 protein, Phleum pratense; D007073:Immunoglobulin E; D010368:Pectins; D011096:Polygalacturonase