Polygalacturonase (pectinase), a new oilseed rape allergen.
BACKGROUND
Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry.


METHODS
Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing and MALDI-TOF mass spectrometry analysis.


RESULTS
Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis.


CONCLUSIONS
The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients.
Chardin
H
H
Institut Pasteur, Paris, France; Université René Descartes, Paris, France.
Mayer
C
C
Sénéchal
H
H
Poncet
P
P
Clément
G
G
Wal
J M
JM
Desvaux
F X
FX
Peltre
G
G
D000485:Allergens / Q000032:analysis / Q000276:immunology*; D015153:Blotting, Western; D029741:Brassica rapa / Q000276:immunology*; D015180:Electrophoresis, Gel, Two-Dimensional; D005229:Fatty Acids, Monounsaturated; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007527:Isoenzymes / Q000032:analysis; D013058:Mass Spectrometry; D010938:Plant Oils; D010940:Plant Proteins / Q000276:immunology; D011058:Pollen / Q000276:immunology*; D011096:Polygalacturonase / Q000276:immunology*; D000074262:Rapeseed Oil
D000485:Allergens; D005229:Fatty Acids, Monounsaturated; D007527:Isoenzymes; D010938:Plant Oils; D010940:Plant Proteins; D000074262:Rapeseed Oil; D007073:Immunoglobulin E; D011096:Polygalacturonase